Registration Dossier
Registration Dossier
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EC number: 220-666-8 | CAS number: 2855-13-2
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
OECD 421 /422 DRF studies
In an OECD 421 reproductive screening study the test item was tested in rats to obtain preliminary information on possible effects on reproduction and/or development and to determine dose levels for the OECD 443 study (LPT, 2019). The test item was administered orally to rats via the drinking water at dose levels of 20, 60 or 160/120 mg test item /kg b.w./day.
Unfortunately, it was not possible to determine adequate dose levels for the OECD 443 study. The animals refused to consummate the drinking water because of the bad smell of the test substance. As a consequence, the reproductive screening study was performed with another form of oral administration. It was chosen to perform an OECD 422 study with rats via oral gavage.
In this OECD 422 reproduction screening study the test item was tested in rats for reproductive (fertility) effects (Provivo, 2022). No influence was noted on the number and length of the estrous cycles, the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period. However, due to mortality, the high dose level was decreased to 240 mg test item /kg b.w./day, starting on test day 28.
The NOAEL for reproductive toxicity was above 300/240 mg/kg/day. The NOAEL for the parental toxicity was 100 mg test item/kg/day. Hence, the dose level of 240 mg/kg/day was considered to be the maximum tolerated dose for the F0 Generation of the following OECD 443 study with no severe systemic effects to be expected in the F1 Generation. However, due to mortality, changes in behaviour and the external appearance of the animals within the OECD 443 study, the high dose level was decreased to 160 mg test item /kg b.w./day on test day 51.
OECD 443 study
Based on the ECHA decision the registrant conducted the OECD 443 study with the basic study design with a 10-week pre-mating (Cohorts 1A, and 1B) without extension to mate the Cohort 1 B animals to produce the F2 generation.
In this OECD 443 study (Provivo, 2022) no adverse effect on mating performance, fertility or gestation length was detected.
The oral exposure to 25, 80, and 240/160 mg test item/kg bw (male and female Sprague-Dawley rats) led to no detectable changes in fertility. In detail, no test item-related influence was noted on the reproductive performance of the parental animals (number and length of estrous cycles, fertility and gestation index, pre-coital time and gestation length).
The prenatal development of the pups (number of resorptions, stillbirths and live born pups) and the postnatal development of the pups (pup body weight, viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights) was also not affected by the test item. No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.
No test item-related influence was noted on the development of the reproductive system (time points of sexual maturations, number and length of oestrous cycles, sperm parameter, detailed histopathological examination of testis and epididymides, number of primordial and growing follicles and number of corpora lutea in the ovaries).
Therefore, the No Observed Adverse Effect Level (NOAEL) for reproductive was considered to be above 240 mg test item/kg/day.
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity in the F0 and in the adult animals of Cohort 1A and 1B was determined to be 80 mg/kg/day.
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- August 2018 - November 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- This OECD 421 study is used as dose range finding study for the OECD 443 study (Provivo, 2021).
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Remarks:
- WI IGS/ Crl:WI
- Details on species / strain selection:
- For this study Wistar rats bred by Charles River Laboratories Germany GmbH were used. The healthy nulliparous adult animals were randomised and assigned to the treatment groups and cages. The body weight range did not exceed 20% of the mean weight for each sex at the time of selection. The rat is a commonly used rodent species for such studies.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld
- Strain: Rat / Wistar WI IGS / Crl:WI
- Sex. male and female
- Age: Young adult rats, approx 10 weeks old (71 days) at the start of the treatment
- Weight at study initiation: Males: 378.9 g - 454.0 g, females: 207.5 g - 258.2 g
- Housing: With the exception of the mating period, the males and females (F0-Generation) were kept singly in MAKROLON cages
- Diet (e.g. ad libitum): ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany
- Water (e.g. ad libitum): tap water
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 °C
- Humidity (%): 55 +/- 15 %
- Air changes (per hr): was at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours daily
- Route of administration:
- oral: drinking water
- Vehicle:
- water
- Remarks:
- tap water
- Details on exposure:
- Route of administration was oral, via the drinking water. The test item-drinking water mixture was prepared once weekly by mixing the respective amounts of the test item with the drinking water. The test item-concentration in the drinking water was adjusted to the relative drinking water consumption of the animals of each group and sex at the beginning of the respective test week.
- Details on mating procedure:
- Sexually mature male and female rats were paired monogamously. One male and 1 female animal were placed together in one cage during the mating period. The female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug. The day of conception (day 0 of gestation or GD0) was considered to be the day on which sperm was found.
With the exception of one female that showed a mating period of 16 test days all other animals were mated within this 14 days. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The concentration and stability of the test item-drinking water mixtures were determined for the first preparation at the start of treatment, at the end of the study after weaning of the first litters and at dose change (group 4 only) for the males and females.
For the analysis of the test item-drinking water mixtures, two aliquots of approximately 5 mL each were taken and stored at ≤-20°C ± 10% until analysis at LPT.
Analysis and reporting of the test item-drinking water mixtures were perhormed by LPT.
With one exception, the concentration of the test item in the test item-drinking water mixtures was between 91.5% and 102.4% of the nominal concentration (see text table 7-25 below). This demonstrated that the test item-drinking water mixtures were correctly prepared and stable for at least 7 days under laboratory conditions. - Duration of treatment / exposure:
- The study animals were treated during the following periods:
Males were treated 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30 until test day 33 at maximum) and during the post-mating period until test day 55.
The females were treated 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30 until test day 33 at maximum) and during the lactation period until test day 65 to 69. - Frequency of treatment:
- continuously via drinking water
- Details on study schedule:
- - Groups of ten male and ten female animals were treated daily at the appropriate dose level throughout the study
- Prior to the start of treatment and once weekly, all animals were observed for signs of functional/behavioural toxicity. Oestrus cyclicity is evaluated followed by daily monitoring of vaginal smears from beginning of treatmrnt until evidence of mating
- On Day 15, all animals were paired on a 1 male:1 female basis within each dose group for a maximum of fourteen days.
- Pregnant females were allowed to give birth and maintain their offspring until Day 4 post partum. Evaluation of each litter size, litter weight, mean pup weight, clinical observations and landmark developmental signs were also performed during this period.
- At the end of the dosing period on day of necropsy 5 males and 5 femals were randomly selected for laboratory examinations (clinical chemistry and haematology)
- Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically on Day 43.
- At Day 5 post partum, all females and offspring were killed and examined macroscopically. - Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- control group
- Dose / conc.:
- 20 mg/kg bw/day (actual dose received)
- Remarks:
- low dose group
- Dose / conc.:
- 60 mg/kg bw/day (actual dose received)
- Remarks:
- intermediate dose group
- Dose / conc.:
- 160 mg/kg bw/day (actual dose received)
- Remarks:
- high dose group, reduction on test day 31 to 120 mg/kg bw d
- No. of animals per sex per dose:
- 10 males and 10 females per dose group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
The dose levels were selected in agreement with the Sponsor based on the results of a 90-day repeated dose toxicity study according to OECD guideline 408.
Nominal dose levels of 20, 60 and 160 mg/kg were selected for this OECD 421 study, since the treatment period was up to 12 weeks. - Positive control:
- no
- Parental animals: Observations and examinations:
- Clinical signs
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was checked twice daily. Animals which died prematurely were necropsied as soon as possible after exitus. However, no premature death was noted.
Body weight
The adult animals were weighed on each day of dosing for dose adjustment and at sacrifice; the individual body weights were recorded.
The report included weekly values for the male animals (starting on test day 15) and the body weight on the day of sacrifice.
Days on which body weight is reported:
Pre-mating period: Test days 15, 22,29
Gestation period: Gestation days 0, 7, 14, 20
Lactation period: Lactation days 1, 4, 13
Food and drinking water consumption
Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week (pre-mating and gestation) or treatment period (lactation). From these data the relative food consumption (in g/kg b.w./day) was determined.
Days on which food consumption is reported:
Study period Males Females
Pre-mating period TD15 - TD 22 TD15 - TD 22
TD 22 - TD 29 TD 22 - TD 29
Mating period None None
Gestation period Not applicable GD7, GD14 and GD21
Lactation period Not applicable LD1, LD8 and LD12
Drinking water consumption was monitored daily by visual appraisal throughout the study.
Reproductive parameters
Pre-coital time and gestation length
-number of pregnant females
-duration of pre-coital time
-gestation length
(The duration of gestation was calculated from gestation day 0 (day of positive sperm detection) until (but not including) lactation day 1 (lactation day 1: morning after littering when no signs of littering were noted anymore)).
Implantation sites
-distribution in the uterine horns (left or right uterus horn)
-total number per dam
-mean number per dam (mean value of the individual values per dam in each group)
-total number per group
Laboratory examinations
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight on the day of necropsy.
All relevant hematological and clinical bichemical parameter were determined.
T4 Determination
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 7.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below.
Blood withdrawal was performed by randomization of the parental male and female animals. The male animals of all test groups were completely randomized in a one- step process, the female animals in different staggers according to their litter day. - Oestrous cyclicity (parental animals):
- During the 14-day pre-exposure period (TD 1 to TD 14), the estrus cycle of the female animals was monitored to yield study groups of at least 10 animals each with a normal estrus cycle. Animals that failed to exhibit typical 4 to 5 day cycles were excluded from the randomization process on test day 14 that was performed to allocate the female animals to the test groups of the main study.
- Sperm parameters (parental animals):
- Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis during histopathology.
- Litter observations:
- As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities.
Number of pups (for both sexes and for male and female pups)
- at birth (alive and dead)
- after 4 and 13 days of life
The above mentioned number of pups is given as total per group, total per dam and mean number per dam (mean value of the individual values per dam in each group)
Number of stillbirths
- total per group
- total number per dam
Number of pups with malformations
- total per group
- per dam
The following examinations/observations were done for the offspring.
Counting
Live pups were counted, sexed and weighed on post-natal days 1, 4 and 13.
Ano-genital distance
On post-natal day 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale. The AGD was normalized to the cube root of body weight.
Litter adjustment on PND 4
After counting on PND 4, the litters were adjusted to 10 pups per litter by eliminating (culling) surplus pups using a randomization scheme generated by Provantis®.
Blood sampling for thyroid hormone (T4) determination
On PND 4 and on PND 13 blood samples for T4 hormone level determination were taken from 2 selected pups per litter, if possible from one male and one female pup. On PND 4 the culled surplus pups were used for blood collection.
Male nipples counting
Nipples were counted in all male pups on PND 13 (shortly before scheduled sacrifice).
T4 Determination
Determination of the pups used for blood withdrawal:
On lactation day 4 (PND4) and lactation day 13 (PND13) the litter sequence of pup blood withdrawal was determined by randomization of the dams. The collection of the pups for blood withdrawal from the individual litters occurred in an ascending order (the male and female pups per dam with the lowest number were used, if possible). - Postmortem examinations (parental animals):
- Gross necropsy
Vaginal smears were examined on the day of necropsy to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.
The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
Males On test day 55
Dams On lactation days 14 - 16
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
During necropsy the number of implantation sites was recorded in the female animals.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
Organs weighed
The organs which were weighed before fixation are listed below. The organ weighing was performed for all adult male and female animals.
Epididymis, Thyroid including parathyroids, Kidney, as a whole: prostate, seminal vesicles, Liver with coagulating glands, Ovary, Uterus including cervix, Testicle
Thyroid weight was determined after fixation; paired organs were weighed individually and identified as left or right.
Histopathology
Histopathological examination was performed on the organs of the animals of the control group and the high dose group (groups 1 and 4).
The organs (labelled with study number, species, animal number, type of sample) were dispatched to AnaPath for histopathological processing.
The histotechnique was performed by AnaPath Services according to all relevant AnaPath SOPs.
Prepared and evaluated sections from the adult animals of group 1 and 4.
Epididymis
Ovary
Testicle
Kidney
Liver
Thyroid
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of all males of groups 1 and 4 following H-E and PAS staining.
The other preserved organs (group 2 and 3 animals) will be examined when necessary, i.e. when changes are seen in the highest dose group. - Postmortem examinations (offspring):
- Histopathological examination was performed on the organs of the pups of the control group and the high dose group (groups 1 and 4).
Prepared and evaluated sections from the pups of group 1 and 4 (1 male and 1 female pup):
Kidney
Liver
Thyroid - Statistics:
- The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 9.4.0.1, Instem LSS Ltd) using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
Significantly different data are indicated in the summary tables of the result sections of the report.
Non-parametrical data
The statistical evaluation of non-parametrical values was done by comparison of the group values using the FISHER or the Chi2 test with the StatXact 4.0.1 software:
FISHER test:
- Fertility index and gestation index of the parental females 'Reproductive Outcomes and Indices per Group - Females'.
Chi2 test:
- Birth and live birth index, post-impantation loss and viability indices of the pups
However, for the birth and live birth index, the post-implantation loss as well as for the viability indices an ANOVA / DUNNETT test was additionally performed using the individual indices per dam. The results are given in the summary tables of the respective sections. - Reproductive indices:
- Gestation Index [%] =Number of dams with live pups / Number of pregnant rats x 100
Female Fertility Index [%] = Number of pregnant females / Number of females used for pairing x 100
For each litter/dam and group the following indices were determined:
Birth Index [%]= Total number of pups born (alive + dead) / Number of implantation scars x 100
Live Birth Index [%] = Number of pups alive on day 0/1 of lactation / Total number of pups (alive + dead) x 100
Viability Index [%] pre-cull = Number of pups alive on day 4 (pre-cull) / Number of pups alive on day 0/1 x 100
Viability Index [%] post-cull = Number of pups alive on day 13 / Number of pups alive on day 4 (post-cull) x 100
Post-implantation loss [%] = Implantations - living fetuses / Implantations x 100
- Offspring viability indices:
- Birth and live birth index, post-impantation loss and viability indices of the pups
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No changes in behaviour, the external appearance or the appearance of the faeces were noted for the male and female animals of the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- No animal of the control group and in the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day) died prematurely.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Males
No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the low and the intermediate dose group (20 or 60 mg test item/kg b.w./day).
A marked reduction in body weight (negative body weight gain) was noted at the high dose level (160 mg test item/kg b.w./day) during the first 2 weeks of treatment (test weeks 3 and 4; test days 15 to 29). This led to a body weight value on test day 29 that was 18.9% (p ≤ 0.01) below the value of the control group.
This reduction in body weight was caused by a severely reduced drinking water consumption as the animals refused to drink the test item-drinking water mixture, most probably due to a bad taste of the test item. After the decrease of the test item concentration in the drinking water on test day 31 to obtain a dose level of 120 mg test item/kg b.w./day, the drinking water consumption and the body weight of the high dosed animals increased. However, the body weight of the high dosed animals remained below the body weight of the the animals of the control group and the low and the intermediate dose group. On test day 54 (one day before sacrifice), the body weight of the high dosed animals was still 9.5% below the value of the control group (p ≤ 0.01).
However, the reduced body weight that was noted for the high dosed animals was due to the reduced drinking water consumption caused by the bad taste of the test item and not caused by a toxic effect of the test item. Hence, the reductions in body weight and body weight gain (see below on the following page) were not considered to be of toxicological relevance.
The percentage of body weight gain was decreased in the high dose group in comparison to the control group and the low and the intermediate dose group
Females
No test item-related differences in body weight and body weight gain were noted between the female rats of the control group and the female rats of the low and the intermediate dose group (20 or 60 mg test item/kg b.w./day) during the pre-mating, the gestation and the lactation period.
At the high dose level (160/120 mg test item/kg b.w./day) a slightly reduced body weight in comparison to the control group was noted during the gestation and the lactation period (between 4.0% and 8.8% below the value of the control group; statistically significant at p ≤ 0.05 on gestation day 0 and lactation day 13).
As for the male animals, the slight reduction in body weight is considered to be a secondary effect of the reduced drinking water consumption of the high dosed females (caused by the bad taste of the test item) and not considered to be of toxicological relevance.
The slight reductions in body weight that were noted for the high dosed animals during the gestation and the lactation period had no effect on the body weight gain of the high dosed females during these periods
The mean body weights of the female rats during the pre-mating period, the gestation and the lactation period are shown in Figures 2 to 4 on the following pages.
BODY WEIGHT AT AUTOPSY
Males
No test item-related differences were noted on the body weight at autopsy between the control group and the treatment groups (20 or 60 mg test item/kg b.w./day).
A slight but statistically not significant reduction was noted at the high dose level (160/120 mg test item/kg b.w./day) (5.9% below the value of the control group). This was in accordance with the last difference in live body weight on test day 55 (6.1% below the value of the control group) after the animals fasted overnight and was considered not to be of toxicological relevance.
Females
No test item-related differences were noted on the body weight at autopsy between the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day). - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- Males: Pre-mating period
During the pre-mating period (test days 15 to 28) no test item-related difference in food consumption was noted between the control group and the low dose group (20 mg test item/kg b.w./day). The statistically significant (p ≤ 0.05) reduction that was noted during test week 4 (test days 22 -29) was considered to be spontaneous, as it was only very small.
The statistically significant reductions that were noted at the intermediate and the high dose group (60 or 160/120 mg test item/kg b.w./day) were considered as a secondary effect on the reduced drinking water consumption of the animals and not considered to be of toxicological relevance.
No food intake of female animals was recorded during the mating period as both sexes were housed together.
Females: Pre-mating, gestation and lactation period
No differences of toxicological relevance were noted between the female rats of the control group and the female rats of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day) during the pre-mating, the gestation and the lactation period.
However, slight but statistically significant reductions in food consumption were noted in test week 4 for the animals of the intermediate and the high dose group. These reductions were considered as a secondary effect of the reduced drinking water consumption. Hence, they were not of toxicological relevance. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- MALES
There was a clear dose dependent tendency of the male rats to refuse the drinking of the test item-drinking water mixtures. The refusal of the drinking water was caused by the properties of the added test item (e.g. bad taste and/or smell) and is not considered as an adverse toxicological effect on the animals of the treatment groups (20, 60 or 160/120 mg/kg b.w./day).
The intake of the test-item drinking water mixture during the course of the study is described in detail below:
At the low dose level (20 mg/kg b.w./day) a statistically significantly reduced drinking water consumption was only noted in test week 7 (13.0% below the value of the control group).
At the intermediate dose level (60 mg/kg b.w./day) similarly reduced levels of drinking water were noted in nearly all test weeks (between 16.5% and 25.6% below the value of the control group; p ≤ 0.05 or 0.01).
At the high dose level (160/120 mg/kg b.w./day) marked and statistically significant (p ≤ 0.01) reductions in drinking water consumption were noted for all test weeks. The maximum reductions in drinking water consumption were noted in test week 4 (test days 22 - 29) with a test item concentration in the drinking water of 3486 mg/L and the beginning of test week 5 (from test days 29 - 31), equivalent to a dose of 160 mg/kg b.w./day.
Test week 5 started with the beginning of the mating period (test day 29). During the mating period the male and female pairs of the high dose group received a test item concentration of 2778 mg/L in the drinking water, which was an average value between the calculated test item concentrations for the male (4324 mg/L) and the female (2362 mg/L) animals alone, to achieve a nominal dose level of 160 mg/kg b.w./day for both sexes.
Between test days 29 - 31 most of the animals were in the mating period and received the averaged test item-concentration of 2778 mg/L to achieve a nominal dose level of 160 mg(kg b.w./day. Though the test item concentration was lower as in test week 4, the drinking water consumption of the male animals was still markedly reduced between test day 29 -31.
Due to this markedly reduced drinking water consumption that caused a marked reduction in body weight the high dose level was reduced from 160 mg/kg b.w./day to 120 mg/kg b.w./day on test day 31.
Due to this dose reduction, the averaged test item concentration during the mating period was reduced from 2778 mg/mL to 2083 mg/mL for both sexes. The test item concentration in the drinking water for the male animals who were sitting alone in their cage again after the mating period was reduced from 4324 mg/mL to 3243 mg/mL. The reduction of the test item concentration in the drinking water led to an increase in the drinking water consumption for the male rats of the high dose group from test day 31 onwards. However, due to the increased drinking water consumption the concentrations used turned out to be too high and led to a too high test item intake in the second part of test week 5. Hence the concentration of the test item was further reduced for the following test weeks. However, from test day 31 onwards, the drinking water consumption that were noted for the male animals of the high dose group remained on a nearly constant level. This level was still below the control group but above the values that were measured for the male animals of the high dose group before the reduction of the dose level.
Though the drinking water consumption of the high dosed animals remained below the control group and the low and the intermediate dose group, the drinking water consumption was now sufficient to support a positive body weight gain for the male rats of the high dose group during the post-mating period.
FEMALES
As for the male rats, a dose-dependent tendency of the rats to refuse the drinking of the test item-drinking water mixtures was also noted for the female animals.
The reductions in drinking water consumption were in the range of those noted for the male animals, with the exception of test weeks 3 and 4, where the reduction in drinking water consumption was much more pronounced for the male animals of the high dose group as for the female animals of the high dose group.
RELATIVE DRINKING WATER CONSUMPTION
Males and females
The reductions in the relative drinking water consumption in comparison to the control group for the male and female rats of the treatment groups were similar to the reductions that were noted for the total drinking water consumption for the male and female animals.
For detailed information on test item uptake via drinking water see section `other effects` - Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related differences were noted between the male and female animals of the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day) for the examined haematological parameters. Decreased Neutrophilic granulocytes and decreased large unstained cells were observed in the high dose group males, however, these findings are considered to be spontanious and not treatment related.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related differences were noted between the male and female animals of the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day) for the examined biochemical parameters. aP was increased on test day 55 in the high dosed males. However, these finding is considered to be spontanious and not treatment related.
T4 hormone determination
Males
No test item-related changes were noted for the T4 levels of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
A slight but statistically significant reduction was noted at the high dose level. However, this slight reduction was considered to be spontaneous. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- The histopathological evaluation of selected organs from the animals of the control and the high dose group (160/120 mg test item/kg b.w./day) was performed and reported by AnaPath GmbH, Switzerland.
No indication of toxicity was noted for the examined organs (epididymides, testes, ovaries, kidneys, liver and the thyroid gland) of the male and female animals of the high dose group (160/120 mg test item/kg b.w./day).
The sperm staging revealed no test item-related alterations. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- effects observed, treatment-related
- Description (incidence and severity):
- TEST ITEM INTAKE
The concentration of the test item in the drinking water was adjusted weekly to the relative drinking water consumption of the male and female animals. The real test item intake of the animals was calculated using the total amount of drinking water and the body weight of the animals and designated as the actual dose level (mg test item/kg b.w./day).
Males and females
In most cases the calculated actual dose level was nearly in the range of the nominal dose level for the male and female animals of all dose group (20, 60 or 160/120 test item mg/kg b.w./day).
However, the following noticeable discrepancies were noted for the male and the female animals:
Males:
At the high dose level (160 test item mg/kg b.w./day), the actual dose levels were clearly below the nominal dose level of 160 mg/kg in test weeks 3 and 4 and the beginning of test week 5 (test days 29 - 31). This was due to the markedly reduced consumption of the test-item drinking water mixtures during this period.
After the dose level reduction to 120 mg test item/kg b.w./day the actual dose level was in the range of the reduced nominal dose level of 120 mg/kg in test weeks 6 to 8. Only between test days 31 and 36 the actual dose level was clearly above the nominal dose level as the test item-concentration in the drinking water turned out to be too high for the period between test days 31 and 36, due to the increased drinking water consumption after the dose level reduction.
Females:
Increased actual dose levels were noted for all dose groups (20, 60 or 160/120 mg test item/kg b.w./day) in test weeks 8 and 9 when most animals where in the lactation period. This was due to the increased absolute and relative drinking water consumption that was noted for all dose groups during the lactation period in comparison to the pre-mating/mating and the gestation period. - Reproductive function: oestrous cycle:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No differences were noted in the mean number of oestrus cycles per dam during the pre-mating and mating period between the female animals of the control group and the female animals of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
However, one animal each of the control group (no. 11), the low dose group (no. 31) and the high dose group (no. 71) was noted with an abnormal cyclus in the form of an elongated diestrus period (between 6 and 12 consecutive days in the diestrus stage). This observation was considered to be spontaneous, moreover as all 3 animals became pregnant. - Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- FERTILITY
No test item-related influence on the fertility index of the female rats was noted for any of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
All female rats that were used for pairing were successfully mated (confirmed by sperm detection). However, one female animal of the control group and one female animal of the high dose group did not become pregnant, leading to a fertility index of 90% for the female animals of the control group and the high dose group. The occurrence of one non-pregnant female at the high dose level was considered to be spontaneous, as one non-pregnant female was also noted at the control group.
GESTATION INDEX
No test item-related influence on the gestation index was noted for the female rats treated with 20, 60 or 160/120 mg test item/kg b.w./day. All pregnant animals of the control group and the treatment groups delivered live pups, leading to gestation indices of 100% for all groups.
PRE COITAL TIME
No test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day). With the exception of female no. 11 of the control group with a pre-coital time of 16 days, the pre-coital time of all other females was between 1 and 4 days. However, live pups were also noted for animal no. 11.
GESTATION LENGTH
No test item-related differences were noted for the length of the gestation period between the rats of the control group and the rats of the treatment groups (20 or 60 or 160/120 mg test item/kg b.w./day).
Birth indices and post-implantation loss
No test item-related differences were noted for the mean number of implantation sites, the mean number of pups born (alive and dead) and the mean number of live born pups between the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
Correlating, the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day). - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- > 160 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects were observed up to the highest tested dose
- Clinical signs:
- not examined
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- Number of live pups
No test item-related difference was noted between the number of live pups per dam of the control group and the number of pups per dam of the low and the intermediate dose groups (20 or 60 mg test item/kg b.w./day).
At the high dose level (160/120 mg test item/kg b.w./day) a reduced number of pups per dam (22.6% below the value of the control group for male and female pups together; p ≤ 0.05) was noted on lactation day 13. This was due to the increased number of prematurely deceased pups at the high dose level during the post-cull period. However, this was not considered as a toxic effect on pup development.
Viability index: Pre-cull period
No difference between the control group and the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
Viability index: Post-cull period
No test item-related difference was noted for the post cull viability index between the control group and the low and the intermediate dose group (20 or 60 mg test item/kg b.w./day).
A statistically significantly decreased viability index was noted at the high dose level (160/120 mg test item/kg b.w./day) for the post-cull period. In detail, 24 pups died prematurely between lactation day 5 and 13 in comparison to 6 prematurely deceased pups in the control group.
The increased number of prematurely deceased pups at the high dose level was considered to be a secondary effect of the reduced drinking water consumption of the dams and not as a toxic effect of the test item on the development of the pups. In detail, the decreased drinking water consumption may have resulted in a decreased milk production of the dams, so that weaker pups failed to receive an adequate amount of milk. However, as 21 of 24 of the prematurely deceased pups during the post-cull period were cannibalized, an examination of the milk status was not possible.
The surviving pups seem to have received an adequate amount of milk as no difference in body weight was noted between the surviving pups of the high dose group and the pups of the control group on lactation day 13. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- BODY WEIGHT
+No test item-related difference was noted between the mean body weight of the pups from the dams of the control group and the mean body weight of the pups from the dams of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day) on lactation days 1, 4 and 13.
No runt was noted in the control group and in the treatment groups.
LITTER WEIGHT
No test item-related difference was noted between the litter weight of the dams of the control group and the litter weight of the dams of the low and the intermediate dose group (20 or 60 mg test item/kg b.w./day).
At the high dose level (160/120 mg test item/kg b.w./day) a reduced litter weight (22.3% below the value of the control group for male and female pups together; statistically not significant) was noted on lactation day 13. This was due to the increased number of prematurely deceased pups that was noted at the high dose level for the post-cull period, leading to a reduced number of live pups on lactation day 13. However, as the premature death of these pups was not considered as a toxic effect on pup development (secondary effect of the reduced consumption of drinking water and a possibly reduced milk production of the dams), the reduced litter weight that was noted at the high dose level was not considered to be of toxicological relevance. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No test item-related changes were noted for the T4 levels of the male and female pups of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- no effects observed
- Description (incidence and severity):
- No test item-related influence was noted on the absolute and the relative ano-genital distance of the male and the female pups of all treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
- Nipple retention in male pups:
- no effects observed
- Description (incidence and severity):
- No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (20, 60 or 160/120 mg test item/kg b.w./day).
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- External examination of the pups
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 20, 60 or 160/120 mg test item/kg b.w./day after terminal sacrifice on lactation day 13 or for the pups that died during the lactation period. - Histopathological findings:
- no effects observed
- Description (incidence and severity):
- The histopathological examination of selected organs of pups from the dams of the control and the high dose group (160/120 mg test item/kg b.w./day) was performed and reported by AnaPath GmbH, Switzerland.
No indications of toxicity were noted for the examined organs (kidneys, liver and the thyroid gland) from 2 selected pups per litter from the dams of the control and the high dose group (160/120 mg test item/kg b.w./day). - Other effects:
- not examined
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- > 160 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects were seen up to the highest tested dose with regard to pre- and postnatal development
- Key result
- Reproductive effects observed:
- no
- Treatment related:
- no
- Conclusions:
- The aim of the study was to obtain preliminary information on possible effects of the test item IPDA on reproduction and/or development according to OECD guideline 421. The test item IPDA was administered orally to rats via the drinking water at dose levels of 20, 60 or 160/120 mg IPDA/kg b.w./day. Unfortunately it was not possible to determine adequate dose levels for the OECD 443 study. The animals refused to consumate the drinking water because of the bad smell of the test substance. As a consequence the OECD 421 study had to be performed with another route of exposure. It was chosen to perform the OECD 422 study with rats and gavage as administration route.
- Executive summary:
The aim of the study was to obtain preliminary information on possible effects of the test item IPDA on reproduction and/or development according to OECD guideline 421. The test item IPDA was administered orally to rats via the drinking water at dose levels of 20, 60 or 160/120 mg IPDA/kg b.w./day.
Parental male and female animals
No premature death and no changes in behaviour, the external appearance and the appearance of the faeces were noted.
Also the laboratory examinations and the examinations at necropsy (examination of haematological and biochemical parameters, determination of the T4 level of the male animals, determination of organ weights, the macroscopical and histopathological examination) revealed no test item-related changes.
However, most probably due to the properties of the test item in the drinking water a dose dependent refusal of intake of the test item-drinking water mixtures was noted for the male and female animals. This was most pronounced for the male animals of the high dose group (160/120 mg IPDA/kg b.w./day) during the first and the second treatment week and caused a reduction in body weight for the male animals. The body weight of the high dosed males recovered as the consumption of drinking water increased after the reduction of the test item concentration in the drinking water (dose level reduction from 160 to 120 mg IPDA/kg b.w./day).
The concentration of the test item in the test item-drinking water mixture was adjusted weekly to the relative drinking water consumption of the male and female animals. The actual dose levels of test item intake were calculated for the male and female animals using the total drinking water consumption and the body weight of the animals:
In most cases the actual dose levels were nearly in the range of the nominal dose levels for the male and female animals of all dose groups.
However, for the male animals of the high dose group (160/120 mg test item/kg b.w./day), the marked reduction in drinking water consumption before the reduction of the dose level revealed a decreased intake of test item via the drinking water. Hence, the calculated actual dose level was below the nominal dose level during the first and second treatment week. After the dose level reduction the actual test item-intake was in the range of the nominal dose level.
For the female animals increased actual dose levels were noted during the lactation period for all dose groups (20, 60or160/120 mg IPDA/kg b.w./day). This was due to increased levels of absolute and relative drinking water consumption during the lactation period in comparison to the pre-mating and gestation period that were noted for all dose groups.
Reproductive toxicity
Parental females
No influence was noted on the estrus cycles, the fertility, the gestation index, the pre-coital time and the gestation length.
Pups
No influence on the pre-natal developmentwas noted (number of implantation sites, birth and live birth index, percentage of post-implantation loss).
During the post-natal development no influence was noted on body weight, the ano-genial distance, the number of nipples of the male pups and the T4 level.
No abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.
A reduced viability index was noted during the post-cull period for the pups of the high dose group (160/120 mg IPDA/kg b.w./day). This was considered as a secondary effect of the reduced consumption of drinking water of the high dosed females in comparison to the females of the control and the low and the intermediate dose group. This lower drinking water consumption led to a reduced milk production and hence, an inadequate feeding of some of the weaker pups. Therefore, the reduced viability index was not considered to be of toxicological relevance.
Though there were no toxicological relevant findings, the maximum dose level for possible further rat studies was considered to be 120 mg IPDA/kg b.w./day for the administration via the drinking water. At this dose level and also at the lower tested dose levels of 20 and 60 mg IPDA/kg b.w./day the test item in the drinking water already evoked a reduced drinking water consumption, but without secondary effects in the form of body weight loss or a possibly reduced milk production of the dams.
Considering these secondary effects that were due to the refusal of intake of the test item-drinking water mixture by the animals and considering the fact that no systemic toxicological effects occured, the study has to be performed with another route of administration. Hence, an OECD 422 study via gavage was performed to be able to determine suitable dose levels for the requested OECD 443 study.
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- Study started on 24th of July 2019 and the end of in-life period was on10 October 2019, final report received on 18th of April 2022.
- Reliability:
- 1 (reliable without restriction)
- Justification for type of information:
- This OECD 422 study is used as dose range finding study for the OECD 443 study (Provivo, 2021).
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- adopted July 29, 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Justification for study design:
- The study was performed as dose range finding study for the EOGRTS (OECD 443).
- Species:
- rat
- Strain:
- Crj: CD(SD)
- Details on species / strain selection:
- Rat / CD® / Crl:CD(SD)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Species / Strain / Stock: Rat / CD® / Crl:CD(SD)
- Breeder: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Body weight (at start of dosing): Males: 411.8 g - 479.2 g, Females: 246.4 g - 278.9 g
- Age (at start of dosing): 70 days (young adults; sexually mature)
- Selection of species: The rat is a commonly used rodent species for such studies.
- Number of animals: Pre-exposure period: 60 female animals will be evaluated pre-exposure for oestrus cyclicity to yield 40 females with a regular oestrus cycle for the study.
Main study: 80 animals (40 males and 40 females) A sufficient number in order to grant at least 8 pregnant females per group for evaluation of the F0 generation.
- Adaption period: 7 days
- Diet (ad libitum): ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany
-Drinking water ad libitum
- Housing: With the exception of the mating period, the males and females (F0-Generation) were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Except during the mating period the animals were placed in the animal room as follows:
Male animals: On one side of the room with each dose group separated by an empty row.
Female animals: On the other side of the room with each dose group separated by an empty row.
Granulated textured wood released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week.
ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C
- Humidity: 55% ± 10%
- Air changes per hour: 15 to 20
- Photoperiod: 12 hours dark/12 hours light - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- Route of administration: Oral, via gavage
Frequency of administration: Once daily
Vehicle: sterile water (Batch no. 184928001, B. Braun Melsungen AG, 34212 Melsungen)
Application volume: 10 mL/kg b.w./day
Dosages: 0, 30, 100, 300 (240) mg/kg bw/d; High dose reduction as of test day 28, due to mortality and poor general condition of the animals.
Selection of administration route: According to OECD guideline 422
The test item formulations were freshly prepared every day and volumes administered were adjusted to the animal's actual body weight daily.
The test item was suspended in the vehicle and was administered orally at a constant volume once daily.
The control animals received the vehicle at the same administration volume daily in the same way. - Details on mating procedure:
- Sexually mature male and female rats are paired monogamously, i.e. 1 male and 1 female animal are placed in one cage during the dark period. The female is placed with the same male until evidence of mating is observed or two weeks have elapsed. The females are examined each morning for the presence of sperm. The day of conception (day 0 of gestation) is considered to be the day on which sperm is found. In case pairing is unsuccessful, re-mating of females with proven males of the same group can be considered after approximately 2 weeks of unsuccessful mating.
- Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- For each test item that is mixed with a vehicle, tests by appropriate analytical methods shall be conducted to determine the concentration, stability and homogeneity of the test item in the formulations.
For the analysis of the test item-vehicle mixtures, samples of 2 x 5 mL are taken as scheduled (and stored at -20°C±10% until analysis:
- At start of the treatment period (first dosing day; test day 15), Analysis of concentration, Immediately after preparation of the administration formulation (1 sample / dose level group; groups 2 - 4). Number of samples: 3
- At dose change (group 4), test day 28, Analysis of concentration, Immediately after preparation of the administration formulation (1 sample of group 4). Number of samples: 1
- Towards the end of the treatment period (when the majority of animals was dosed, test day 48), Analysis of concentration, During treatment always before administration to the last animal/dose level group (1 sample / dose level group; groups 2 - 4) Number of samples: 3
- Sum of all samples: 7
Results of test item formulation analysis:
Sampling /Percent of nominal concentration [%]
- immediately after preparation at the start of dosing on test day 15: 100.1 % - 102.6 %
- at dose change on test day 28: 101.7 %
- before administration to the last animal on test day 48: 100.2 % - 103.4 %
These results indicated correctly prepared test item vehicle mixtures. - Duration of treatment / exposure:
- The study animals will be treated during the following periods:
- Males+ Females: Pre-mating (test days 15 to 29 (pairing was in the evening of test day 29)), Mating (test days 30 to 43 (first mating day, confirmed by positive sperm detection, was in the morning of test day 30))
- Males: Post-mating (until test day 48 (one day before necropsy on test day 49))
- Females: Gestation and lactation period (until test days 64 to 78 (corresponding to lactation days 13 to 15). The last dosing was always one day before necropsy (necropsy was between test days 65 to 79).) - Frequency of treatment:
- daily
- Details on study schedule:
- study schedule according to OECD 422 guideline, s. also "duration of treatment/ exposure"
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- control group
- Dose / conc.:
- 30 mg/kg bw/day (nominal)
- Remarks:
- low dose group
- Dose / conc.:
- 100 mg/kg bw/day (nominal)
- Remarks:
- intermediate dose group
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Remarks:
- high dose group; 240 mg/ kg bw/ day as of test day 28, due to mortality and poor general condition of the animals.
- No. of animals per sex per dose:
- 10 males and 10 females
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Justification for dose selection
The dose levels were selected in agreement with the Sponsor based on the results of a 14-day dose range finding study.
In this 14-day dose range finding study the test item was administered orally to rats by gavage at dose levels of 250 or 500 mg/kg b.w./day for 2 weeks. Due to mortality and a reduced body weight, the high dose level was reduced to 350 mg/kg b.w./day on test day 6.
One male animal of the high dose group died at a dose level of 500 mg/kg b.w./day and another male animal after the reduction of the high dose level to 350 mg/kg b.w./day.
Changes in behaviour in the form of salivation (in many cases consistently during the whole day) and breathing sounds were noted for some male and female animals of the low dose group and / or the high dose group (250 or 500/350 mg/kg b.w./day).
Statistically significant reductions in body weight at the end of the first test week (test day 8) were noted for the female animals of the low dose group (250 mg/kg b.w./day) and for the male and female animals of the high dose group (500/350 mg/kg b.w./day). During the second test week a slight recovery of the body weight was noted for all affected sexes and dose levels. However, the values of body weight still remained below the control values.
Necropsy revealed test item related gastrointestinal changes for some male and female animals of the high dose group (500/350 mg/kg b.w./day).
No changes of toxicological relevance were noted for food consumption and no test item-related changes were noted for the haematological or biochemical parameters and the organ weights. Based on these results, the rats of the OECD 422 study were treated orally with 30, 100 or 300 mg IPDA/kg b.w./day. - Positive control:
- not needed
- Parental animals: Observations and examinations:
- CLINICAL SIGNS
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was recorded twice daily. Animals which died prematurely were necropsied as soon as possible after exitus.
Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded for each individual animal.
Cage-side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
Detailed clinical observations (parental animals):
Additionally, once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals of the parental generation. These detailed clinical observations were performed at least 2 hours after dosing.
These observations were made outside the home cage in a standard arena and at the same time, each time preferably by observers unaware of the treatment. Signs observed included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypy (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
VAGINAL SMEARS
Daily monitoring of vaginal smears will continue throughout the premating period until evidence of mating. A vaginal smear will also be taken in the morning of the day of scheduled necropsy.
NEUROLOGICAL SCREENING
Screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on Gad ), as well as the assessment of grip strength (Meyer ) and motor activity assessment were conducted as described on the following pages in five males and five females randomly selected from each study group.
The screening was conducted between approx. 2.0 hours and 4.0 hours after dosing and before any blood sampling for laboratory examinations:
5 male F0 animals per group (randomly selected) On test days 44 and 45.
5 female F0 animals per group (randomly selected) Between lactation days 10 to 13.
OBSERVATIONAL SCREENING
Righting reflex
The animal was grasped by its tail and flipped in the air approximately 60 cm above the cart surface so that it turned head over heels. The normal animal should land squarely on its feet; that means zero (0) points were scored. If it landed on its side, 1 point was scored; if it landed on its back, 2 points were scored. This test was repeated five times and the total scores were recorded.
Body temperature
An electronic probe thermometer with a blunt probe was used to take the rectal temperature, being allowed to equilibrate for 30 seconds before the reading was recorded.
Salivation
Discharge of clear fluid from the mouth is most frequently seen as beads of moisture on lips in rats. The normal state is to see none, in which case a zero (0) was recorded in the blank space of the scoring sheet. If present, a plus sign (+) was recorded in the blank.
Startle response
With the animal on the cart, the metal cage was struck with the blunt probe. The normal animal should exhibit a marked but short-lasting response, whereby a zero (0) was recorded in the blank space of the scoring sheet. If there was no response, a plus sign (+) was recorded.
Respiration
While at rest on the cart, the animal's respiration cycle was observed and evaluated in terms of a scale from 1 (reduced) to 5 (increased), with 3 being normal.
Mouth breathing
Rats are normally obligatory nose-breathers. Each animal was observed whether or not it was breathing through its mouth. If the rat was breathing through its nose, a zero (0) was recorded; mouth breathing was documented by a plus sign (+).
Urination
When an animal was removed from its cage, the pan beneath the animal's cage was examined while returning the animal to its cage. The signs of urination were evaluated on a scale of 0 (lacking) to 5 (polyuria) with 3 being normal.
Convulsions
If clinic or tonic convulsions were observed, their intensity was graded on a scale of 1 (minor) to 5 (marked) and the type was recorded. In the normal animal no convulsions should be observed, in which case a score of zero (0) was recorded.
Piloerection
The fur of the animal's back was observed whether it was raised or elevated. In the normal animal no piloerection should be observed and a score of zero (0) was recorded. If piloerection was present, a plus sign (+) was recorded.
Diarrhoea
In examining the pan beneath an animal's cage, it was noted if there were any signs of loose or liquid stools. The normal state is for there to be none (0); in case of diarrhoea the intensity was recorded on a scale of 1 (slight) to 5 (much increased).
Pupil size
It was determined if the pupils were constricted or dilated and the observations were evaluated in terms of a scale from 1 (constricted) to 5 (dilated), with 3 being normal.
Pupil response
The beam of light from the pen light was played across the eyes of the animal and the changes in pupil size were noted. In the normal animal, the pupil is constricted when the beam is on it and then dilates back to normal when the light is removed, whereby a score of zero (0) was recorded. If there was no pupil response, a minus sign (-) was recorded in the blank space.
Lacrimation
The animal was observed for the secretion and discharge of tears. The tears of rats contain a reddish pigment. No discharge is normal, whereby a score of zero (0) was recorded in the blank space of the scoring sheet. If a discharge was present, a plus sign (+) was recorded.
Impaired gait
The occurrence of abnormal gait was evaluated. The most frequent impairments are waddling (W), hunched gait (H), or ataxia (A, the inability of all the muscles to act in unison). The extent of any impairment was recorded on a scale of 1 (slight) to 5 (marked). A normal gait was documented by a score of zero (0).
Stereotypy
Each animal was evaluated for stereotypic behaviour (isolated motor acts or partial sequences of more complex behavioural patterns occurring out of context and with an abnormally high frequency). These were graded on a scale of 1 (slight) to 5 (marked). Normal behaviour was documented by a score of zero (0).
Toe pinch
The blunt probe was used to bring pressure to bear on one of the digits of the hind limb. This should evoke a response from the normal animal. The response or lack thereof was graded on a scale from 1 (absent) to 5 (exaggerated) with 3 being the normal response.
Tail pinch
The toe pinch procedure was utilized with the animal's tail instead of its hind limb and was graded on the same scale from 1 (absent) to 5 (exaggerated), with 3 being the normal response.
Wire maneuver
The animal was placed on the metal rod suspended parallel to the cart approximately 60 cm above the cart's surface. The animal's ability to move along the rod was evaluated. If impaired, a score from 1 (slightly impaired) to 5 (unable to stay on wire) was recorded. Normal movement was documented by a score of zero (0).
Hind leg splay
The hind paws were marked with ink using an ink pad. The rat was then held 30 cm above a sheet of blotting paper on the cart. The animal was dropped and the distance between the prints of the two hind paws was measured (in cm).
Positional passivity
The animal was observed after being placed in an awkward position, such as on the edge of the top of the wire-bottomed cage on the cart surface. If the animal immediately moved into a normal position, a score of zero (0) was recorded. If not, a score was recorded on a scale of 1 (slightly impaired) to 5 (cataleptic).
Tremors
Periods of continued fine movements, usually starting in the limbs and perhaps limited to them. The normal case is to have none, in which case a score of zero (0) was recorded. If tremors were present, they were graded on a scale of 1 (slight and infrequent) to 5 (continuous and marked).
Positive geotropism
The animal was placed on the inclined (approximately 30°) top surface of the wire cage with its head facing downward. It should turn 180° and face "uphill", in which case a score of zero (0) was recorded in the blank space of the scoring sheet. If this did not occur, a negative sign was recorded in the blank.
Limb rotation
One of the animal's hind limbs was taken and moved through its normal plane of rotation. In the normal state, it should rotate readily but there should be some resistance. The variations from normal were from no resistance (1) to markedly increased resistance or rigidity (5), with 3 being normal.
Auditory function
Each animal was placed in a container and observed for Preyer's reflex (twitching of the pinna) in response to a high frequency sound stimulus. The stimulus was repeated, if necessary, up to 3 times. A normal response was recorded with a plus sign (+); if there was no response a zero (0) was recorded.
FUNCTIONAL TESTS
Grip strength
Prior to testing, the gauge (Chatillon, Modell DPP - 1.0 kg) was calibrated with a set of known weights and the apparatus adjusted for the size of the animal (about 1 cm clearance on both sides of the animal). After the strain gauge was zeroed and set in the record mode, the animal was placed into the trough with the forepaws inside the triangular grasping ring. Using one hand, the animal was grasped about 2.5 cm of the way up toward the base of the tail and steadily pulled (approx. 2.5 cm/sec) away from the ring until the grip was broken. It was continued to pull the animal along the trough until the hind limbs grasp the T-bar. The trial was completed when grip of the hind limbs was broken. Three successive readings were taken for each animal with an intertrial interval long enough to record the data and zero both meters for the next trial.
Locomotor activity
The motility was measured using the TSE InfraMot system . The infrared sensor was placed on the cage and any movements were measured for a duration of 12 min by sensing the body heat image, i.e. the infrared radiation, and its spatial displacement over time.
Any movements within the cage, even brief movement events of only a few milliseconds duration, were detected and included in the activity data.
MORTALITY
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m..
BODY WEIGHT
The adult animals will be weighed on each day of dosing for dose adjustment and at sacrifice; The report included weekly values for the male animals (starting on test day 15) and the body weight on the day of sacrifice.
Days on which the female body weights are reported:
Pre-mating period: Test days 15, 22, 29
Gestation period: Gestation days 0, 7, 14, 20
Lactation period: Lactation days 1, 4, 8, 13
FOOD AND DRINKING WATER CONSUMPTION
Food intake per rat (g) was calculated using the total amount of food given to and left by each rat in each group on those days:
Study period/ Males /Females
Pre-mating period/ TD15(#1), TD22 and TD29 (#2)/ TD15 (#1), TD22 and TD29 (#2)
Mating period/ None/ None
Gestation period/ Not applicable/ GD0 (#1), GD7, GD14 and GD20 (#2)
Lactation period/ Not applicable/ LD1 (#1), LD8 and LD13 (#2)
#1: Days on which only the amount of food at food start was weighed.
#2: Days on which only the amount of food at food residue was weighed.
On the other days, the amount of food at food residue, followed by food start was weighed.
From these data the relative food consumption (in g/kg b.w./day) was determined using the following formula:
Relative food consumption (g/kg b.w./day)= ((Total food given (g) - Total food left (g))/ (Number of animal days# x Body weight (kg))
# The term 'animal days' counts one animal day for each animal alive for a whole
day; it is assumed that on the day of death an animal does not eat.
Drinking water consumption was monitored daily by visual appraisal throughout the study.
REPRODUCTIVE PERFORMANCE
The following parameters and indices were evaluated:
Reproductive parameters
- stages of the estrous cycle
- pre-coital time
- gestation length calculated from day 0 of pregnancy
Implantation sites
- number per dam
- distribution in the uterine horns (left or right)
- absolute number per group
- mean per group
Number of pups per group and per dam
- at birth (live and dead)
- on postnatal days 1, 4 and 13
Number of male and female pups per group and per dam
- at birth (alive and dead)
- on postnatal days 1, 4 and 13
Number of stillbirths
- per group
- per dam
Number of pups with malformations (see Appendix 4)
- per group
- per dam
LABORATORY EXAMINATIONS
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight at the following times:
At study termination (before necropsy) 5 male and 5 female F0 animals randomly selected from each group.
The blood samples collected were divided into tubes as follows:
- EDTA anticoagulant (whole blood) -> for haematological investigations
- Citrate anticoagulant (plasma) -> for coagulation tests
- Li-Heparin anticoagulant (plasma) -> for clinical chemistry tests
HAEMATOLOGY
The parameters listed below were determined (Instrument: ADVIATM 120, Siemens Diagnostics GmbH, 35463 Fernwald, Germany):
Parameter/ Units
Differential blood count (relative)/ %
Differential blood count (absolute)11/ 10^3/µL
Erythrocytes (RBC)/ 10^6/µL
Leucocytes (WBC)/10^3/µL
Haematocrit value (PCV or HCT)/ %
Haemoglobin content (HGB)/ mmol/L
Platelets (PLT)/ 10^3/µL
Reticulocytes (Reti)/ % of erythrocytes
Mean corpuscular volume (MCV)/ fL
Mean corpuscular haemoglobin (MCH)/ fmol/L
Mean corpuscular haemoglobin concentration (MCHC)/ mmol/L
Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings (see section 4.2 'Histopathology').
COAGULATION
The parameters listed below were determined (Instrument: Amax Destiny Plus™, Tcoag Deutschland GmbH, 32657 Lemgo, Germany):
Parameter/ Units
Prothrombin time (PT)/ sec
Activated partial thromboplastin time (aPTT)/ sec
BIOCHEMISTRY
The parameters listed below were determined (Instrument: KONELAB 30i, Thermo Fisher Scientific, 63303 Dreieich, Germany):
Parameter/ Units
Sodium/ mmol/L
Potassium/ mmol/L
Calcium/ mmol/L
Chloride/ mmol/L
Albumin/ g/L
Total bilirubin/ µmol/L
Total cholesterol/ mmol/L
Glucose/ mmol/L
Total protein/ g/L
Blood urea (BUN)/ mmol/L
Creatinine/ µmol/L
Alanine aminotransferase (ALAT/GPT)/ U/L
Alkaline phosphatase (aP)/ U/L
Aspartate aminotransferase (ASAT/GOT)/ U/L
Bile acids/ µmol/L
Lactate dehydrogenase (LDH)/ U/L
Globulin/ g/L -> by subtraction
Albumin / Globulin ratio/ on-dimensional-> by calculation
THYROID HORMONE (T4) DETERMINATION
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 7.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below.
Blood withdrawal was performed by randomization of the parental male and female animals. The male animals of all test groups were completely randomized in a one- step process, the female animals in different staggers according to their litter day.
Animals/ Time of sampling/ Number of samples/ Feeding status/ Analysis T4/ Sample Volume
All evaluated dams/ At scheduled sacrifice/ 32/ Fasted/ Not yet/ 6x 100 µl
All adult males/ At scheduled sacrifice/ 38/ Fasted/ Yes/ 6x 100 µl
Blood samples were processed for serum, divided into aliquots and stored at - 20°C ± 10 % at the Test Facility.
The T4 ELISA (Total Thyroxine (T4) ELISA, IBL INTERNATIONAL cat. no. RE55261; batch no. 304K079; Instrument: Tecan Sunrise) was conducted at the test institute.
Additional serum samples will be analysed for different hormones (T3 and / or TSH) based on findings and only upon agreement with the Sponsor. - Oestrous cyclicity (parental animals):
- During a 14-day pre-exposure period, the oestrus cycle of the female animals will be monitored to select 40 animals with regular oestrus cycles. Animals that fail to exhibit typical 4-5 day cycles will not be included in the study.
Daily monitoring of vaginal smears will continue throughout the premating period until evidence of mating.
A vaginal smear will also be taken in the morning of the day of scheduled necropsy. - Sperm parameters (parental animals):
- Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of groups 1 and 4 following H-E and PAS staining.
- Litter observations:
- CLINICAL SIGNS
Daily observations (parental animals and pups)
Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity were recorded. Mortality was recorded twice daily. Animals which died prematurely were necropsied as soon as possible after exitus. Individual animals were observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded for each individual animal.
Cage-side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.
In addition, animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
EXAMINATION OF THE PUPS
As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (pups were considered as runts if their weight was less than 70% of the mean litter weight) and the presence of gross abnormalities. Any abnormal behaviour of the offspring would have been recorded. However, no abnormal behaviour was noted for the pups.
The following examinations/observations were done for the offspring:
COUTING, SEXING AND WEIGHING
Live pups were counted, sexed and weighed on post-natal days (lactation days) 1, 4 and 13.
ANO-GENITAL DISTANCE
On post-natal day 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale.
LITTER ADJUSTMENT ON PND 4
After counting on PND 4, the litters were adjusted to 10 pups per litter by eliminating (culling) surplus pups using a randomization scheme generated by Provantis®.
BLOOD SAMPLING FOR THYROID HORMONE (T4) DETERMINATION
On PND 4 and on PND 13 blood samples for T4 hormone level determination were taken from 2 selected pups per litter, if possible from one male and one female pup. On PND 4 the culled surplus pups were used for blood collection.
Determination of the pups used for blood withdrawal:
On lactation day 4 (PND4) and lactation day 13 (PND13) the litter sequence of pup blood withdrawal was determined by randomization of the dams. The collection of the pups for blood withdrawal from the individual litters occurred in an ascending order (the male and female pups per dam with the lowest number were used, if possible).
Animals/ Time of sampling/ Number of samples/ Feeding status/ Analysis T4 /Sample Volume
Pups (at least 2 surplus pups per litter, all litters)/ PND 4/ 57/ Non-fasted/ Not yet/ 1x 75 µl
Pups (at least 2 per litter, all litters)/ PND 13/ 64/ Non-fasted/ Yes/ 1x 75 µl (for T4), 1x 70 µl, 1x 100 µl
#1: 57 culled pups from altogether 32 litters (dam no. 17 with only 1 culled pup and dam nos. 60, 74 and 78 with zero culled pups).
Blood samples were processed for serum, divided into aliquots and stored at - 20°C ± 10 %.
The T4 ELISA (Total Thyroxine (T4) ELISA, IBL INTERNATIONAL cat. no. RE55261; batch no. 304K079; Instrument: Tecan Sunrise) was conducted at the test institute.
Additional serum samples will be analysed for different hormones (T3 and / or TSH) based on findings and only upon agreement with the Sponsor.
MALE NIPPLES COUNTING
Nipples were counted in all male pups on PND 13 (shortly before scheduled sacrifice). - Postmortem examinations (parental animals):
- GROSS NECROPSY
Vaginal smears were examined on the day of necropsy to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.
The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times:
Males: On test day 49
Dams: On test days 65 to 79 (corresponding to lactation days 14 to 16)
DISSECTION OF ADULT ANIMALS
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
During necropsy the number of implantation sites was recorded in the female animals.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
ORGANS WEIGHED
The weight of the following organs of all adult male and female animals was determined. Paired organs were weighed individually and identified as left or right.
Adrenal gland (left and right)
Spleen
Brain
Testicle (left and right)
Epididymis (left and right)
Thyroid (left)
Heart
Thymus
Kidney (left and right)
As a whole: prostate, seminal vesicles
Liver with coagulating glands
Ovary (left and right)
Uterus including cervix
The weights of the organs were determined before fixation. Only the weight of the thyroid glands was determined after fixation.
ORGANS FIXED FOR PRESERVATION
The following organ(s) or parts thereof of all adult male and female animals were fixed in modified Davidson's solution or 7% buffered formalin:
Fixative: modified Davidson's solution
Epididymis (left and right)
Testicle (left and right)
Fixative: 7 % buffered formalin
Gross lesions observed
Thyroid (including parathyroids)
Ovary and oviduct (left and right)
Uterus (including cervix)
Prostate Vagina
Seminal vesicles with coagulating glands
Any other organs displaying macroscopic changes were also preserved.
SELECTION OF ANIMALS AND ORGANS FOR HISTOPATHOLOGY
The 5 male and female animals from each group were randomly selected for histopathology examination.
Telected parental animals for histopathological examination.
Group Randomly selected animals
and prematurely deceased or sacrificed animals
Group Males no. Females no.
1 2, 3, 5, 6, 9 12, 13, 15, 17, 19
2 23, 24, 27, 28, 30 34, 35, 36, 37 , 38
3 41, 42, 43, 44, 45 52, 54, 56, 58, 60
4 61, 64#, 65, 66, 67#, 68, 69 71, 72, 73, 74, 75#, 77#, 79#, 80
#: Prematurely deceased or sacrificed animals (no histopathological examination could be performed for animal no. 75, as no organs were preserved at necropsy, see section 2.13 ‘Study Plan deviations‘).
The organs or parts thereof from the animals listed above that were fixed for histopathology examination are listed below:
Fixative: Davidson's solution
Eye with optic nerve (2)
Fixative: modified Davidson's solution
Epididymis (2)
Testicle (2)
Fixative: 7 % buffered formalin
Adrenal gland (2)
Nerve (sciatic)
Bone
Oesophagus
Bone marrow (os femoris)
Ovary and oviduct (2)
Brain (cerebrum, cerebellum, brain stem (pons))
Pituitary
Gross lesions observed
Prostate
Heart (3 levels: right and left ventricle, septum)
Seminal vesicles with coagulating glands
Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method)
Spinal cord (3 sections)
Intestine, large (colon, rectum)
Spleen
Kidney and ureter (2)
Stomach
Liver
Thyroid (including parathyroids)
Lungs (with mainstem bronchi and bronchioles, preserved by inflation with fixative and then immersion)
Thymus
Lymph node (1, cervical)
Trachea (including larynx)
Lymph node (1, mesenteric)
Urinary bladder
Mammary gland
Uterus (including cervix)
Muscle (skeletal)
Vagina
HISTOPATHOLOGY
ANIMALS TO BE EXAMINED AND PREPARATION OF SLIDES
Full histopathology was performed on the preserved organs of the selected parental animals of groups 1 and 4, and the thyroids of the selected pups.
Due to test item-related findings, stomachs of the selected male and female animals and the kidneys of the selected male animals of groups 2 and 3 were additionally examined histopathologically.
The organs listed above were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they are noted were grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O and examined histologically.
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of groups 1 and 4 following H-E and PAS staining.
The blood smears prepared from all animals during the haematological examination were available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Sponsor. So far, no examination was performed.
HISTOPATHOLOGICAL EVALUATION
The histotechnique was performed by the test institute. The slides (labelled with study number, species, animal number, block number) were dispatched to AnaPath Services GmbH on November 26, 2019 and on February 27, 2020 (additional examinations). The transport of the slides for the histopathology work to AnaPath Services GmbH was arranged by the Test Facility, whereas the return transport to the Test Facility will be arranged by AnaPath Services GmbH. - Postmortem examinations (offspring):
- The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically (gross necropsy) at the following times: Pups: On PND 13
Dead pups and pups sacrificed at day 13 post-partum were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
On lactation day 13, the thyroid of 1 male and 1 female pup from each litter was fixed in 7% formalin. In summary, 64 thyroids were selected from 32 litters. The same pups were used for T4 ELISA sampling. - Statistics:
- DATA ACQUISITION
The following data were captured or calculated by the departmental computerized system (Provantis® integrated preclinical software, version 10.2.1, Instem LSS Ltd):
Parental clinical signs, body weight, body weight gain, food consumption, haematological and biochemical parameters.
Raw data not fully compatible with the computerized system (e.g. data from the neurological screening or pup data) were maintained on paper according to the appropriate SOPs.
Data maintained on paper (e.g. data from the neurological screening or pup data) was entered in Provantis in a retrospective manner using the laboratory records according to the appropriate SOPs.
STATISTICS
PARAMETRICAL DATA
The statistical evaluation of the parametrical values was done by Provantis (Provantis® integrated preclinical software, version 10.2.1, Instem LSS Ltd) using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
NON-PARAMETRICAL DATA
No statistical evaluation of non-parametrical values (reproductive group indices or macroscopic and microscopic findings) was performed.
Significantly different data are indicated in the summary tables of the result sections of the report. - Reproductive indices:
- The following indices were calculated for each group:
Female Fertility Index [%] = (Number of pregnant rats/ Number of rats paired with a male) x 100
The female fertility index reflects the total number of dams that had achieved pregnancy, including dams which delivered at term, aborted or had fully resorbed litters.
Gestation Index [%] = (Number of dams with live pups/ Number of pregnant rats) x 100
For each litter and group the following indices were determined:
Birth Index [%] #1 = (Total number of pups born (alive + dead)/Number of implantation sites) x 100
Live Birth Index [%] = (Number of pups alive on day 0/1 of lactation/ Total number of pups (alive + dead)) x 100
Viability Index [%] pre-cull = (Number of pups alive on day 4 (pre cull)/ Number of pups alive on day 0/1) x 100
Viability Index [%] post cull = (Number of pups alive on day 13/ Number of pups alive on day 4 (post cull)) x 100
Post-implantation loss [%]#1 = ((Implantations - number of pups born alive)/ Implantation sites) x 100
#1: Twins (shared placentae) are considered as additional implantation sites in the calculation of the birth index and the post-implantation loss, to avoid a birth index above 100% or a negative post-implantation loss. - Offspring viability indices:
- Birth and live birth index, post-impantation loss and viability indices of the pups
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Males: No observations were noted for the surviving male animals. Females: At 300/240 mg/kg b.w./day salivation, haemorrhagic nose/snout, a reduced motility, breathing sounds and / or piloerection were noted for 3 of 7 surviving females during the gestation and / or lactation period on one or several test days. Start and duration: Salivation started immediately to 5 min after administration and disappeared again between 20 and 60 min after administration. The reduced motility which was noted for female no. 71 during the gestation period started in the first 5 min after administration and disappeared between 20 and 60 min after administration.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Males: None of the males treated with 30 or 100 mg/kg b.w./day died prematurely. At 300/240 mg/kg b.w./day two premature deaths were noted. Male no. 67 was found dead on test day 41 and male no. 64 was found dead on test day 44 (12 or 15 days after dose reduction). Both premature deaths were considered to be test item-related. A loss of body weight, breathing sounds, a reduced motility and / or reduced faeces were noted on several days before death. Histopathology revealed test item-related changes in the stomach (e.g. erosion/ulcer) and the kidneys (e.g. tubular cell degeneration, tubular basophilia) for male no. 67 (but not for male no. 64).
Females: None of the females treated with 30 or 100 mg/kg b.w./day died prematurely.
At 300/240 mg/kg b.w./day two females (no.75 and no. 77) were prematurely sacrificed on test days 27 or 29 (one day before or one day after dose reduction on test day 28) due to a poor general condition. A loss of body weight, breathing sounds, reduced motility, piloerection, laboured breathing and / or gasping were noted several days before death. Histopathology revealed test item-related changes in the stomach (e.g. erosion/ulcer) and the kidneys (e.g. tubular cell degeneration, tubular basophilia) for female no. 77. No organs were examined for no. 75. A further high dosed female (no. 79) was prematurely sacrificed on test day 48 after misgavage. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Males: At 300/240 mg/kg b.w./day, a slightly, statistically not significantly, reduced body weight was noted during the course of the study (at maximum 6.4 % below the control group on test day 36). This was due to the 2 prematurely deceased high dosed males (premature death on test days 41 and 44). On test day 48 (last live body weight before necropsy), when only the surviving high dosed males were left, no differences between the high dose group and the control group were noted anymore. Body weight gain: A reduced body weight gain was noted at the high dose level for the period between test days 15 and 36, when both prematurely deceased animals were still in the study. For the period between test days 15 and 48, when both prematurely deceased animals were no longer present, no differences in body weight gain were noted between the animals of the high dose group and the control group.
Females:
At 300/240 mg/kg b.w./day, a minimal reduction in body weight was noted during the pre-mating period on test day 22 (3.9 % below the control; statistically not significant), due to the 2 females that were prematurely sacrificed at the end of the pre-mating period on test days 27 and 29. The surviving high dosed females revealed reduced body weights on gestation day 7 and lactation days 8 and 13 (5.9 %, 11.7 % and 5.7 % respectively below the control; statistically not significant). Body weight gain: Accordingly, slightly reduced body weights were noted at the high dose level during the gestation and the lactation period. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Males: No adverse effects on food consumption were noted. Slight, but statistically significant reductions in food consumption that were noted at the high dose level during the first treatment week were considered to be test item-related but not adverse. Females: No test item-related effect on food consumption was noted.
- Food efficiency:
- no effects observed
- Description (incidence and severity):
- s. "food consumption"
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- No test item-related changes in the consumption of drinking water was noted for the male and female rats treated with 30, 100 or 300/240 mg/kg b.w./day by visual appraisal.
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Males: No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (30, 100 or 300/240 mg/kg b.w./day).
Statistically significantly increased values were noted for the percentage of reticulocytes at the low and at the intermediate dose level (62.2% or 51.5% above the control; p ≤ 0.05). However, with one exception from the low dose group (no. 24 with 4.6% reticulocytes), all individual values for the reticulocytes of the low dose group (2.8 to 4.6%) and the intermediate dose group (2.6 to 3.6%) were within the range of the background data (1.6% to 3.9%). Therefore, and because no dose response relationship was noted (the mean value of the high dose group was only 7.3% above the control, statistically not significant) the changes were considered to be spontaneous.
Females: No test item-related differences for the examined haematological parameters were noted between the control group and the low and the intermediate dose group (30 or 100 mg/kg b.w./day).
Decreased values were noted for the number of lymphocytes in the low, the intermediate and the high dose group (35.3%, 39.9% or 21.4% below the control, statistically significant at the low and the intermediate dose group at p ≤ 0.05). However, with one exception at the low dose level, all individual values were within the range of the background data. Furthermore, as no dose response-relationship was noted, the decreased numbers of lymphocytes were considered to be spontaneous.
At the high dose level (300/240 mg /kg b.w./day), a statistically significantly increased number was noted for neutrophilic granulocytes (186.1% above the control, p ≤ 0.01). The numbers of neutrophilic granulocytes from 4 (2.74 to 5.28 x10E3 cells/µL) of 5 high dosed females were above the background range (0.41 to 1.68 x10E3 cells/µL). In contrast, with one exception at the low dose level, all individual values of the control group, the low and the intermediate dose group were within the range of the background data.
Hence, the increased number of neutrophilic granulocytes that was noted for the females of the high dose group was considered to be test item-related. This was considered a consequence of the severe stomach erosion/ulcer due to the corrosivity of the test item. Hence, the increased number in neutrophilic granulocytes was considered as a secondary effect to the test item treatment. - Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- Males: No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (30, 100 or 300/240 mg/kg b.w./day).
Increased cholesterol concentrations were noted at the low, the intermediate and the high dose level (53.1%, 29.2% or 16.5% above the control; statistically significant at p ≤ 0.01 at the low dose level). However, only one individual value each of the low and the high dose level was above the background data. All other individual values were within the background range. Furthermore, no dose response-relationship was noted. Hence, the statistically significantly increased cholesterol concentration at the low dose level was considered to be spontaneous.
Females:
No test item-related differences for the examined biochemical parameters were noted between the control group and the low and the intermediate dose group (30 or 100 mg/kg b.w./day).
A statistically significantly increased LDH activity was noted at the high dose level (300/240 mg /kg b.w./day). In detail, 2 of 5 individual values (no. 73 with 211 U/L and no. 80 with 267 U/L) were above the background range (32 – 159 U/L). For both animals (nos. 73 and 80) the histopathological finding in the stomach with necrotic cellular/tissue debris (no. 73) or intestinal metaplasia (no. 80) can be regarded as the cause for the LDH increase for these animals. Hence, the increased LDH activity was considered a secondary effect to the test item treatment. - Endocrine findings:
- no effects observed
- Description (incidence and severity):
- T4 serum level (test day 49), Males: No test item-related changes were noted between the control group and the treatment groups (30, 100 or 300/240 mg/kg b.w./day) for the T4 levels of the parental males.
A statistically significantly decreased T4 concentration was noted at the intermediate and the high dose level (18.4% or 17.9% below the control; p ≤ 0.01 or 0.05).
One individual value of the low (no. 27 with 37.684 nmol T4/L), one of the intermediate (no. 43 with 38.123 nmol T4/L) and 2 values of the high dose group (nos. 65, 70 with 42.529 or 41.446 nmol/L) were slightly below the background data range (42.661 to 90.506 nmol T4/L).
However, as from the high dose group only 2 values were slightly below the background range and no changes were observed for the thyroids during the histopathological examination, the statistically significantly decreased T4 concentrations that were noted at the intermediate and the high dose levels were considered to be spontaneous.
Furthermore, no influence was noted on the thyroid weight and no changes were noted during the histopathological examination of the thyroid glands from the high dosed animals. It is generally known that histopathological examination of the thyroid is usually more sensitive than thyroid weight and hormone levels . The validation report of OECD 407 states that “thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weigth was reliable, but was somewhat less sensitive when compared to thyroid histopathology. Circulating thyroid hormone levels (T3, T4, and TSH) were not always reliable and sensitive, but the standard operating procedures for blood sampling and for thyroid hormone analyses were not standardized to reduce stress induced variability and to reduce analytical variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormonal values. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- Males and females: No test item-related observations of abnormal behaviour, no adverse effects on motoric skills, changes in the external appearance and the appearance of the faces were noted for the male and female animals of all treatment groups (30, 100 or 300/240 mg/kg b.w./day), approx. 2 hours after treatment.
Furthermore, no test item-related differences were noted in body temperature or the hind-leg splay in comparison to the control group.
Grip strength, Males and females:
No test item-related influence on the fore- and hindlimb grip strength was noted for the male and female animals of all treatment groups (30, 100 or 300/240 mg/kg b.w./day), approx. 2 hours after treatment.
spontaneous motility, Males and females: No test item-related influence on the spontaneous motility was noted for the male and female animals of all treatment groups (30, 100 or 300/240 mg/kg b.w./day), approx. 2 hours after treatment. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Males and females: At the high dose level (300/240 mg/kg b.w./day), microscopic changes that could be attributed to treatment with the test item were observed in the stomach (male and female animals) and in the kidneys (male animals only).
Stomach (male and female animals):
In both sexes of group 4, forestomach and/or glandular stomach lesions were observed, which included forestomach erosion/ulcer, glandular stomach erosion, and inflammation in the forestomach or the glandular stomach. Intestinal metaplasia of glandular stomach, glandular dilatation and squamous cell hyperplasia were also observed as the secondary changes to the ulcerative and inflammatory lesions.
The stomach lesions recorded in both sexes of the high dose group were most likely due to local effect associated with the corrosive property of the test item, and were considered not to be due to systemic effects of the test item.
No stomach lesions were observed for the additionally examined stomachs from the male and female animals of the low and the intermediate dose group (30 or 100 mg/kg b.w./day).
Kidneys (male animals only):
Tubular basophilia and mononuclear cell foci with a minimal severity grade were noted for a few or several male and female animals of the control group and the high dose group, as well as the additionally examined kidneys from the male animals of the low and the intermediate dose groups. However, an increased severity grade was noted for the male animals from the high dose group. Due to the increased severity grade (slight instead of minimal) the kidney observations were considered as test item-related for the male animals of the high dose group.
As described above, only a minimal severity grade was noted for the above mentioned kidney findings for the surviving female animals. Only for the prematurely deceased high dosed female no. 77 tubular basophilia of a moderate grade was noted and considered to be test item-related
In addition, pyelonephritis was observed in 2 males of group 4. Pyelonephritis could arise occasionally in rats as spontaneous lesions. However, in the present study, this lesion was found in 2 of 5 males (40%) in the high dose group. Due to higher incidence of it, the possible relationship of pyelonephritis with the treatment could not be denied.
No test item-related kidney changes were noted for the additionally examined kidneys from the male animals of the low and the intermediate dose group (30 or 100 mg/kg b.w./day).
Reproductive organs (males and females):
There was no histological evidence of toxicity in the reproductive organs including testes, epididymides, prostate gland, seminal vesicles, coagulating glands, ovaries, uterus, cervix and vagina.
Detailed examination for testes:
Testes were also checked on completeness of cell populations and stages, while taking account into the interstitial cell structure and presence/absence of any degenerative changes. As a result, no treatment-related effects on the testicular histomorphology were observed. - Histopathological findings: neoplastic:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- No test item-related influence was noted during the examination from test day 15 until a positive mating sign was noted. Estrous stage at necropsy: A cycle stage of diestrus was mostly noted for the females of the control group and the treatment groups
- Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- Testes were checked on completeness of cell populations and stages, while taking account into the interstitial cell structure and presence/absence of any degenerative changes. As a result, no treatment-related effects on the testicular histomorphology were observed.
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- FERTILITY: No test item-related influence on the fertility index of the female rats was noted for any of the treatment groups (30, 100 or 300/240 mg /kg b.w./day).
One non-pregnant female each was noted at the low (no. 32) and the high dose level (no. 76), though a positive mating sign (sperm detection) was noted for both animals. A non-pregnant animal despite a positive sperm detection per group is in the range of normal variability and considered to be spontaneous.
At the intermediate dose level two non-pregnant animals were noted. For one of these non-pregnant animals a positive mating sign was noted (no. 51), whereas for the other non-pregnant animal (no. 55) no positive mating sign was noted after a mating period of 14 days. A non-pregnant animal without a positive mating sign (non-mated animal) is rare in comparison to non-pregnant animals with a positive mating sign. This could be related to the male animal or the behaviour of the female animal. However, as only one such case was noted and without a dose response-relationship, the occurrence of this non-mated animal was considered to be spontaneous. The number of 7 pregnant females is below the recommendation in the OECD guideline 422 of at least 8 pregnant females. However, the OECD guideline 422 states, that the number of pregnant females may be lower 'in case of marked toxic effects'. As two female animals were noted in a moribund condition that led to a decrease of the high dose level, this exception was met. Therefore, the number of 7 pregnant females out of the remaining 8 females does not affect the validity of the study.
GESTATION INDEX:
No test item-related influence on the gestation index was noted for the female rats of the treatment groups (30, 100 or 300/240 mg/kg b.w./day).
All pregnant females of the treatment groups delivered live pups (the pregnant high dose female no. 79 which deceased on gestation day 17 due to a misgavage was excluded), leading to a gestation index of 100% for all treatment groups.
Only in the control group one female was noted with a total resorption of all implantation sites, leading to a gestation index of 90 % in the control group.
PRE-COITAL TIME: No test item-related influence was noted.
GESTATION LENGTH: No test item-related influence was noted. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- > 240 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- female
- Basis for effect level:
- reproductive performance
- Remarks on result:
- other: 300/ 240 mg/ kg bw/ day
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical signs
- mortality
- gross pathology
- histopathology: non-neoplastic
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 240 mg/kg bw/day (nominal)
- Organ:
- kidney
- stomach
- Treatment related:
- yes
- Clinical signs:
- no effects observed
- Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- no mortality observed
- Description (incidence and severity):
- VIABILITY INDEX: Pre- and post-cull period: No test item-related differences between the control group and the treatment groups (30, 100 or 300/240 mg/kg b.w./day) were noted for the viability indices between lactation days 0/1 and 4 (pre-cull) and lactation days 5 and 13 (post-cull).
Overall, 4 prematurely deceased pups were noted during the pre-cull period (3 prematurely deceased pups in the control group and 1 in the high dose group. No prematurely deceased pups were noted during the post-cull period.
NUMBER OF LIVE PUPS: A slightly (statistically not significantly) reduced mean number of live pups per dam was noted at the high dose level (300/240 mg/kg b.w./day) on lactation days 1 and 4.
This was due to the high number of stillbirths at the high dose level (altogether 9 stillbirths from dam nos. 78 and 80 in comparison to 1 stillbirth in the control group). This led to a reduction from 14.3 pups born alive and dead per dam to 12.8 live born pups per dam in the high dose group. In comparison, in the control group 14.6 live born pups per dam were noted from 14.7 pups that were born alive and dead per dam. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- BODY WEIGHT: No test item-related difference was noted between the mean body weight of the pups from the dams of the control group and the mean body weight of the pups from the treatment groups (30 or 100 mg/kg b.w./day) on lactation days 1, 4 and 13.
However, on PND13 a decreased pup body weight was noted at the high dose level (300/240 mg/kg b.w./day). In detail, the mean pup body weight (mean value from the mean pup body weights from each dam) of the male and female pups combined was 7.2% below the control group (statistically not significant). This was due to the decreased pup body weights of the high dose female nos. 74 and 80 with 8 pups each on PND 13. The mean pup body weights of these 2 females (22.93 g for no. 74 and 19.83 g for no. 80) were clearly below the mean pup body weights of the remaining 4 high dose dams (between 28.10 g and 34.30 g). Furthermore, the mean pup body weights of these 2 dams (nos, 74, 80) were also clearly below the range of the control group (27.48 g to 32.00 g) and the background range (24.34 to 32.75 g).
The observation, that 2 of 6 high dosed females showed a clearly reduced mean pup body weight was considered to be not test item-related. The decreased pup body weight of the dams nos. 74 and 80 is a secondary effect, due to the observed maternal toxicity. Both dams showed an overall lower body weight between the end of the gestation and the lactation period compared to the other four dams of the high dose group. Furthermore, the mean pup body weights of the other 4 females of the high dose group (28.10 to 34.40 g; each dam with 10 pups on PND13) were clearly in the range of the background data on PND13 or even above (24.34 to 32.75 g). RUNTS: One runt was noted in the control group (no. 12-17).
LITTER WEIGHT: No test item-related differences in litter weight were noted between the control group and the low and the intermediate dose groups (30 or 100 mg/kg b.w./day).
At the high dose level (300/240 mg/kg b.w./day) a reduced litter weight (male and female pups combined) was noted on lactation days 1, 4 and 13 (20.5%, 19.2% and 13.7%, respectively below the control value, statistically not significant).
In detail, on lactation day 1 the litter weights from 2 of 6 high dosed dams (nos. 74 with 63.7 g and no. 78 with 66.9 g) were below the lower range of the background data on lactation day 1 (70.3 g – 122.6 g). This was due to a slightly reduced number of live pups on lactation day 1 for dam nos. 74 and 78 (8 or 9 pups in comparison to 12 to 19 pups for the remaining high dosed dams).
After the adjustment of the pup number per dam due to the culling process, the remaining difference in litter weight between the high dose group and the control group was due to the reduced pup body weight that was noted for the pups of the high dose group on lactation day 13. As discussed above, this was due to the reduced pup body weights of dam nos. 74 and 80.
Due to the increased male to female ratio, the difference in litter weight between the control group and the high dose group was mostly pronounced for the female pups. - Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- No test item-related differences between the control group and the treatment groups (30, 100 or 300/240 mg/kg b.w./day) were noted for the T4 levels of the male and female pups.
However, at the intermediate and the high dose level statistically significantly increased T4 levels were noted for the female pups (16.7% or 20.5% above the control, p ≤ 0.05), whereas no statistically significant changes were noted for the male pups. For the male and female pups combined a statistically significantly increased T4 level was only noted at the high dose level (15.7% above the control, p ≤ 0.05).
As all individual values were in the range of the background data, the observed increased T4 concentrations at the intermediate and the high dose level were considered to be spontaneous. Furthermore, no changes were noted during the microscopic examination of the pup thyroids. - Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- no effects observed
- Description (incidence and severity):
- No test item-related difference in the absolute and the relative ano-genital distance (ano-genital distance normalized to the cube root of pup body weight) of the male and the female pups was noted between the control group and the treatment groups (30, 100 or 300/240 mg/kg b.w./day).
- Nipple retention in male pups:
- no effects observed
- Description (incidence and severity):
- No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (30, 100 or 300/240 mg/kg b.w./day).
However, a spontaneously increased number of nipples was noted in the control group in comparison to the high dose group. In detail, 7 pups with nipples were noted in the control group, 5 in the low dose group and 2 in the intermediate dose group. No pup with nipples was noted at the high dose level.
However, male pups are supposed to have no nipples and the number of pups with nipples in the control group is at the upper limit of the background data. Therefore, the absence of male pups with nipples in the high dose group was considered to be of no toxicological relevance but due to spontaneously high number of male nipples in the control group.
Mammary gland development begins similarly in male and female rats, but only female rats have nipples, whereas male rats possess only rudimentary mammary glands but no nipples, as in male pups locally produced testosterone causes the regression or apoptosis of the nipple anlagen. Therefore, the retention of nipples in male pups is an indicator of impaired androgen action during the development. - Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with 30, 100 or 300/240 mg/kg b.w./day after terminal sacrifice on lactation day 13.
Also no gross abnormalities were noted for the pups that prematurely deceased during the lactation period (stillbirth or found dead). - Histopathological findings:
- no effects observed
- Description (incidence and severity):
- No test item-related changes were noted for the thyroid glands from the pups of the treatment groups (30, 100 or 300/240 mg/kg b.w./day).
- Other effects:
- no effects observed
- Description (incidence and severity):
- BIRTH INDICES AND POST-IMPLANTATION LOSS:
No test item-related influence on prenatal development (number of implantation sites per dam, number of resorptions, number of stillbirths) was noted at the low and the intermediate dose level (30 or 100 mg/kg b.w./day). The increased number of resorptions that was noted at the low dose level (17 resorptions in comparison to 12 in the control group and 5 in the intermediate dose group) was considered to be spontaneous, as no dose response-relationship was noted.
A test item-related effect on prenatal development was noted at the high dose level (300/240 mg/kg b.w./day).
In detail, 9 stillbirths were noted at the high dose level in comparison to 1 stillbirths each in the control group and the intermediate dose group. This led to a markedly decreased live birth index in the high dose group (89.5 % in comparison to 99.3 % in the control group, 100.0 % in the low dose group and 99.2 % in the intermediate dose group; group values).
The 9 stillbirths of the high dose group were noted from two (no. 78 with 5 stillbirths and no. 80 with 4 stillbirths) of 6 high dosed females that had littered. Furthermore, 5 resorptions were noted for dam no. 78 and 2 resorptions for dam no. 80. No resorptions and no stillbirths were noted for the remaining 4 females of the high dose group which littered live pups.
Due to the high number of stillbirths from 2 of 6 females with live pups, an adverse effect of the test item on prenatal development has to be considered. However, compared to the OECD 443 study of the test item the increased number of stillbirths in the high dose group can be regarded to be within the range of biological variability as also 9 stillbirths were noted in the low dose group of the OECD 443 study. No dose response-relationship was observed.
MALE TO FEMALE RATIO OF THE PUPS
No test item-related influence on the male to female ratio was noted for any treatment group (30, 100 or 300/240 mg/kg b.w./day).
However, a male to female ratio that markedly differed from the expected value (around 1.0) was noted at the intermediate dose group and the high dose group (0.70 or 1.41). However, as no dose-response relationship was noted (the values at the intermediate and the high dose group have opposite directions), the reduced male to female ratio that was noted at the intermediate dose group, was not considered to be test item-related but spontaneous. - Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- no abnormal behaviour was noted for the pups.
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- > 240 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other:
- Remarks on result:
- other: > 300/ 240 mg/ kg bw/day
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item was administered orally to rats at dose levels of 30, 100 or 300 mg/kg b.w./day. Due to mortality, the high dose level was decreased to 240 mg/kg b.w./day, starting on test day 28.
Additionally, this OECD 422 serves as dose range finding study for the OECD 443 study. The NOAEL for general toxicity is considered to be 100 mg/ kg bw/day. The NOAEL for reproductive toxicity based on adverse effects on the reproductive parameters of the parental females is considered to be above 300/ 240 mg/ kg bw/day. Based on adverse effects on prenatal development (conceptus to birth) the NOAEL for reproductive toxicity is above 300/240 mg/ kg bw/ day and based on adverse effects on postnatal development (pup) above 300/240 mg /kg b.w./day. - Executive summary:
Conclusion
The aim of the study was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development according to OECD guideline 422. The test item was administered orally to rats at dose levels of 30, 100 or 300 mg/kg b.w./day. Due to mortality, the high dose level was decreased to 240 mg/kg b.w./day, starting on test day 28.
Additionally, this OECD 422 serves as dose range finding study for the OECD 443 study.
General toxicity
Parental male and female animals
At 300 mg/kg b.w./day 2 females were prematurely sacrificed due to a poor general condition, one female one day before and the other female one day after dose reduction.
At 300/240 mg/kg b.w./day 2 male animals were found dead 12 or 15 test days after dose reduction.
At 300/240 mg/kg b.w./day a few of the surviving females showed salivation, a haemorrhagic nose/snout, piloerection, breathing sounds and / or reduced motility during the gestation and or lactation period. No changes in behaviour, the external appearance or the faeces were noted for the surviving male animals of the high dose group.
A marked body weight loss was noted at 300/240 mg/kg b.w./day for the prematurely deceased or sacrificed male and female animals.
The surviving female animals showed a slightly reduced body weight in comparison to control during the gestation and the lactation period. This and the observed general bad condition of the females are signs of maternal toxicity. No differences in body weight were noted for the surviving high dosed male animals.
At 300/240 mg/kg b.w./day an increased number of neutrophilic granulocytes and an increased LDH activity were noted for the female animals. Both were secondary effects due to the corrosivity of the test item, which is manifesting in the very severe histopathological observations of erosions/ulcer in the female animals. No test item-related differences for the haematological and biochemical parameters were noted for the male animals.
The macroscopic examination at necropsy revealed kidney changes for 2 of the 8 surviving male animals of the high dose group (300/240 mg/kg b.w./day) that could possibly be test item-related.
Kidney changes that were considered to be test item-related were noted during the microscopic examination for the surviving male animals of the high dose group (300/240 mg/kg b.w./day), as well as for one of the prematurely deceased male animals and one of the prematurely sacrificed female animals, but not for the surviving female animals.
Test item-related changes for the surviving animals from both sexes were noted during the microscopic examinations of the stomachs from the male and female animals of the high dose group (300/240 mg/kg b.w./day) in form of an erosion of the stomach wall and an inflammatory response. These stomach changes were considered as local and secondary effects that were caused by the corrosive properties of the test item.
The additional microscopic examinations of the kidneys from the male animals and the stomachs from the male and female animals of the low and the intermediate dose groups revealed no test item-related changes.
No test item-related influence or adverse effects were noted for the male and female animals on food consumption, on the parameters of the neurological screening, for the organ weights and on the T4 serum levels of the male animals.
Reproductive toxicity
Parental females
No influence was noted on the number and length of the estrous cycles, the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period.
Pups
At 300/240 mg/kg b.w./day an effect was noted on the prenatal development in the form of an increased number of stillbirths. However, compared to the OECD 443 study of the test item the increased number of stillbirths in the high dose group can be regarded to be within the range of biological variability as also 9 stillbirths were noted in the low dose group of the OECD 443 study. No dose response-relationship was observed.
During the postnatal development a reduced pup body weight was noted at 300/240 mg/kg b.w./day, which was a secondary effect due to maternal toxicity.
No influence was noted on the viability of the pups, the ano-genital distance, the number of nipples per male pup and on the T4 concentration. No changes were noted for the thyroid glands from the pups during the microscopic examination.
No external gross abnormalities (malformations or variations) were noted for any of the pups.
The following no-observed-adverse-effect levels (NOAEL) were established:
General toxicity
NOAEL (for systemic toxicity)
100 mg/kg b.w./day, p.o.
Reproductive toxicity
a) adverse effects on the reproductive parameters of the parental females
NOAEL
above 300/240 mg/kg b.w./day, p.o.
b) adverse effects on pre- and postnatal development
- b1) adverse effects on prenatal development (conceptus to birth)
NOAEL
above 300/240 mg/kg b.w./day, p.o.
- b2) adverse effects on postnatal development (pup)
NOAEL
above 300/240 mg/kg b.w./day, p.o.
- Endpoint:
- extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Date of Study Plan: 19 November 2019
Start of pre-estrous: 20 November 2019
Start of pre-mating administration: 04 December 2019
Start of mating: 12 February 2020
End of In-life: 18 June 2020
Date final report 17 February 2022 - Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- According to the ECHA final decision on a compliance check from 2017-04-05 (Decision number: CCH-D-2114356498-35-01/F and submission number: BQ538638-17) the study design of the EOGRTS according to OECD 443 is as follows:
Based on Article 41 of Regulation (EC) No 1907/2006 (the ‘REACH Regulation’), ECHA requests you to submit information on:
Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation. - Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
- Version / remarks:
- adopted 25 June 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Justification for study design:
- The study design is based on final decision on a compliance check from ECHA dated 5th of April 2017. An Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance specified as follows, was requested:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation; - Species:
- rat
- Strain:
- Crj: CD(SD)
- Details on species / strain selection:
- Rat / CD® / Crl:CD(SD)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS:
- Species / Strain / Stock: Rat / CD® / Crl:CD(SD)
- Breeder: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Body weight (at start of dosing): Males: 399.1 g - 463.5 g, Females: 230.0 g - 287.7 g
- Age (at start of dosing): Males and females: 69 days
- Selection of species: The rat is a commonly used rodent species for such studies and required by the guideline.
- Number of parental animals: Pre-exposure period:125 female animals were evaluated pre-exposure for estrous cyclicity to yield 96 females (i.e. 24 per group) with a regular estrous cycle for the study. Main study: 192 (96 male and 96 female) animals in order to grant at least 20 pregnant females per group for evaluation of the F0 Generation.
- Adaptation period: 7 days
- Housing: With exception of the mating period, the male and female animals (F0 Generation) are kept singly in MAKROLON cages (type III plus) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) was used as bedding material in the cages. The cages were changed and cleaned once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted by LUFA-ITL.
The animals received one piece of wood (certified for animal use) to gnaw on once weekly at change of the cages. Octagon-shaped red-tinted huts (polycarbonate) were placed in the cages to offer the animals a resting and hiding place.
- Diet (ad libitum): ssniff® R-Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany, Food residue was removed and weighed.
Periodic analysis of the food for contaminants based on EPA/USA is conducted at least twice a year by LUFA-ITL. Certificates of analysis of the composition and for contaminants are provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.
- Water: Tap water was offered ad libitum. Samples of the drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung, Bundesgesetzblatt 2001' [German Regulations on drinking water, public notice of the law, 2001 ]. In addition, drinking water samples taken at Provivo are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trinkwasserverordnung 2001. No contaminants above the limitations were noted.
ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 2 °C (maximum range)
- Humidity: 55% ± 10% (maximum range)
- Photoperiod: The rooms are alternately lit (about 150 lux at approx. 1.50 m room height) and darkened in a 12 hours dark/12 hours light cycle.
- Air changes per hour: 15 to 20 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- purified water
- Details on exposure:
- Route of administration: Oral, via gavage
Frequency of administration: Once daily
Vehicle: Aqua ad injectabilia (Batch nos. 192838161, 192738002, 180958001 Braun Melsungen AG; Carl-Braun-Str. 1, 34212 Melsungen, Germany.)
Administration volume: 10 mL/kg b.w.
Dosages: 0, 25, 80, 240 (160) mg/kg bw/d; Dose reduction since 9th of January 2020
Frequency of administration: Once daily (from test day 15 until one day before sacrifice)
Selection of route of administration: According to OECD guideline 443.
The test item formulations were administered at a constant administration volume of 10 mL/kg b.w. once daily. The control animals received the vehicle at the same administration volume in the same way. - Details on mating procedure:
- Sexually mature male and female rats of the F0 Generation were randomly paired for mating. Mating was monogamous: 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until evidence of mating was observed or 2 weeks had elapsed. Each morning the females were examined for the presence of sperm or a vaginal plug.
The day of conception (day 0 of gestation or GD0) was considered to be the day on which sperm was found.
Females without a positive mating sign were separated from their male partner after 2 weeks without further opportunity for mating.
If there would have been insufficient males, for example due to male death before pairing, then males which had already mated would have been paired with a second female such that all females would have been paired. However, as 3 males and 3 females prematurely deceased before the start of mating, sufficient male animals were always available in this study. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Preparation of test item administration formulations: The administration formulations were freshly prepared every day. The test item was diluted in the vehicle to the appropriate concentrations. The amount of the test item was adjusted to the animal's current body weight before oral administration at a constant volume of 10 mL/kg b.w. once daily.
Test item formulation analysis: For the analysis of the test item-vehicle formulations, two aliquots of approximately 5 mL were taken at the following times and stored at -20°C ± 10% until analysis.
Test item formulation sampling schedule: F0 Generation – Groups 2 to 4:
- At start of the treatment period (first administration day): Analysis of concentration and homogeneity, At the start, during (middle) and before administration to the last animal of each dose level group. (3 samples / dose level group; groups 2 - 4). Number of samples (aliquots): 3 x 3 = 9 (18)
- At the end of the premating period Analysis of concentration, During treatment always before administration to the last animal/dose level group. (1 sample / dose level group; groups 2 - 4).
Number of samples (aliquots): 3 x 1 = 3 (6)
- At dose reduction of group 4 Analysis of concentration and homogeneity, At the start, during (middle) and before administration to the last animal of dose level group 4. (3 samples / dose level group 4). Number of samples (aliquots): 3 x 1 = 3 (6)
- At termination of the F0 treatment period (when the majority of animals was dosed), Analysis of concentration, During treatment always before administration to the last animal/dose level group.
(1 sample / dose level group; groups 2 - 4) Number of samples (aliquots): 3 x 1 = 3 -> (6) Total number of samples (aliquots): 18 (36)
Test item formulation sampling schedule: F1 Generation – Groups 2 to 4:
- At start of the treatment period (first administration day), Analysis of concentration and homogeneity, At the start, during (middle) and before administration to the last animal of each dose level group. (3 samples / dose level group; groups 2 - 4). Number of samples (aliquots): 3 x 3 = 9 (18)
- At termination of the Cohort 1 A treatment period (when the majority of animals was dosed), Analysis of concentration, During treatment always before administration to the last animal/dose level group. (1 sample / dose level group; groups 2 - 4) Number of samples (aliquots): 3 x 1 = 3 (6)
-> Total number of samples (aliquots): 12 (24)
The samples were labelled with study number, test species, generation, cohort no. type of sample, aliquot number, group, concentration, sampling time and date.
The samples were analysed according the method validated by the test institute.
Three months after the issuance of the Final Report, any samples or aliquots still remaining at the testing facility will be destroyed unless the Sponsor requests otherwise.
Test item-formulation analysis: The measured concentrations of the test substance in the test item-formulations were between 101.6 % and 109.6 % of the nominal concentration, indicating correctly prepared and homogeneous test item-formulations. - Duration of treatment / exposure:
- The animals were treated with the test item during the following periods:
F0 GENERATION:
- Males: 10 weeks prior to mating, during the mating period and at least until weaning of the F1 Generation (up to and including the day before sacrifice).
- Females: 10 weeks prior to mating, during the mating and lactation period and until termination of weaning of their litters (up to and including the day before sacrifice).
F1 GENERATION:
- F1 Pups: Until weaning, the pups were indirectly exposed to the test item through the breast milk. During the last week of lactation the pups additionally received the test item directly when they commence eating for themselves.
After weaning, each F1 Pup selected for the F1 Cohorts was dosed via gavage.
- Cohort 1A: The male and female animals were dosed for 10 weeks up to and including the day before sacrifice (males and females: PNDs 90 to 92).
- Cohort 1B: The male and female animals were dosed for 11 weeks up to and including the day before sacrifice (males and females: PNDs 98 to 102). - Frequency of treatment:
- daily
- Details on study schedule:
- The animals were treated with the test item during the following periods:
F0 Generation
Males 10 weeks prior to mating, during the mating period and at least until weaning of the F1 Generation (up to and including the day before sacrifice).
Females 10 weeks prior to mating, during the mating and lactation period and until termination of weaning of their litters (up to and including the day before sacrifice).
F1 Generation
F1 Pups Until weaning, the pups were indirectly exposed to the test item through the breast milk. During the last week of lactation the pups additionally received the test item directly when they commence eating for themselves.
After weaning, each F1 Pup selected for the F1 Cohorts was dosed via gavage.
Cohort 1A The male and female animals were dosed for 10 weeks up to and including the day before sacrifice (males and females: PNDs 90 to 92).
Cohort 1B The male and female animals were dosed for 11 weeks up to and including the day before sacrifice (males and females: PNDs 98 to 102). - Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- control group
- Dose / conc.:
- 25 mg/kg bw/day (nominal)
- Remarks:
- low dose group
- Dose / conc.:
- 80 mg/kg bw/day (nominal)
- Remarks:
- intermediate dose group
- Dose / conc.:
- 240 mg/kg bw/day (nominal)
- Remarks:
- high dose group; dose reduced on test day 51 (to 160 mg/kg bw/day)
- No. of animals per sex per dose:
- 20 males and females in P generation and 20 males and females in Cohort 1A and Cohort 1B
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The dose levels for this study were selected by the Sponsor based on the results of a 14 day dose range finding study in rats (LPT study no. 37378, 2020) and an OECD 422 study in rats (Provivo study no. 37482, 2021).
In 14-day dose range finding study, the rats were treated orally with 250 or 500/350 mg IPDA/kg b.w./day. The rats of the OECD 422 study were treated orally with 30, 100 or 300/240 mg IPDA/kg b.w./day.
In the OECD 422 study, two female animals of the high dose group treated with 300/240 mg IPDA /kg b.w./day had to be sacrificed on test days 27 or 29 (one day before or one day after dose reduction on test day 28) due to significant body weight loss. Furthermore, piloerection, reduced motility and breathing sounds were noted for these two animals. Two male animals of the high dose group were found dead on test day 41 or 44 (12 or 15 days after dose reduction). Prior to death, breathing sounds, reduced motility and body weight loss occurred.
On test day 48 (i.e. gestation day 17), one further female animal of the high dose had to be prematurely sacrificed due to severe signs of toxicity, i.e. salivation, reduced motility, laboured breathing and prone position.
During the first lactation week, slight reductions in food consumption were noted for the female animals of the intermediate (100 mg IPDA/kg b.w./day) and high dose group (300/240 mg IPDA/kg b.w./day). Furthermore, the remaining 6 females of the high dose group (with live pups) revealed slightly reduced body weights on lactation day 13. However, both findings were statistically not significant. Breathing sounds occurred in 3 of 6, salivation in 2 of 6 and piloerection in 1 of 6 female animals.
Hence, the dose level of 240 mg/kg/day was considered to be the maximum tolerated dose for the F0 Generation with no systemic effects to be expected in the F1 Generation of the present OECD 443 study.
Deviations:
The study was conducted in accordance with the Study Plan and 10 Study Plan Amendments agreed upon.
The following deviations were noted which were not covered by an Amendment:
Test item formulation
Aqua ad iniectabilia was used as vehicle not purified water as stated in the Study Plan.
Clinical observation
Animal no 182 f was excluded from dosing from test day 22 onwards until premature sacrifice on test day 24, due to marked laboured breathing noted during the daily cage-side observation.
Body Weight
Animal no. 454 m (Group 3, Cohort 1B) was overdosed by approximately 17 % on postnatal day 54 (i.e. 01 May 2020) due to a faulty weighing process.
The body weight was not plausible when compared to the body weights noted on the day before or the day after as follows:
PND 53 (i.e. 30 April 2020): 295.8 g
PND 54 (i.e. 01 May 2020): 351.5 g
PND 55 (i.e. 02 May 2020): 310.5 g
Therefore, the body weight on postnatal day 54 of animal no. 454 should have been excluded from reporting. However, this was impossible due to technical reasons.
All deviations were assessed by the Study Director and do not affect the validity and integrity of the scientific results obtained in this study. - Positive control:
- no
- Parental animals: Observations and examinations:
- CLINICAL SIGNS
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for each animal. Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded. In case signs of toxicity occurred the frequency of observations was increased. Each animal was observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded. In addition, the animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays, the animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m. Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns.
A more detailed examination of all F0 animals was conducted on a weekly basis. F0 animals were examined once before the first test item treatment on test day 14 to allow for within-subject comparisons. Thereafter, the examination was performed weekly until termination. Detailed clinical observations were carried out for all animals outside the home cage in a standard arena at approximately the same time of day, each time preferably by observers unaware of the treatment. The observations included in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.
MORTALITY
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m. If necessary, these provisions allowed to record any premortal symptoms in detail and a post mortem examination to be carried out during the working period of a day.
BODY WEIGHT
The body weight the animals was recorded as follows:
Pre-mating period: Daily, starting on the first day of dosing, i.e. test day 15# - report of weekly values
Mating period: Daily - report of weekly values
Post-mating period: Daily - report of weekly values for the males
Gestation period: - report on GD 0, 7, 14, 21 for the females
Lactation period: - report PND 4, 7, 14, 21 for the females
Termination of in-life: Day of sacrifice
FOOD AND DRINKING WATER CONSUMPTION:
Food intake per rat (g) was calculated using the total amount of food given to and left by each rat in each group on those days that are listed below.
Pre-mating period: Weekly
Mating period: None
Post-mating period: Weekly values for the males#
Gestation period: GD 0, 7, 14, 21
Lactation period: PND 1, 7, 14, 21
#: Starting on a suitable day after the mating period to consolidate all male animals (test day 91)
From these data the relative food consumption (in g/kg b.w./day) was determined using the following formula:
Relative food consumption (g/kg b.w./day) = (Total food given (g) - Total food left (g))/ (Number of animal days# x Body weight (kg))
# The term 'animal days' counts one animal day for each animal alive for a whole
day; it is assumed that on the day of death an animal does not eat.
Drinking water consumption was monitored daily by visual appraisal throughout the study.
REPRODUCTIVE PERFORMANCE
The reproductive parameters and reproductive indices listed below were determined to evaluate the reproductive performance.
Reproductive parameters:
- stages of the estrous cycle
- pre-coital time
- number of pregnant females
- gestation length calculated from day 0 of pregnancy
Implantation sites
- number per dam
- distribution in the uterus horns
- absolute number per group
- mean per group
Number of pups per group and per dam
- at birth (live and dead)
- on postnatal days 1, 4 and 13
Number of male and female pups per group and per dam
- at birth (alive and dead)
- on postnatal days 1, 4 and 13
Number of stillbirths
- per group
- per dam
Number of pups with malformations
- per group
- per dam
LABORATORY EXAMINATIONS
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight and collected into tubes as follows:
- EDTA anticoagulant (whole blood) for haematological examinations
- Citrate anticoagulant (plasma) for coagulation tests
- Serum for clinical chemistry
The following sampling times and animals were employed:
Time of blood sampling: At necropsy
Animals: 10 males and 10 females randomly selected from each group
HAEMATOLOGY
The following parameters were determined (Instrument: ADVIATM 120, Siemens Diagnostics GmbH, 35463 Fernwald, Germany):
Parameter (in blood)/ Unit
Haemoglobin content (HGB)/ mmol/L
Erythrocytes (RBC)/ 10^6/µL
Leucocytes (WBC)/ 10^3/µL
Differential blood count : - relative/ % - absolute 10^3/µL
Reticulocytes (Reti)/ ‰ of the erythrocytes
Haematocrit value (HCT)/ %
Platelets (PLT)/ 10^3/µL
Mean corpuscular volume (MCV)/ fL
Mean corpuscular haemoglobin (MCH)/ fmol
Mean corpuscular haemoglobin concentration (MCHC)/ mmol/L
Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.
COAGULATION PARAMETERS
The following parameters were determined (Instrument: Amax Destiny Plus™, TCoag Deutschland GmbH, 32657 Lemgo, Germany):
Parameter (in plasma)/ Unit
Prothrombin time (PT)/ sec
Activated partial thromboplastin time (aPTT)/ sec
BIOCHEMISTRY
The following parameters were determined (Instrument: KONELAB 30i, Thermo Fisher Scientific , 63303 Dreieich, Germany):
Parameter (in serum)/ Unit
Albumin/ g/L
Bile acids/ µmol/L
Bilirubin (total)/ µmol/L
Cholesterol (total)/ mmol/L
Creatinine/ µmol/L
Glucose/ mmol/L
Protein (total)/ g/L
Blood urea nitrogen (BUN)/ mmol/L
Calcium/ mmol/L
Chloride/ mmol/L
Potassium/ mmol/L
Sodium/ mmol/L
Alanine aminotransferase (ALAT)/ U/L
Alkaline phosphatase (aP)/ U/L
Aspartate aminotransferase (ASAT)/ U/L
Lactate dehydrogenase (LDH)/ U/L
Globulin/ g/L -> by substraction
Albumin/globulin ratio/ non-dimensional -> by calculation
Sodium/Potassium ratio/ non-dimensional -> by calculation
BUN/creatinine ratio/ non-dimensional -> by calculation
DETERMINATION OF THYROID HORMONES (T4 and TSH)
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 6.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below:
10 males and 10 females per group#1; fasted; Test days 127/129 (at sacrifice)
#1 Animals also selected for laboratory examinations
Blood samples were processed for serum, divided into aliquots and stored -20°C ± 10% until analyses using commercial ELISA kits.
URINALYSIS
The urine was collected for 16 hours in URIMAX funnel cages. The collection of urine was terminated immediately prior to start of blood withdrawals for the haematological and clinical chemistry examinations. The following sampling times and animals were employed:
At the end of dosing period (prior to blood withdrawal), 10 males and 10 females randomly selected from each group
The following parameters were determined by using the instruments given below:
Parameter/ Unit/ Instrument
Volume/ mL/ Graduated vessel
pH/ non-dimensional/ Digital pH meter, type WTW InoLab pH 720
Specific gravity/ g/mL/ Kern Refractometer, type ORA 2PA Sample compared with water (nominal value of 1.000)
The following examinations were also performed using Combur 9 Test (semi-quantitative/qualitative indicators) for determination of analyte concentration in the urine:
Proteins
Glucose
Bilirubin
Urobilinogen
Ketones
Haemoglobin (Hb) (approximate values)
Nitrite
Microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of the following parameters:
E Epithelial cells
L Leucocytes
R Erythrocytes
B Organisms
C Further constituents (i.e. sperm, casts)
A Crystalluria
The frequency of the above parameters were recorded as follows:
0 None found in any field examined
+ Few in some fields examined
++ Few in all fields examined
+++ Many in all fields examined
The colour and the turbidity of the urine were examined visually. - Oestrous cyclicity (parental animals):
- Vaginal smears were taken and the oestrus cycle stages were determined at the following time points:
F0 animals during the 14-day pre-exposure period to select 96 animals with regular oestrus cycles (4-5 days) and during 10 weeks of premating until evidence of mating.
F1 animals, Cohort 1A starting after onset of vaginal patency until first appearance of cornified cells and two weeks starting around PND 75.
F0 and F1 animals On the day of sacrifice, Shortly before necropsy. - Sperm parameters (parental animals):
- Sperm viability and morphology (spermiogram)
• All F0 males
• All F1 Cohort 1A males
One epididymis and one testicle will be used for the sperm count. The sperm viability is determined and the sperm morphology is examined according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001). - Litter observations:
- CLINICAL SIGNS
Dated and signed records of appearance, change, and disappearance of clinical signs were maintained on clinical history sheets for each animal. Throughout the test period, each animal (parental animals and pups) was observed for clinical signs at least once daily. Behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity were recorded. In case signs of toxicity occurred the frequency of observations was increased. Each animal was observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded. In addition, the animals were checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays, the animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approximately 3:30 p.m. Cage side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, locomotor activity and behaviour patterns.
A more detailed examination of all F1 Cohort 1B animals was conducted on a weekly basis. The F1 animals of Cohort 1B were examined weekly after weaning until termination. Detailed clinical observations were carried out for all animals outside the home cage in a standard arena at approximately the same time of day, each time preferably by observers unaware of the treatment. The observations included in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.
MORTALITY
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. On Saturdays and Sundays, a similar procedure was followed with a final check at approximately 3.30 p.m. If necessary, these provisions allowed to record any premortal symptoms in detail and a post mortem examination to be carried out during the working period of a day. For the prematurely deceased pups a post mortem examination was performed.
BODY WEIGHT
The body weight the animals was recorded as follows:
Lactation period: PND 1, 4, 7, 14, 21
Time after weaning: Daily, starting on PND 22 - report of weekly values
Termination of in-life: Day of sacrifice
FOOD AND DRINKING WATER CONSUMPTION:
Food intake per rat (g) was calculated using the total amount of food given to and left by each rat in each group on those days that are listed below.
End of weaning: Weekly
From these data the relative food consumption (in g/kg b.w./day) was determined using the following formula:
Relative food consumption (g/kg b.w./day) = (Total food given (g) - Total food left (g))/ (Number of animal days# x Body weight (kg))
# The term 'animal days' counts one animal day for each animal alive for a whole
day; it is assumed that on the day of death an animal does not eat.
Drinking water consumption was monitored daily by visual appraisal throughout the study.
EXAMINATION OF THE PUPS (F1 Generation)
As soon as possible after delivery, each litter was examined to establish the number and sex of pups, stillbirths, live births, runts (i.e. body weight less than 70% of mean litter weight) and the presence of gross abnormalities. Abnormal behaviour or changes in the external appearance of the pups noted during the daily cage side inspections were recorded.
The following examinations/observations were done for the offspring:
COUNTING, SEXING AND WEIGHING
Live pups were counted, sexed and weighed on post-natal days (PND) 1, 4, 7, 14 and 21.
ANO-GENITAL DISTANCE
On PND 4 before litter adjustment the ano-genital distance (AGD) of all pups was determined using a scale. The AGD was normalised to the cube root of body weight.
LITTER ADJUSTMENT
After counting on PND 4 (lactation day 4), the litters were adjusted to 10 pups per litter (5 pups/sex/litter) by eliminating (culling) surplus pups using a randomization scheme generated by Provantis® . Selective elimination of pups, e.g. based upon body weight was not appropriate. In case of unequal gender distribution, a partial litter size adjustment was performed (e.g. 6 male and 4 female pups).
COUTING OF MALE NIPPLES/ AREOLAE
Nipples/areolae were counted in all male pups on PND 13.
SEXUAL MATURATION
All F1 Pups (Cohorts 1A and 1B) were evaluated daily for balano-preputial separation or vaginal opening which indicate sexual maturity of the animals. The genitals were examined for any abnormalities. The body weight was recorded at the time point of balano-preputial separation or vaginal opening.
Balano-preputial separation:
During this examination the time point of the onset of the function of the preputial glands is determined. A soft pressure is exerted against the root of the penis. If this leads to the observation of small drops of secretion from the preputial glands on both sides of the foreskin, the postnatal day of this observation is determined as the time point of the onset of preputial gland function.
LABORATORY EXAMINATIONS
Blood samples were taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight and collected into tubes as follows:
- EDTA anticoagulant (whole blood) for haematological examinations
- Citrate anticoagulant (plasma) for coagulation tests
- Serum for clinical chemistry
The following sampling times and animals were employed:
Time of blood sampling: At necropsy
Animals: 10 males and 10 females randomly selected from each group
HAEMATOLOGY
The following parameters were determined (Instrument: ADVIATM 120, Siemens Diagnostics GmbH, 35463 Fernwald, Germany):
Parameter (in blood)/ Unit
Haemoglobin content (HGB)/ mmol/L
Erythrocytes (RBC)/ 10^6/µL
Leucocytes (WBC)/ 10^3/µL
Differential blood count : - relative/ % - absolute 10^3/µL
Reticulocytes (Reti)/ ‰ of the erythrocytes
Haematocrit value (HCT)/ %
Platelets (PLT)/ 10^3/µL
Mean corpuscular volume (MCV)/ fL
Mean corpuscular haemoglobin (MCH)/ fmol
Mean corpuscular haemoglobin concentration (MCHC)/ mmol/L
Following the haematological examinations using the ADVIA system, blood smears were prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.
COAGULATION PARAMETERS
The following parameters were determined (Instrument: Amax Destiny Plus™, TCoag Deutschland GmbH, 32657 Lemgo, Germany):
Parameter (in plasma)/ Unit
Prothrombin time (PT)/ sec
Activated partial thromboplastin time (aPTT)/ sec
BIOCHEMISTRY
The following parameters were determined (Instrument: KONELAB 30i, Thermo Fisher Scientific , 63303 Dreieich, Germany):
Parameter (in serum)/ Unit
Albumin/ g/L
Bile acids/ µmol/L
Bilirubin (total)/ µmol/L
Cholesterol (total)/ mmol/L
Creatinine/ µmol/L
Glucose/ mmol/L
Protein (total)/ g/L
Blood urea nitrogen (BUN)/ mmol/L
Calcium/ mmol/L
Chloride/ mmol/L
Potassium/ mmol/L
Sodium/ mmol/L
Alanine aminotransferase (ALAT)/ U/L
Alkaline phosphatase (aP)/ U/L
Aspartate aminotransferase (ASAT)/ U/L
Lactate dehydrogenase (LDH)/ U/L
Globulin/ g/L -> by substraction
Albumin/globulin ratio/ non-dimensional -> by calculation
Sodium/Potassium ratio/ non-dimensional -> by calculation
BUN/creatinine ratio/ non-dimensional -> by calculation
DETERMINATION OF THYROID HORMONES (T4 and TSH)
Blood samples were taken under isoflurane anaesthesia from animals fasted overnight always at the same time of day (in the morning between 6.30 a.m. and 9.30 a.m. for the adult animals) as scheduled below:
F1 Generation: 2 surplus pups per litter#2; non-fasted; PND 4; T4 only
F1 Generation: 2 surplus pups per litter#2#3; non-fasted; PND 21/22; T4 + TSH
F1 Generation: 10 males and 10 females per Cohort 1 A group#1; fasted; PND 90 - 92 (at sacrifice); T4 + TSH
#1 Animals also selected for laboratory examinations
#2 If the individual volume obtained from the pups was insufficient for analysis, the samples may be pooled by litter.
#3 Pups were selected from all available litters.
Blood samples were processed for serum, divided into aliquots and stored -20°C ± 10% until analyses using commercial ELISA kits.
URINALYSIS
The urine was collected for 16 hours in URIMAX funnel cages. The collection of urine was terminated immediately prior to start of blood withdrawals for the haematological and clinical chemistry examinations. The following sampling times and animals were employed:
At the end of dosing period (prior to blood withdrawal), F1 Generation Cohort 1A 10 males and 10 females randomly selected from each group
The following parameters were determined by using the instruments given below:
Parameter/ Unit/ Instrument
Volume/ mL/ Graduated vessel
pH/ non-dimensional/ Digital pH meter, type WTW InoLab pH 720
Specific gravity/ g/mL/ Kern Refractometer, type ORA 2PA Sample compared with water (nominal value of 1.000)
The following examinations were also performed using Combur 9 Test (semi-quantitative/qualitative indicators) for determination of analyte concentration in the urine:
Proteins
Glucose
Bilirubin
Urobilinogen
Ketones
Haemoglobin (Hb) (approximate values)
Nitrite
Microscopic examination of urine samples was carried out by centrifuging samples and spreading the resulting deposit on a microscope slide. The deposit was examined for the presence of the following parameters:
E Epithelial cells
L Leucocytes
R Erythrocytes
B Organisms
C Further constituents (i.e. sperm, casts)
A Crystalluria
The frequency of the above parameters were recorded as follows:
0 None found in any field examined
+ Few in some fields examined
++ Few in all fields examined
+++ Many in all fields examined
The colour and the turbidity of the urine were examined visually. - Postmortem examinations (parental animals):
- NECROPSY
On the day of necropsy, vaginal lavages of the adult animals were obtained and examined to determine the stage of estrous cycle and allow correlation with the histopathology of the female reproductive organs. The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis.
A gross or full necropsy of the animals was carried out. At gross necropsy the animals were inspected externally and/or internally for gross abnormalities. The full necropsy additionally included sampling and weighting of selected organs.
Blood samples for determination of haematological and biochemical parameter as well as for thyroid hormone determination were taken. The animals were weighed, dissected and inspected macroscopically (gross necropsy) as follows:
Animals/ Examination after sacrifice/ Time of necropsy/ Vaginal lavage
All males#1/ Full necropsy/ TD 127 - 129/ No
All dams#1/ Full necropsy /TD 129 - 134/ Yes
#1 After weaning of F1 Generation
In the case the time of dissection was fallen on a weekend or bank holiday, necropsy would have been postponed to the next working day and dosing would have been continued up to and including the day before sacrifice.
Animals which were prematurely sacrificed or died during the study were necropsied as soon as possible after exitus.
NECROPSY OF ADULT ANIMALS
At the time of sacrifice or premature death during the study, all adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
WEIGHING/ PRESERVATION OF ORGANS - F0 Generation
During necropsy the number of implantation sites in the uteri was recorded in the female animals and used to evaluate reproductive performance. Apparently non-pregnant uteri of the F0 animals were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI The following organs of the male and female F0 animals were weighed before fixation except for the thyroid. Paired organs were weighed individually and identified as left or right:
Adrenal gland (2)
Ovary (2)
Prostate (dorsolateral and ventral parts combined)
Brain
Oviducts (2)
Seminal vesicles with coagulating glands
Epididymis (2)
Spleen
Pituitary
Heart
Testis (2)
Uterus incl. cervix
Kidney (2)
Thyroid, parathyroids (after fixation) (1, left)
Liver
Thymus
# Weighing before fixation except for thyroids. Paired organs were weighed individually and identified as left or right.
The following organs or parts thereof obtained from the male and female animals of the F0 Generation were preserved in an appropriate fixative:
Fixative: Davidson's solution
Eye with optic nerve (2)
Fixative: modified Davidson's solution
Epididymis (1)#
Testis (1)#
Fixative: 7%buffered formalin
Adrenal gland (2)
Ovary (2)
Bone
Oviducts
Bone marrow (os femoris)
Pituitary
Brain (cerebrum, cerebellum, pons)
Prostate
Gross lesions observed
Seminal vesicles with coagulating glands
Heart (3 levels: right and left ventricle, septum)
Spinal cord (3 sections)
Intestine, small (duodenum, jejunum, ileum, incl. Peyer’s patches, Swiss roll method)
Spleen
Stomach
Intestine, large (colon, rectum)
Thyroid (2, incl. parathyroids)
Kidney and ureter (2)
Thymus
Liver
Trachea (incl. larynx)
Lungs (with mainstem bronchi and bronchioles)
Urinary bladder
Mammary gland
Uterus (incl. cervix)
Muscle (skeletal)
Vagina
Nerve (sciatic)
Vas deferens
Oesophagus
# The second epididymis and testis was not preserved but used for the spermiogram.
Any other organs displaying macroscopic changes were also be preserved. In addition, sperm viability and morphology were evaluated for all male F0 animals and bone marrow smears were prepared for selected F0 animals (same as used for laboratory examinations).
SPEERMIOGRAM
One epididymis and one testis of all F0 males were used for the sperm count. The sperm viability was determined and the sperm morphology was examined according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).
BONE MARROW EXAMINATION
During dissection fresh bone marrow was obtained from the os femoris (3 air-dried smears/animal) of male and female F0 animals and stained according to PAPPENHEIM. The myeloid : erythroid ratio was determined by cell differentiation (counting of 200 nuclei-containing cells) for the animals of groups 1 and 4.
HISTOPATHOLOGY
The blood smears prepared from all animals during the haematological examination are available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Sponsor.
Full histopathology was performed on the preserved organs of the control and high dosed animals of groups 1 and 4:
- F0 Generation (parents): 20 male and 20 female animals per group
A full histopathology was also performed for all deceased or prematurely sacrificed animals, from the F0 Generation.
The organs "Organs/Tissues to be preserved - F0 Generation"and "Organs/Tissues to be preserved - F1 Generation Cohort 1A" were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin (H&E) staining. Parathyroids could not always identified macroscopically. They were examined microscopically if in the plane of section. In addition, frozen sections of the heart, liver and one kidney were examined after staining with Oil Red O.
Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testis and one epididymis of the control and high dosed animals of groups 1 and 4 following H&E and PS staining:
- F0 Generation (parents): 20 male animals per group
In case of test item-related changes in the high dose level group 4, the Sponsor would have given sufficient notice before the corresponding organs of the animals of the F0 Generation of the intermediate and low dose level groups (group 2 and 3) are sectioned and examined histopathologically.
HISTOPATHOLOGICAL EVALUATION
Histotechnique was performed by the test institute. The slides were labelled with study number, test species, animal number and block number and were dispatched on August 07, 2020 to the Test Site for histopathological evaluation.
The transport of slides to the histopathology Test Site (TS) was arranged by the test institute, whereas the return transport to the Test Facility will be arranged by the TS. The study phase was recorded under the TS reference number 12848C. All observations upon final assessment were reported per animal and the findings considered to be relevant for the treatment were recorded in incidence and occurrence tables. All microscopic findings are recorded, reported and archived with the with the PathData system .
The report of this study phase comprises a description of objective, materials, methods and results (macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report was presented electronically to the Study Director for comments/review. Comments will be addressed and the Draft Final Phase Report of the histopathology phase of the study will be presented to the TS Quality Assurance Unit (TSQAU) for auditing.
The audited Draft Final Phase Report of the histopathology phase of the study will be sent electronically to the Study Director who will provide documented approval on the contents of the Phase Report by e-mail to the Principal Investigator. An original of the final, signed Phase Report will be sent (on paper and electronically) to the Study Director; a copy will be archived by the Test Site. - Postmortem examinations (offspring):
- NECROPSY
On the day of necropsy, vaginal lavages of the adult animals were obtained and examined to determine the stage of estrous cycle and allow correlation with the histopathology of the female reproductive organs. The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis. The pups were euthanized by decapitation (PND4) or by carbon dioxide (CO2) inhalation (PND 21/22). A gross or full necropsy of the F1 animals was carried out. At gross necropsy the animals were inspected externally and/or internally for gross abnormalities. The full necropsy additionally included sampling and weighting of selected organs.
Blood samples for determination of haematological and biochemical parameter as well as for thyroid hormone determination were taken. The animals were weighed, dissected and inspected macroscopically (gross necropsy) as follows:
Animals/ Examination after sacrifice/ Time of necropsy/ Vaginal lavage
All 'surplus' (= culled) pups/ Gross necropsy/ PND 4/ No
10 ‘surplus’ pups/sex/group/ Gross necropsy, partial organ/tissue preservation/ PND 21 - 22/ No
All remaining 'surplus' pups/ Gross necropsy/ PND 21 - 22/ No
Cohort 1A All animals#2/ Full necropsy/ PND 90-92/ Yes
Cohort 1B All animals#2/ Full necropsy/ PND 98-102/ Yes
#1 After weaning of F1 Generation
#2 At the end of the dosing period
In the case the time of dissection was fallen on a weekend or bank holiday, necropsy would have been postponed to the next working day and dosing would have been continued up to and including the day before sacrifice.
Animals which were prematurely sacrificed or died during the study were necropsied as soon as possible after exitus.
NECROPSY OF DEAD OR "SURPLUS PUPS"
External inspection for gross abnormalities:
Dead pups and culled F1 Pups on PND 4 were carefully examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.
External and internal inspection for gross abnormalities:
Pups not selected for the F1 Cohorts were sacrificed on PND 21/22 and examined macroscopically for any abnormalities or pathological changes.
NECROPSY OF ADULT ANIMALS
At the time of sacrifice or premature death during the study, all adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
WEIGHING / PRESERVATION OF ORGANS "SURPLUS PUPS"
Ten ‘surplus’ pups per sex and group were used for organ weighing and preservation. The following organs/tissues of the F1 Pups were weighed and/or preserved in 7% formalin:
Brain#
Spleen#
Gross Abnormalities
Stomach
Kidney (left, right) #
Thymus#
Mammary gland
# Organs were weighed before fixation. Paired organs were weighed individually and identified as left or right.
WEIGHING / PRESERVATION OF ORGANS - F1 Generation Cohort 1A
The following organs of all adult male and female F1 Cohort 1A animals were weighed before fixation except for the thyroid. Paired organs were weighed individually and identified as left or right.
Adrenal gland (2)
Lymph node (1, cervical)
Thymus
Brain
Lymph node (1, mesenteric)
Prostate (dorsolateral and ventral parts combined)
Epididymis (2)
Ovary (2)
Seminal vesicles with coagulating glands
Heart
Oviducts (2)
Pituitary
Kidney (2)
Spleen
Uterus incl. cervix
Liver
Testis (2)
Thyroid incl. parathyroids (after fixation) (1, left)
# Weighing before fixation except for thyroids. Paired organs were weighed individually and identified as left or right.
Organs/Tissues of 'surplus' F1 Pups (PND 4) to be preserved and/or weighed
Weighing and preservation of the following organs/tissues (fixative 7% formalin)
Brain#
Intestine, large (colon, rectum)
Mammary gland
Gross Abnormalities
Kidney (left, right) #
Spleen#
Heart#
Liver#
Stomach
Intestine, small (duodenum, jejunum, ileum)##
Lungs#
Thymus#
# Organs were weighed before fixation. Paired organs were weighed individually and identified as left or right.
## Swiss roll method
The following organs or parts thereof obtained from the male and female animals of F1 Generation Cohort 1 A were preserved in an appropriate fixative:
Fixative: Davidson’s solution
Eye with optic nerve (2)
Fixative: modified Davidson’s solution
Epididymis (1)#1
Testis (1)#1
Fixative: 7%buffered formalin
Adrenal gland (2)
Oesophagus
Bone
Ovary (2)#4
Bone marrow (os femoris)
Oviducts
Brain (cerebrum, cerebellum, pons)
Pituitary
Gross lesions observed
Prostate
Heart (3 levels: right and left ventricle, septum)
Seminal vesicles with coagulating glands
Intestine, small (duodenum, jejunum, ileum, incl. Peyer’s patches, Swiss roll method) Spinal cord (3 sections)
Spleen#3
Intestine, large (colon, rectum)
Stomach
Kidney and ureter (2)
Thyroid (2, incl. parathyroids)
Liver
Thymus
Lungs (with mainstem bronchi and bronchioles)
Trachea (incl. larynx)
Lymph node (1, cervical)#2
Urinary bladder
Lymph node (1, mesenteric)#2
Uterus (incl. cervix)
Mammary gland
Vagina
Muscle (skeletal)
Vas deferens
Nerve (sciatic)
#1 The second epididymis and testis was not preserved but used for the spermiogram
#2 Lymph nodes were preserved for all Cohort 1A animals, however, histopathology was only carried out for 10 animals/sex/group (1 animal per litter, all litters represented by at least 1 pup; randomly selected).
#3 For 10 animals/sex/group of Cohort 1A, randomly selected (same as selected for laboratory examination): One half of the spleen was preserved for histopathoogical evaluation, the second half was used for splenic lymphocyte subpopulation analysis
#4: One ovary of the F1 Cohort 1A females of groups 1 and 4 ( 20 females per group) was processed for detailed histopathological examination as follows:
- Ten (10) 2-4-μm step sections with 50 μm steps in between;
- Each slide was labelled with the slide number to follow the sequence.
Any other organs displaying macroscopic changes were also be preserved. In addition, sperm viability and morphology were evaluated for all male animals of F1 Cohort 1A and bone marrow smears were prepared for selected animals of F1 Cohort 1A animals.
WEIGHING/ PRESERVATION OF ORGANS - F1 Generation Cohort 1B
The following organs of male and female F1 Cohort 1B animals were weighed before fixation except for the vagina. Paired organs were weighed individually and identified as left or right.
Organ/ Weigh/ Fixative/ Block
Endocrine system:
Adrenal gland (2)/ Yes/ 7% formalin/ No
Pituitary/ Yes/ 7% formalin/ Yes
Thyroid (2) (including parathyroids)/1, post-fixation/ 7% formalin/ No
Reproductive system:
Epididymis (2)/ Yes/ Modified Davidson’s/ Yes
Ovary (2)/ Yes/ 7% formalin/ Yes
Prostate/ Yes/ 7% formalin/ Yes
Seminal vesicles with coagulating glands/ Yes/ 7% formalin/ Yes
Testicle (2)/ Yes/ Modified Davidson’s/ Yes
Uterus (including oviducts and cervix)#/ Yes/ 7% formalin/ Yes
Vagina/ No/ 7% formalin/ Yes
Vas deferens/ No/ 7% formalin/ No
# The oviducts of Cohort 1B were weighed separately as in Cohort 1A.
The adrenals, thyroids and vas deferens are not processed to block stage as this is not required by the current OECD TG 443 (paragraph 67) adopted 25 June 2018.
In the case of test item-related changes in the corresponding organs of the F0 and F1 Cohort 1A animals, the Study Monitor will be given sufficient notice before the respective organs of F1 Cohort 1B animals are sectioned and examined histopathologically, if possible.
SPERMIOGRAM - F1 Generation Cohort 1A
One epididymis and one testis of all F0 and F1 Cohort 1A males were used for the sperm count. The sperm viability was determined and the sperm morphology was examined according to the method described by I. Chahoud and R. Franz (1993) as well as by S. Plassmann and H. Urwyler (2001).
BONE MARROW EXAMINATION - F1 Generation Cohort 1A
During dissection fresh bone marrow was obtained from the os femoris (3 air-dried smears/animal) of male and female F0 and F1 Generation Cohort 1 A animals and stained according to PAPPENHEIM. The myeloid : erythroid ratio was determined by cell differentiation (counting of 200 nuclei-containing cells) for the animals of groups 1 and 4.
PHENOTHYPIC ANALYSIS OF SPLEEN CELLS - F1 Generation Cohort 1A
The spleens of the male and female F1 Cohort 1A animals were split in two halves. The portion of the spleen not preserved for histopathology was minced using a mechanic dissociator to prepare single cell suspensions. The following parameters were determined in the samples( Instrument: MACSQuant® Analyzer 10/16, Miltenyi Biotec GmbH, Friedrich-Ebert-Str. 68, 51429 Bergisch Gladbach, Germany):
CD4+ T-Lymphocytes
CD8+ T-Lymphocytes
Pan-T-lymphocytes (CD3+)
B-lymphocytes (CD45RA+)
Natural killer cells (CD161+)
Evaluation was performed by the test institute.
HISTOPATHOLOGY
The blood smears prepared from all animals during the haematological examination are available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Sponsor.
Full histopathology was performed on the preserved organs of the control and high dosed animals of groups 1 and 4:
- F1 Generation (Cohort 1A): 20 male and 20 female animals per group
A full histopathology was also performed for all deceased or prematurely sacrificed animals, from Cohort 1A.
The organs listed in"Organs/Tissues to be preserved - F1 Generation Cohort 1A" were examined histopathologically after preparation of paraffin sections and haematoxylin-eosin (H&E) staining. Parathyroids could not always identified macroscopically. They were examined microscopically if in the plane of section.
In addition, frozen sections of the heart, liver and one kidney were examined after staining with Oil Red O.
Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testis and one epididymis of the control and high dosed animals of groups 1 and 4 following H&E and PS staining:
- F1 Generation (Cohort 1A): 20 male animals per group
Detailed histopathological examination with quantitative evaluation of primordial and small growing follicles as well as corpora lutea was performed on one ovary of the following control and high dosed animals of groups 1 and 4:
- F1 Generation (Cohort 1A): 20 female animals
In case of test item-related changes in the high dose level group 4, the Sponsor would have given sufficient notice before the corresponding organs of the animals of the F1 Generation Cohort 1A of the intermediate and low dose level groups (group 2 and 3) are sectioned and examined histopathologically.
HISTOPATHOLOGICAL EVALUATION
Histotechnique was performed by the test institute.
The slides were labelled with study number, test species, animal number and block number and were dispatched on August 07, 2020 to the Test Site for histopathological evaluation.
The transport of slides to the histopathology Test Site (TS) was arranged by Provivo, whereas the return transport to the Test Facility will be arranged by the TS. The study phase was recorded under the TS reference number 12848C. All observations upon final assessment were reported per animal and the findings considered to be relevant for the treatment were recorded in incidence and occurrence tables. All microscopic findings are recorded, reported and archived with the with the PathData system .
The report of this study phase comprises a description of objective, materials, methods and results (macroscopic and microscopic changes) and conclusions. The unaudited Draft Phase Report was presented electronically to the Study Director for comments/review. Comments will be addressed and the Draft Final Phase Report of the histopathology phase of the study will be presented to the TS Quality Assurance Unit (TSQAU) for auditing.
The audited Draft Final Phase Report of the histopathology phase of the study will be sent electronically to the Study Director who will provide documented approval on the contents of the Phase Report by e-mail to the Principal Investigator. An original of the final, signed Phase Report will be sent (on paper and electronically) to the Study Director; a copy will be archived by the Test Site. - Statistics:
- DATA ACQUISITION: The following data were captured or calculated by the departmental computerized system Provantis: Clinical signs, body weight, body weight gain, food consumption, haematological and biochemical parameters. Data maintained on paper (e.g. pup data, ELISA experiments) were entered in Provantis in a retrospective manner using the laboratory records according to the appropriate SOPs.
STATISTICS: The statistical evaluation of the parametrical values was done by Provantis® using the following settings: Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
For the statistical evaluation of the histopathological data FISHER's exact test was used.
The mean values and standard deviations were calculated to the highest possible degree of accuracy and then rounded to the reported number of decimal places. Hence, deviations to the last decimal place of up to +/- 1 may occur caused by rounding. - Reproductive indices:
- The following indices were calculated for each group:
Female Fertility Index [%] = (Number of pregnant rats/ Number of rats paired with a male) x 100
The female fertility index reflects the total number of dams that had achieved pregnancy, including dams which delivered at term, aborted or had fully resorbed litters.
Gestation Index [%] = (Number of dams with live pups/ Number of pregnant rats) x 100
For each litter and group the following indices were determined:
Birth Index [%] #1 = (Total number of pups born (alive + dead)/Number of implantation sites) x 100
Live Birth Index [%] = (Number of pups alive on day 0/1 of lactation/ Total number of pups (alive + dead)) x 100
Viability Index [%] pre-cull = (Number of pups alive on day 4 (pre cull)/ Number of pups alive on day 0/1) x 100
Viability Index [%] post cull = (Number of pups alive on day 13/ Number of pups alive on day 4 (post cull)) x 100
Post-implantation loss [%]#1 = ((Implantations - number of pups born alive)/ Implantation sites) x 100
#1: Twins (shared placentae) are considered as additional implantation sites in the calculation of the birth index and the post-implantation loss, to avoid a birth index above 100% or a negative post-implantation loss. - Offspring viability indices:
- Birth and live birth index, post-impantation loss and viability indices of the pups.
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Male animals
No test item-related changes in behaviour and the external appearance were noted for the male animals of the low dose group (25 mg IPDA/kg b.w./day). Nevertheless, in this dose group a haemorrhagic nose/snout (no. 71) or pultaceous faeces (no. 64) were noted for one animal each on one test day each. Furthermore, an increased water consumption (no. 52) was noted for one animal on seven test days. Due to the low incidence of only one affected animal for the observed symptoms they were considered to be spontaneous.
At the intermediate dose level (80 mg IPDA/kg b.w./day) a post-dosing salivation was noted for 17 of 24 male animals. The observation of a post dosing salivation was considered to be test item-related but not adverse (or of toxicological relevance), as in nearly all cases it did not last longer than 60 min (see section 7.3.1. ‘Start and duration of observations’). Additionally, an increased water consumption was noted for one animal (no. 120) on 4 test days.
At the high dose level (240/160 mg IPDA/kg b.w./day) a post-dosing salivation was noted for all surviving male animals. Furthermore, breathing sounds were noted for 10 of 22 animals, piloerection for 5 of 22 animals, an increased water consumption for 2 animals (nos. 146 and 158) and haemorrhagic urine (no. 159) and pultaceous faeces (no. 145) for one animal each.
The observations of breathing sounds and piloerection that were noted at the high dose level were considered to be test item related, as they were noted for several animals. Furthermore, breathing sounds were in most cases noted at the start of the working day before dosing and lasted consistently for the whole day.
Female animals
No signs of toxicological relevance were noted at the low and the intermediate dose level (25 or 80 mg IPDA/kg b.w./day) during the pre-mating, the gestation and the lactation period. The observation of post dosing salivation for one animal of the low dose group and several animals of the intermediate dose group was considered to be of no toxicological relevance.
At the high dose level (240/160 mg IPDA/kg b.w./day) 4 female animals had to be prematurely sacrificed due to poor health condition. In detail 3 of the 4 prematurely sacrificed animals were sacrificed during the pre-mating period on test days 49, 59, 24 (nos. 174, 178 and 182, respectively) and no. 187 was prematurely sacrificed at the end of the gestation period (gestation day 20).
At the high dose group post dosing salivation was noted for all animals, piloerection and / or breathing sounds were observed for 5 and 10 animals, respectively. Furthermore, an increased respiratory rate (no. 181) and an increased water consumption (no. 188) were noted during the pre-mating / mating period for altogether 2 females. Female no. 187, which was prematurely sacrificed on gestation day 20 revealed gasping, a haemorrhagic nose/snout, laboured breathing and a thickened abdomen during the pre-mating / mating period.
The 4 prematurely sacrificed high dose females (nos. 174, 178, 182 and 187) all showed breathing sounds before death. A haemorrhagic nose/snout was additionally noted for nos. 182 and 187. No. 182 furthermore revealed laboured breathing and no. 187 piloerection on the day and two days before the sacrifice, respectively (see Text Tables 7-3 and 7-4B in section 7.2 ‘Mortality’).
As a result of the increased number of recorded observations the 4 females were prematurely sacrificed. The later histopathology assessment revealed an accident during the process of dosing (influx of test item formulation into the respiratory tract by accidental influx or incidental regurgitation) for female no.187 (prematurely sacrificed on gestation day 20). This can also be the cause of gasping, laboured breathing and the thickened abdomen that were noted for female no. 187 during the pre-mating / mating period.
The observation of breathing sounds are in nearly all cases long lasting observations which lasted for several hours or for the whole day.
The observations at the high dose level that were noted for the female animals were considered to be test item-related and, with the exception of post-dosing salivation, of toxicological relevance. - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Males and females
No test item-related premature death was noted in the control group, low dose group (25 mg IPDA/kg b.w./day) and intermediate dose group (80 mg IPDA/kg b.w./day) for the male and female animals.
Three test item-related deaths were noted at the high dose level (240/160 mg IPDA/kg b.w./day).
At the high dose level (240/160 mg IPDA/kg b.w./day) one male (no. 153) and 2 female animals (nos. 178 and 182) had to be prematurely sacrificed due to poor health condition on test day 73, 59 and 24, respectively (see Text Table 7-2). The noted observations during the daily cage side observations before deaths are listed in Text Table 7-3.
All recorded microscopic findings were stress-related changes or non-specific changes that are frequently observed in animals with deteriorated general conditions, and the findings that could be specific to the test item treatment were not identified. No signs of misgavage, an accidental influx or incidental regurgitation of the test item-formulation into the respiratory tract were observed for these 3 animals. Thus, histomorphologically, a direct cause of animals morbidity could not be established (see section 3.4.1. ‘Microscopic Findings in Decedents’ of the Histopathology Phase Report).
The macroscopic findings that were noted for the prematurely deceased animals (no gross lesions were considered to be specific to the test item) are listed in Text Table 5 in section 3.1 ‘Mortality’ of the Histopathology Phase Report.
Not test item-related premature deaths
The premature death of six animals (two male (nos. 151, 164) and 4 female (nos. 40, 126, 174, 187)) was caused by an accident during the process of dosing (see text table 7-4A).
Among other findings, reddened lungs were noted for 3 (nos. 126, 151, 174) of these six animals at necropsy.
The microscopic lesions that were noted for these animals were considered to be produced by accidental influx or incidental regurgitation of the test item-formulation into the respiratory tract and not to be adverse, but deemed to be the direct cause(s) of animals death or morbidity (see section 3.4.1. ‘Microscopic Findings in Decedents’ of the Histopathology Phase Report).
The male animal no. 164 of the high dose group (240/160 mg IPDA/kg b.w./day) was found dead on TD 16 after a misgavage. No changes in behaviour, the external appearance of the faeces were noted in the days prior to the premature sacrifice.
The animal was excluded from the evaluation and replaced by animal no. 193. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Males: Pre-mating-, mating- and post-mating period
No test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Females: Pre-mating-, gestation- and lactation period
No test item-related changes in body weight and body weight gain were noted for the female rats between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Males and females:
No test item-related differences between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) were noted for the body weight at autopsy for the male and female animals. - Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Males & Females
No test item-related difference in food consumption was noted between the control group and in the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day). - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Water consumption was performed by visual appraisal during the daily cage side observations. For nearly all animals, no decreased or increased water consumption was noted.
However, for 4 males and 1 female an increased water consumption was noted during the daily cage side observations on a few test days. Due to the low incidence this was considered spontaneous. - Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Males
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Females
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Decreased values in comparison to the control group were noted for the number of white blood cells for the female animals of the low ( 12.2 %, statistically not significant) and the intermediate dose group (27.2 % below the control; p ≤ 0.05).
However, these changes were considered spontaneous, as no dose response-relationship was noted considering all treatment groups and no changes were noted for the male animals. Additionally, the opposite picture was noted for the number of white blood cell in female animals of the F1 Generation (Cohort 1A), which were exposed to the test item during their whole life time. Finally, with one exception, all individual values of the low dose group (5.80 to 10.43 x10E3 cells/µL) and the intermediate dose group (4.70 to 9.91 x10E3 cells/µL) were in the range of the Provivo background data (4.65 to 11.38 x10E3 cells/µL).
In the number of eosinophilic granulocytes, statistically significant reductions were noted for the female animals of the low, the intermediate and the high dose group (52.3 %, 46.1 % and 51.5 %, respectively, below the control, p ≤ 0.05 or 0.01). No dose response-relationship was noted and the values in all three treatment groups were similar to each other. The noticed reductions in the number of eosinophilic granulocytes in the treated groups can be considered to be due to an increased value in the control group.
Furthermore, only two animals (one control and one intermediate dosed female) exhibit eosinophilic granulocytes values outside of the Provivo background range (0.05 to 0.42 x10E3 cells/µL). Hence, the decreased values in the treatment groups are considered as not relevant.
A statistically significantly reduced number of basophilic granulocytes was noted at the intermediate dose level (50.0 % below the control, p ≤ 0.05).
However, firstly all individual values of the control group and the treatment groups were within the Provivo background range. Secondly, no dose response-relationship was noted and thirdly no changes were noted for the male animals. Therefore, the statistically significantly decreased value that was noted at the intermediate dose level was considered to be spontaneous. - Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- Males
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) for the male animals.
Females
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) for the female animals.
However, a statistically significantly increased bilirubin concentration was noted at the low dose level (15.1 % above the control, p ≤ 0.05). However, as no dose response-relationship was noted and all individual values of the low dose group (2.5 to 4.4 µmol/L) were within the Provivo backround range (2.4 to 4.5 µmol/L). The increased bilirubin concentration that was noted at the low dose level was considered to be spontaneous.
A statistically significantly decreased glucose concentration was noted at the high dose level (18.7 % below the control, p ≤ 0.05). The glucose concentrations of 5 individual females of the high dose level (5.43 to 5.72 mmol/L) were marginally below the Provivo background range (5.88 to 9.72 mmol/L). However, no changes were noted for the high dosed male animals of the F0 Generation (2.2 % below the control). Also in Cohort 1A, no changes were noted for the high dosed male and female animals (6.8 % above or 5.0 % below the control, statistically not significant), which were exposed to the test item during their whole life time. Hence, the decreased glucose concentration that was noted for the female animals of the high dose group was considered to be spontaneous. - Endocrine findings:
- no effects observed
- Description (incidence and severity):
- Males and females
No test item-related differences for the examined thyroid hormone levels (T4 and TSH) were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day). Nearly all T4 concentrations of the individual animals were within the Provivo background range. Four animals one control male and female, one female of the low and the intermediate dose each showed a value slightly above the Provivo background range.
Differences between the control group and the treatment groups were noted for the TSH concentration of the female animals. The male animals treated with 25, 80 or 240/160 mg IPDA/kg b.w./day showed reductions in TSH concentration of about 51.5 %, 38.7 % and 14.2 %, respectively. The changes in the female animals were for the low dose ( 7.0 %) below and for the intermediate (+39.4 %) and high dose (+30.4 %) above the control. A comparison with the Provivo background data revealed that for the male animals a few individual values from the treatment groups were below the Provivo background range (1.506 to 6.964 ng/mL, 25 % to 95 %). In this case 25 % percentile was used as the lower value of the background range, as the 5 % percentile was at the low limit of detection (LOD = 0.081 ng/mL). Nearly all individual female animals of the treatment groups were within the Provivo background range (0.238 to 4.123 ng/mL, 25 % to 95 % percentile) (see Text Table 7 16A). As this was the case and the observed TSH differences were not statistically significant due to a high variability of the individual values, no dose response relationship was noted, and the changes of the male and female animals showed opposite directions, the observations were not considered to be test item-related.
Furthermore, no influence was noted on the thyroid weight in the male animals. For the female animals a statistically significantly increased relative organ weight was noted at the low dose level. However, this difference was considered to be spontaneous (see section ‘Organ weights (relative and absolute).
Finally, no changes were noted during the histopathological examination of the thyroid glands from the high dosed males and females. It is generally known that histopathological examination of the thyroid is usually more sensitive than thyroid weight and hormone levels ( Beekhuijzen et. al., 2016). The validation report of OECD 407 states that “thyroid histopathology was consistently the most reliable and most sensitive endpoint for the detection of thyroid modulation. Thyroid weigth was reliable, but was somewhat less sensitive when compared to thyroid histopathology. Circulating thyroid hormone levels (T3, T4, and TSH) were not always reliable and sensitive, but the standard operating procedures for blood sampling and for thyroid hormone analyses were not standardized to reduce stress induced variability and to reduce analytical variability, respectively. Circulating T4 levels were the most promising of the three thyroid hormonal values. As observed in this study the T4 values are nearly all in the range of the Provivo background range for the males and female animals. - Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- Males and females
No test item-related differences for the examined urinalysis parameters were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day). - Behaviour (functional findings):
- not examined
- Immunological findings:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- Males and females
No test item-related changes were observed for the examined male and female animals of the control and high dose group (240/160 mg IPDA/kg b.w./day) during the histopathological examination.
Among the dead animals, the lesions considered to be produced by accidental influx or incidental regurgitation of the test item formulation into the respiratory tract were observed in a female of control (no. 40), a female of intermediate dose group (no. 126), and two males (nos. 151, 164) and two females of high dose group (nos. 174, 187). The respiratory lesions are not considered to be adverse, but were deemed to be the direct cause(s) of animals’ death or morbidity.
The remainders of microscopic findings were stress-related changes or non-specific changes that are frequently observed in animals with deteriorated general conditions, and the findings that could be specific to the test item treatment were not identified.
Thus, histomorphologically, the direct cause(s) of animals’ morbidity was not established in male no. 153 and female nos. 178 and 182 of high dose group, and the effects to animal’s general conditions by the exposure with higher dose level of the test item were not excluded.
In conclusion, since the animals’ deaths occurred in this study were due to accidental/ incidental events, it was considered that there was no lethal effect in the dose levels of the test item selected for this study. However, the high dose level (240/160 mg/kg b.w./day) may affect the animals' general conditions, because of presence of stress-related microscopic findings in decedents.
In survivors, toxicologically relevant treatment-related changes were not observed in any organs and tissues in the control and high dose animals of the F0 generation.
In the previous study (OECD 422 study) in which the treatment in the high-dose group was started at a dose of 300 mg/kg b.w./day (thereafter it was reduced to 240 mg/kg during the treatment period), the treatment-related microscopic changes were observed in the stomach and kidneys. However, the histopathological examination of the present study revealed that the test item did not produce treatment-related microscopic changes not only in the stomach and kidneys but also in any other organs including male and female reproductive system of the high dose animals that were treated at 240/160 mg/kg b.w./day in F0 generation
.
Male reproductive organs:
No test item-related changes were noted. No histomorphological changes to be toxicologically concerned in the view of reproductive performance were found in the male reproductive organs including testes, epididymides, prostate glands, seminal vesicles and coagulating glands in the control and high dose group.
All testes showed completeness of stages and cell population. All the other findings recorded in the male reproductive organs were within the range of normal background changes that may be observed in this type of study or animals of this strain and age, and hence, deemed not to be treatment-related.
Female Reproductive Organs:
No test item-related histomorphological changes were noted in the female reproductive organs including ovaries, oviducts, uterus, uterine cervix and vagina. In summary no histopathological changes was observed which would have lead to an test-item induced change in the female reproductive performance.
All findings recorded were within the range of normal background lesions or physiological alterations that may be observed in this type of study or animals of this strain and age. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- bone marrow
Males and females
No test item-related differences for the myeloid/erythroid ratio of the bone marrow were noted between the control group and the high dose group (240/160 mg IPDA/kg b.w./day). - Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- ADULT P0
Monitoring of estrous cycles - Exposure period
After the allocation of the animals to the test groups and the start of treatment on test day 15, the estrous cycles were further monitored during the pre-mating and mating period until one day before a positive mating sign (verification of copulation) was noted.
No test item-related differences were noted for the mean length and the mean number of estrous cycles per dam during the pre-mating period between the female animals of the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
A slightly but statistically significantly increased mean cycle length of 5.05 days in comparison to 4.50 days in the control group was noted at the high dose level.
However, this difference was only small and only one female of the high dose group (the high dosed female no. 187 with a mean cycle length of 7.7 test days) was outside the range of the mean cycle length from the females of the control group (4.0 to 6.8 days).
A comparison with the Provivo background range revealed that the mean cycle length from one female of the control group (6.8 days) and one female of the low dose group (6.6 days) were slightly above the Provivo background range (3.8 to 6.4 days). In the high dose group 2 females were noted with mean cycle lengths that were above the Provivo background range (no. 190 with 6.7 days and the already mentioned no. 187 with 7.7 days).
Hence, the isolated observation of the elevated mean cycle length of female no 187 can be considered spontaneous. Secondly, no increase of the mean cycle length was observed in the F1 Co1A and lastly no differences in the mating (pre-coital time) were observed in the F0 generation.
Stage of estrous cycle at necropsy
No test item-related differences were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) in the distribution of the stages of the estrous cycle. A diestrus stage was noted for more than half of the animals of each group after examination of the vaginal lavage taken at necropsy form the females of the control and the treatment groups. This was in compliance with the results of the histopathological examination of the vagina.
ADULT F1 Cohort 1A
The stages of the estrous cycle of the cohort 1A animals were monitored on 14 test days between test days 49 and 62 of the F1 Generation Study.
No test item-related differences were noted for the mean length and the mean number of estrous cycles per female animal between the females of the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day) (see Text Table 8-15 on the following page).
A slight, but statistically significantly elongated mean cycle length was noted at the high dose level (4.5 days compared to 4.12 days in the control group, p ≤ 0.05). However, the difference was only small and the mean cycle length of the individual females of the high dose group were in the range of the mean cycle length of the individual females of the control group. Secondly, the physiological cycle length of a rat lies between 4 and 5 days. Hence, the observed increase in the mean cycle length is still in physiological range. Therefore, this observation was considered to be spontaneous. - Reproductive function: sperm measures:
- no effects observed
- Description (incidence and severity):
- Sperm number
No test item-related difference was noted between the rats of the control group and the rats treated with 25, 80 or 240/160 mg IPDA/kg b.w./day for the number of ultrasound-resistant sperm heads (sperm count) per gram testicular tissue.
Sperm motility
No test item-related differences were noted between the rats of the control group and the treatment groups (25, 80 or 260/140 mg IPDA/kg b.w./day) for the percentage of motile spermatozoa in the epididymal cauda on the total number of motile and non-motile spermatozoa.
A slightly increased percentage of motile spermatozoa in comparison to the control group was noted at the intermediate dose level (75.8 % motile spermatozoa in comparison to 71.3 % in the control group). As the difference was only small and no dose-response relationship was noted, the difference was not considered to be test item-related. Furthermore, an increased number of motile spermatozoa is not an adverse effect.
Finally, the percentages of sperm motility of all individual males of the control group, the low dose group and the high dose group were within the Provivo background range. With a motility range between 51 to 86 %. In the intermediate dose group one male animal (no. 105) showed 87 % motile spermatozoa, which was marginally above the Provivo background range.
Sperm morphology
The examination of spermatozoa from the epididymal cauda revealed no increased numbers of spermatozoa with a malformation in the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) in comparison to the control group.
In detail, the only observed malformation that was noted was banana-like sperm heads (7 were noted in the control group, 11 in the low dose group, 3 in the intermediate dose group and 14 in the high dose group).
Males that did not inseminate their female partner
No verified copulation (no sperm detection in the vaginal smear) was noted for 3 pairs. However, the investigated sperm parameter for the 3 male animals did not show any changes that could explain the missing sperm detection during the mating period and the resulting not pregnant females. - Reproductive performance:
- no effects observed
- Description (incidence and severity):
- Female fertility
No test item-related influence on the fertility index was noted for the female rats of the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
One non-pregnant female was noted in the control group (no. 28; with a verified copulation) and the low dose group (no. 95, without a verified copulation). Two non-pregnant females each in the intermediate (nos. 137, 140, both with a verified copulation) and the high dose group (nos. 177, 189, both without a verified copulation) were additionally observed.
Gestation index
No test item-related influence on the gestation index was noted for the female rats of the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
One pregnant female each in the control (no. 40), intermediate (no. 126) and high dose (no. 187) deceased during the gestation period were excluded in the calculation of the gestation index. All other pregnant females delivered live pups leading to a gestation index of 100% in all groups.
Pre-coital time
No test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Gestation length
No test item-related differences were noted for the length of the gestation period between the rats of the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day). - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 80 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical signs
- mortality
- Key result
- Critical effects observed:
- no
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- Cohort 1A: Males
No changes in behaviour, the external appearance and the consistency of the faces were noted for the male animals of the control group and the low dose group (25 mg IPDA/kg b.w./day).
At the intermediate dose level (80 mg IPDA/kg b.w./day) a post dosing salivation was noted for 3 of 20 animals on 1 or 3 test days.
At the high dose level (160 mg IPDA/kg b.w./day) post dosing salivation was noted for 11 of 19 animals and breathing sounds for 2 of 19 animals. Animal no. 326 revealed breathing sounds, laboured breathing and hunched position for several days.
The observation of a post dosing salivation was considered to be not of toxicological relevance, as in nearly all cases it did not last longer than 60 min.
Females
No changes in behaviour, the external appearance and the consistency of the faces were noted for the female animals of the control group and the low dose and the intermediate dose group (25 or 80 mg IPDA/kg b.w./day).
At the high dose level (160 mg IPDA/kg b.w./day) post dosing salivation was noted for 15 of 20 animals and breathing sounds for 2 of 20 animals. The observation of a post dosing salivation was considered to be not of toxicological relevance, as in nearly all cases it did not last longer than 60 min.
Males and females: Start and duration of observations
Salivation started immediately to 5 min after the administration and disappeared between 5 to 60 min after administration.
Breathing sounds are in nearly all cases long lasting observations which lasted for several hours or for the whole day.
Cohort 1B: Males
No changes in behaviour, the external appearance and the consistency of the faces were noted for the male animals of the control group.
In the low dose group (25 mg IPDA/kg b.w./day) one (no. 406) of 20 males showed a post dosing salivation on one test day.
At the intermediate dose level (80 mg IPDA/kg b.w./day) one animal each showed post dosing salivation (no. 452), piloerection (no. 456) or a haemorrhagic urine (no. 460) on one test day.
At the high dose level (160 mg IPDA/kg b.w./day) post dosing salivation was noted for 16 of 20 animals. Breathing sounds were noted for 2 (nos. 492, 493) animals and a haemorrhagic urine for one (no. 488) animal on one test day each.
The observation of a post dosing salivation was considered to be not of toxicological relevance, as in nearly all cases it did not last longer than 60 min.
Females
No changes in behaviour, the external appearance and the consistency of the faces were noted for the female animals of the control group and the low dose group (25 mg IPDA/kg b.w./day).
At the intermediate dose level (80 mg IPDA/kg b.w./day) a post dosing salivation was noted for 2 of 20 animals on one test day each.
At the high dose level (160 mg IPDA/kg b.w./day) a post dosing salivation was noted for all animals. One animal (no. 502) showed breathing sounds on 16 consecutive test days and animal no. 506 showed piloerection on one test day.
The observation of a post dosing salivation was considered to be not of toxicological relevance, as in nearly all cases it did not last longer than 60 min. - Dermal irritation (if dermal study):
- not examined
- Mortality / viability:
- mortality observed, treatment-related
- Description (incidence and severity):
- PUP
Viability index - F1 Pups
Pre- and post-cull period
No test item-related differences between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) were noted for the viability indices of the pre- and the post-cull period. In detail, the viability index for the intermediate dose group was the lowest with 97.44% compared to the control group 99.36%.
ADULT
Cohort 1A: Males and females
One prematurely sacrificed male animal was noted at the high dose level (160 mg IPDA/kg b.w./day) (see Text Table 8-1 below). There were no signs of an accident during the dosing process. A direct cause of animals morbidity could not be established by histopathology as it was also stated for 3 prematurely sacrificed high dosed animals of the F0 Generation. The microscopic findings were stress-related changes or non-specific changes that are frequently observed in animals with deteriorated general conditions, and histopathological findings that could be specific to the test item treatment were not identified.
Cohort 1B: Males and females
No test item-related premature death was noted in any dose group (25, 80 or 160 mg IPDA/kg b.w./day) for the male and female animals.
However, male no. 443 of the intermediate dose group was found dead on its postnatal day 84. Piloerection and a pale body were noted on several test days before death. Furthermore, body weight loss was noted. Necropsy revealed an increased spleen, beige liquid in the abdomen, reddish liquid in the thorax, reddened lungs and a haemorrhagic nose/snout.
An accidental death (caused by the accidental influx or incidental regurgitation) was considered as the cause of death due to the observation of reddened lungs at necropsy, though no histopathological examination was performed (only requested for prematurely deceased animals of the F0 Generation and Cohort 1A in the Study Plan) to verify that observation. - Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- PUP
No test item-related influence on the body weight of pups was noted for all treatment groups (25, 80 or 240/160 mg IPD/kg b.w./day).
Slightly reduced pup body weights were noted for all treatment groups in comparison to the control group on lactation day 14 (between 3.7 % and 4.5 % below the control for the male and the female animals combined; statistically not significant) (see Text Table 7-30 on the following page).
However, as the differences that were noted on lactation day 14 were only small and the differences between the control group and the treatment groups were smaller on lactation day 21 as on lactation day 14, the differences that were noted on lactation day 14 were considered to be spontaneous.
Statistically significant differences in pup body weight were noted for the female pups on lactation day 14 for the low dose with 4.8 % and for the high dose at 5.1 % below the control (p ≤ 0.05). As the differences were only small and no dose response-relationship was noted (on lactation day 21 the pup body weight of the female animals was 3.9 % at the low dose and 3.0 % at the high dose below the control, statistically not significant), they were considered to be spontaneous.
No test item-related influence on the litter weight was noted for all treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Slight, but statistically significantly reduced litter weights were noted for the female pups of the low (12.2 % below the control, p ≤ 0.05) and intermediate dose group (13.3 % below the control, p ≤ 0.05) on lactation day 14. This was due to the slightly reduced body weights on lactation day 14 that were considered to be spontaneous and due to a slightly reduced number of female pups per dam. Hence, the reduced litter weights of the female pups on lactation day 14 were considered to be spontaneous. On LD 21, no statistically significant difference was noted anymore between the treatment groups and the control group.
ADULT
Cohort 1A:
Males and females
No test item-related differences in body weight and body weight gain were noted for the male and female animals between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
A marginally reduced body weight was noted for the female animals on postnatal day 26, which remained during the whole study period of Cohort 1A until postnatal day 89. However, these marginal differences that failed statistical significance were considered to be spontaneous.
Cohort 1B: Males
No test item-related differences in body weight and body weight gain were noted for the male animals between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day). Only marginal and statistically not significant differences in body weight were noted between the control group and the treatment groups at start of Cohort 1B on PND 26 and near the end of Cohort 1B on PND 98.
No test item-related differences in body weight gain were noted between the control group and the treatment groups for the male animals.
Females
No test item-related differences in body weight and body weight gain were noted for the female animals between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
However, marginal reductions in body weight were noted for the female animals of the three treatment groups compared to the control group animals from postnatal day 26 on until near the end of the study on postnatal day 98 (see Text Table 9-8A below). These marginal differences were considered to be non-advers, as there was no correspondence in the body weight gain noticed.
Furthermore, the differences temporarily decreased for the female animals during the course of the study between postnatal days 40 and 96. Statistically significant differences were noted in this period in all dose groups (at maximum 8.4 % below the control on postnatal day 47 in group 4; p ≤ 0.01). As these slight but statistically significant differences were without any influence on body weight gain they were considered to be spontaneous. Moreover, no statistically significant differences in body weight were noted for the female animals of Cohort 1A.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Cohort 1A: Males
No test item-related difference in food consumption was noted in the control group and in the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
A slight but statistically significant increased food consumption was noted for the high dose males between the test days 43 to 50 and 50 to 57 (6.5 % and 6.4 % below the control, respectively, p ≤ 0.05). These slight and transient differences were considered to be spontaneous.
Females
No test item-related difference in food consumption was noted in the control group and in the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
Cohort 1B:
Males
No test item-related difference in food consumption was noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day) for the male animals.
However, a statistically significant differences in food consumption was noted for the males of the high dose group (5.0 % below the control, p ≤ 0.05) between test days 57 and 64. This was considered not to be test item-related but spontaneous.
Females
No test item-related difference in food consumption was noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day) for the female animals.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- Cohort 1A & 1B:
No decreased or increased water consumption was noted by visual appraisal during the daily cage side observations. - Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Description (incidence and severity):
- Cohort 1A: Males and females
No test item-related differences for the examined haematological parameters were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day) for the male and female animals.
However, a statistically significantly increased concentration was noted for the male animals of the high dose group for the number of platelets (28.3 % above the control, p ≤ 0.01). These is only due to the increased number of platelets of 2 individual high dosed males (no. 329 with 1386 and no. 330 with 1727 platelets x10E3/µL). The values they both exhibit were above the Provivo background range of 562 to 1268 platelets x10E3/µL.
As only 2 individual values of the high dose group were slightly above the Provivo background data and no statistically significant changes were noted for the female animals, the observed increased platelet concentration that was noted for the male animals of the high dose group was considered to be spontaneous. Finally, no changes were noted for the other haematological parameters for the male animals.
For the high dosed female animals a marginally but statistically significantly decreased MCV value was noted (2.6 % below the control, p ≤ 0.05). Nevertheless, only the MCV value from one high dosed female (49.9 fL) was marginally below the Provivo background range (50.7 to 55.8 fL). As furthermore no statistically significantly decreased MCV value was noted for the male animals, the decreased MCV value that was noted for the female animals of the high dose group was considered to be spontaneous.
Lymphocyte typing in spleen: Males and females
No test item-related influence was noted in the proportion of the examined lymphocyte subtypes to each other between the male and female animals of the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day). - Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- PUP
Thyroid hormone determination - F1 Pups
T4 level (PND 4 and PND 21/22)
No test item-related differences between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) were noted for the T4 level on lactation days 4 and 21/22 for the male and female pups.
Post-natal day 4:
On lactation day 4 increased T4 levels were noted at the low dose level and the high dose level. This was the case for the male and the female pups alone (statistically significant and not, respectively) and for the male and female pups combined (18.6 % or 26.0 %, p ≤ 0.05 / 0.01). In detail, the mean T4 concentrations of the pups (both sexes) from 6 individual dams of the low dose group, 4 individual dams of the intermediate dose group and 8 individual dams of the high dose group were above the Provivo background range.
However, no dose response relationship was noted.
Furthermore, no increased T4 levels were noted for the pups on lactation days 21/22 (see text below)
Hence, the increased T4 levels that were noted for the pups on lactation day 4 were considered to be spontaneous.
Post-natal days 21/22:
On lactation day 22 statistically significantly decreased T4 levels were noted at the intermediate dose level for the male and female pups (15.4 % and 16.7 % below the control, respectively, p ≤ 0.01). This was also noted for the low dose female pups alone (11.5 % below the control, p ≤ 0.05). For the male and female pups combined, a statistically significantly decreased T4 level was noted at the intermediate and the high dose level ( 15.7 % and 9.6 %, respectively, p ≤ 0.05 / 0.01) (see Text Table 7-38A below).
However, the mean T4 concentrations of the pups from the individual dams from all treatment groups were within the Provivo background range (see Text Table 7-38B below).
Hence, as no dose response relationship was noted, and all individual values of the treatment groups were within the background range, the decreased T4 concentrations that were noted on lactation days 21/22 are not considered to be test item-related, but spontaneous.
ADULT
Cohort 1A:
Clinical biochemistry: Males and females
No test item-related differences for the examined biochemical parameters were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
A statistically significantly decreased sodium concentration was noted for the female animals of the intermediate dose group (1.1 % below the control, p ≤ 0.5). In detail, the sodium concentrations from 4 individual females of the intermediate dose group (134 to 135 mmol/L) were slightly below the Provivo background range (136 to 141 mmol/L). Additionally, one female each in the control and low dose and two females in the high dose groups also show similar sodium values, which were also slightly below the Provivo background range. Therefore, as this was observed in all female groups, no statistically significant changes were noted for the male animals and no dose-response relationship was noted, this observation was considered to be spontaneous.
Thyroid hormone levels: Males
No test item-related differences for the examined thyroid hormone levels (T4 and TSH) were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
All individual T4 concentrations of the control group and all individual T4 concentrations of the intermediate and the high dose level, with one exception each, were within the Provivo background range. Only in case of the low dose group 4 individual values (79.006 to 93.925 nmol/L) were above the Provivo background range (42.528 to 76.219 nmol/L), which was considered to be spontaneous as no statistical significance and no dose response-relationship was noted.
An increased TSH concentration was observed for the male animals of the intermediate and the high dose group (50.0 % and 80.8 % above the control, respectively, statistically not significant). The rise appeared to be dose dependent but in the treatment groups the values are highly variable. In detail, 2 or 3 of the individual male TSH values (group 2: 1.732 and 2.589 ng/mL, group 3: 1.790 and 5.126 ng/mL, group 4: 2.416, 3.219 and 4.043 ng/mL) were above the range of the control group (0.081 to 1.530 ng/mL). Additionally, none of individual value was above the upper range of the Provivo background data (upper range = 5.699 ng/mL). Finally, an opposite trend was noted for the female animals with decreased TSH values in all treatment groups in comparison to the control. Hence, the observed increased values that were noted for the TSH values of the male animals at the intermediate and the high dose level were considered to be spontaneous.
Females
No test item-related differences for the T4 and TSH thyroid hormone levels were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
Nearly all T4 concentrations of the individual animals were within the Provivo background range.
A dose dependent decrease of the TSH concentration was noted for all treatment groups (group 2: 31.5 %, group 3: 66.6 % and group 4: 79.5 % (p ≤ 0.05) below the control). At the intermediate and the high dose level the TSH concentrations from 7 of 10 females each were below the lower limit of detection (LOD = 0.081 ng/mL). Corresponding to the hormonal decrease, a weight increase of the pituitary gland (absolute and relative), the gland which produces TSH, was noted at the low and the high dose level. But no weight increase of the pituitary gland was noted at the intermediate dose level. Nevertheless, the histopathology observations did not report any histopathological changes in the pituitary gland compared to the control.
The female animals of the F0 generation showed a direct opposite trend. The females of the F0 generation show increased values in comparison to the control group. There the males on the other hand exhibited decreased values in comparison to the control. Due to this opposite trend, the high variability between the individual values, no histopathological changes noted for the pituitary gland and the males of the Co1A also showed a direct opposite development a decreased TSH, the differences that were noted between the control group and the treatment groups were considered to be spontaneous and toxicological not relevant. - Urinalysis findings:
- no effects observed
- Description (incidence and severity):
- Cohort 1A: Males and females
No test item-related differences for the examined urinalysis parameters were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/day). - Sexual maturation:
- no effects observed
- Description (incidence and severity):
- Males
Cohort 1A
No test item-related differences between the control group and the test item-treated groups (25, 80 or 160 mg IPDA/kg b.w./day) were noted for the time point of balano-prepuital separation and the body weight at the time point of balano-prepuital separation.
However, a marginally and statistically not significantly earlier time point of balano-preputial separation was noted at the intermediate and the high dose level. The intermediate dosed males showed a balano-preputial separation on PND 22.2 ± 0.4, the high dosed males at PND 22.3 ± 0.7 in comparison, the control group exhibit it at PND 22.6 ± 0.7.
As these differences were only small and as slightly earlier sexual maturation is not an adverse effect, these observations were considered to be spontaneous and not toxicological relevant.
Cohort 1A and 1B combined
A combined analysis of the time-points of balano-preputial separation from animals Cohort 1A and 1B together results in similar values as the analysis for Cohort 1A alone. However, due to the increased number of analyzed animals, the earlier time points of balano-preputial separation that were noted at the intermediate and the high dose level were now statistically significant. In detail, for Cohorts 1A and 1B combined, the time point of balano-preputial separation was 22.2 ± 0.5 for the intermediate and the high dose level, p ≤ 0.05 and p ≤ 0.01, respectively. In the control group the time-point of balano-preputial separation was on post-natal day 22.6 ± 0.7.
However, as stated for Cohort 1A alone, these differences were considered to be spontaneous, as the difference was only marginal and a slightly earlier sexual maturation is not an adverse effect, these observations were considered to be spontaneous and not toxicological relevant.
Females: Females
No test item-related differences between the control group and the test item-treated groups (25, 80 or 160 mg IPDA/kg b.w./day) were noted for the time point of vaginal opening and the body weight at the time point of vaginal opening.
Time point (postnatal day) of vaginal opening:
Cohort 1A
A slight delay for the time point of vaginal opening was noted at the low dose level (post-natal day 34.5 ± 3.4, statistically not significant) and the high dose level (post-natal day 34.9 ± 2.7, p ≤ 0.05) in comparison to the control group (postnatal day 33.1 ± 3.0) for the females of Cohort 1A. However, no dose response relationship was noted, as the time point of vaginal opening. Then the time point for the vaginal opening at the intermediate dose group (postnatal day 33.1 ± 1.7) was again in the range of the control group.
Cohort 1B:
Males
No test item-related differences between the control group and the test item-treated groups (25, 80 or 160 mg IPDA/kg b.w./day) were noted for the time point of balano-preputial separation and no differences were noted for the body weight at the time point of balano-preputial saparation.
However, for the male animals of Cohort 1B the time point of balano-preputial separation was slightly, but statistically significantly earlier for the high dose (postnatal day 22.1 ± 0.2) than for the control group (postnatal day 22.6 ± 0.7). As this difference was only small and is non-adverse, the slightly earlier time point of balano-preputial separation that was noted for the males of cohort 1B was considered to be spontaneous.
Females
No test item-related differences between the control group and the test item-treated groups (25, 80 or 160 mg IPDA/kg b.w./day) were noted for the time point of vaginal opening and no differences were noted for the body weight at the time point of vaginal opening.
Cohort 1A and 1B combined
Evaluation of the time points of vaginal opening from Cohort 1A and 1B combined resulted in nearly the same time points as for Cohort 1A alone. However, the combination showed an increased statistical significance at the high dose level. In detail, the time points of vaginal opening were on post-natal day 33.1 ± 2.5 for the control, 34.2 ± 2.8 for the low dose group, 33.1 ± 1.9 for the intermediate dose group and 34.6 ± 2.3 for the high dose group. Statistically significant at the high dose level (p ≤ 0.01).
Nevertheless, also in the combination of both cohorts, no dose response relationship was noted. Secondly, nearly all individual values were within the Provivo background range. In detail, only the time points of vaginal opening of one female each of the control group (postnatal day 43), high dose group (postnatal day 43) and 2 females of the low dose group (postnatal days 41 and 44) were above the range of the Provivo background data (postnatal days 30 to 37).
Hence, the observed delays that were noted for the time points of vaginal opening at the low and the intermediate dose groups were considered to be spontaneous.
Body weight at the time point (postnatal day) of vaginal opening:
Cohort 1A and 1B combined
A slightly increased body weight was noted at the time point of vaginal opening for the females of the low and the high dose group (4.3 % or 3.9 % above the control, statistically not significant). These slight differences were considered to be spontaneous.
Appearance of cornified cells in the vaginal lavage
(first time the cycle of the animals reaches the stage of estrus)
In the control group, the low dose group and the intermediate dose group the interval between the time point of vaginal opening and the time point the rat cycle reaches the stage of estrus for the first time (identified by the appearance of cornified cells in the vaginal lavage) was between 1.9 and 1.7 test days. This time interval was reduced to 0.7 test days in the high dose group. In detail, 15 of 20 females of the high dose group were in the stage of estrus at the day of vaginal opening in comparison to 5 of 20 females from the control group.
However, the reduced time interval between the time point of vaginal opening and the time point for the appearance of cornified cells was not considered to be test item-related. The reason is that there is no dose-response relationship for the time point for the appearance of cornified cells. The postnatal day for the appearance of cornified cells at the low dose level was later as at the high dose level (postnatal day 36.3 in comparison to postnatal day 35.6). Hence, the reduced time interval that was noted at the high dose level was only due to a delay for the time point of vaginal opening at the high dose level, which was considered as spontaneous. - Anogenital distance (AGD):
- no effects observed
- Description (incidence and severity):
- PUP
No test item-related difference in the absolute and the relative ano-genital distance (value normalized to cube root of pup body weight) of the male and the female pups was noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Slightly reduced absolute and relative values (statistically significant or not) were noted at the intermediate and the high dose level for the male and female animals. However, the similar distance reduction in the ano-genital distance for male and female pups are only showing a developmental delay at LD 4, which normalised until LD21. Hence, these slight differences were considered to be spontaneous and have no toxicological relevance. - Nipple retention in male pups:
- no effects observed
- Description (incidence and severity):
- No test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Number and pups that were noted with nipples are given in Text Table 7-34 on the following page. For most of the affected male pups only 1 nipple was noted per pup. The number of these pups (male pup with one nipple) is nearly identical in each group (8 in the control group and between 7 and 8 in the treatment groups).
Male pups with 2 nipples were noted in all treatment groups (2 pups in each group) but not in the control group.
One male pup with 3 nipples was noted in the control group and 2 pups with 3 nipples each in the high dose group.
The maximum number of nipples that was noted for a male pup were 4 nipples. Pups with 4 nipples were noted in the low dose group (4 pups) and in the high dose group (1 pup), but not in the control group and in the intermediate dose group.
In summary, there is a slightly increased number of more severely affected pups (pups with 3 or 4 nipples) in the low and the high dose group (4 or 3 pups with 3 or 4 nipples) than in the control group and in the intermediate dose group (1 or no pup with 3 or 4 nipples). However, as no dose response relationship was observed, these slight differences were considered to be spontaneous. - Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- PUP
Pup organ weights (absolute) - F1 Pups
No test item-related differences for the examined absolute organ weights were noted between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
ADULT
Cohort 1A:
Males
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
Nevertheless, decreased relative and absolute prostate weights were noted at the intermediate and the high dose level. In detail, at the intermediate dose level, the relative prostate weight was 9.2 % below the control (statistically not significant) and at the high dose level 13.9 % below the control at the high dose level (p ≤ 0.05). The absolute prostate weight at the intermediate dose level was 8.9 % below the and 8.4 % below the control at the high dose level (both not statistically significant).
A comparison with the Provivo background data revealed that the relative prostate weights from 2 individual animals of the control group, 5 individual animals of the low dose group, 6 individual animals of the intermediate dose group and 11 individual animals of the high dose group were below the Provivo background range.
However, only marginally reduced relative and absolute prostate weights were noted for the high dosed males of Cohort 1B (5.5 % and 4.3 % below the control, respectively, statistically not significant).
Furthermore, the absolute prostate weights did not show a dose dependent reduction in neither Cohort and also for the combination of Cohorts 1A and 1B.
Finally, there were no qualitative and semi-quantitative differences in the histomorphology between Groups 1 and 4.
The combination of the organ weights of Cohorts 1A and 1B revealed a statistically significantly decreased relative weight of the left epididymis in the low dose group ( 7.7%, p ≤ 0.05). However, no dose-response relationship and no histopathological changes were noted.
Hence, the differences observed in the prostate and epididymis weight were considered to be spontaneous and not test item-related.
Females
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
Statistically significant weight changes were detected for the pituitary, the heart and the mesenteric lymph node.
For the pituitary gland statistically significantly increased relative and absolute weights were noted at the low dose level (17.5 % and 18.9 % above the control, respectively, p ≤ 0.05) and at the high dose level (23.2 % and 20.0 % above the control, respectively, p ≤ 0.05). However, all absolute weights of the pituitary glands from the individual animals of the low dose group were within the Provivo background range. At the high dose level only one animal with a pituitary gland weight of 0.025 g was marginally above the Provivo background range (0.010 to 0.022 g). Furthermore, only slightly increased weights were noted at the intermediate dose level. Hence, there was no dose response-relationship for the changes of the pituitary weights. Also the combination of the relative and absolute pituitary weights of Cohorts 1A and 1B, did not reveal a dose-response relationship.
Statistically significant reductions in the relative and absolute heart weight were noted at the intermediate dose level (9.1 % and 11.1 % below the control, respectively, p ≤ 0.05 / 0.01). The absolute heart weights of 4 individual animals of the intermediate dose group were below the Provivo background range. The reductions in the relative and absolute heart weight were declined at the high dose level but not as much as in the intermediate dose (4.5 % or 7.2 % below the control, statistically not significant). Hence, there was no dose-response-relationship present for the changes in the heart weights.
At the intermediate dose group a statistically significant reduction was also noted for the relative and absolute weight of the mesenteric lymph nodes (37.3 % and 36.8 % below the control, respectively, p ≤ 0.05). The absolute mesenteric lymph node weights from 3 females of the intermediate dose group were below the Provivo background range. The differences in the mesenteric lymph node weights between the control group and the high dose group were like with the heart weights smaller in comparison to the intermediate dose group and were no longer statistically significant. Therefore, also in this case no dose-response relationship was observed.
The combination of the relative and absolute organ weights of Cohort 1A and 1B, led to a statistically significantly decreased absolute weight of the left adrenal gland in the high dose group (-10.7%, p ≤ 0.05) and a statistically significantly increased absolute weight of the left ovary in the intermediate and high dose group (+12.8% and +15.6%, respectively, p ≤ 0.05). However, for the right adrenal gland, no dose-response relationship was present and for the left ovary, the weights were within the range of the Provivo background data.
Finally, the histopathological examination of the animals of group 4, revealed no histomorphological findings that could correlate with the weight changes that were noted for the pituitary gland, the heart, the mesenteric lymph nodes, the adrenal glands and the ovaries.
Therefore, it was considered that there is no toxicological significance in the weight changes detected in the pituitary gland, the heart, the mesenteric lymph nodes, the adrenal glands and the ovaries.
Cohort 1B:
Males
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
Increased values were noted for the relative and absolute organ weights of the pituitary gland at the low dose level (5.6 % and 8.0% above the control, respectively, statistically not significant) and at the high dose level (21.9 % and 22.6 % above the control, respectively, statistically significant at p ≤ 0.05 / 0.01). However, as no dose response-relationship was noted and no such observations were recorded for the male animals of Cohort 1A, the changes that were noted for the males of Cohort 1B were considered to be spontaneous.
Females
No test item-related differences for the examined absolute and relative organ weights were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
Decreased relative and absolute organ weights were noted for the right adrenal gland from the female animals of the low dose group (14.5 % (p ≤ 0.05) and 12.4 % (not statistically significant) below the control, p ≤ 0.05 or not statistically significant) and the high dose group (15.4 % (p ≤ 0.01) and 11.5 % (not statistically significant) below the control, respectively).
However, no significant changes were noted for the left adrenal weight from the females of Cohort 1B and the right and the left adrenal weights from the females of Cohort 1A. Furthermore, no dose-response relationship was noted. Hence, the decreased weights of the right adrenal gland, noted for the females of Cohort 1B were considered to be spontaneous.
Statistically significantly increased values were noted for the relative organ weights of the left ovary at all dose levels (16.6 %, 20.1 % or 20.8 % above the control value at the low, the intermediate or the high dose level, respectively; p ≤ 0.05 / 0.01).
The absolute organ weights of the left ovary were also increased but failed statistical significance (13.8 %, 15.2 % or 15.0 % above the control at the low, the intermediate or the high dose level; respectively).
However, no statistically significant changes were noted for the relative and absolute organ weights of the right ovary of the females from Cohort 1B and the left and the right ovary weights from the females of Cohort 1A. Additionally, no histomorphological changes to be toxicologically concerned in the view of reproductive performance were found in the female reproductive organs including ovaries in the F1 Cohort 1A. All findings recorded were within the range of normal background lesions or physiological alterations that may be observed in this type of study or animals of this strain and age.
Hence, the increased weights of the left ovary that were noted for the females of Cohort 1B were considered to be spontaneous and not of toxicological relevance. - Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- PUP
External and internal examination of the pups – F1 Pups
No gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the pups that were terminally sacrificed after weaning, the pups that were found dead or for the stillbirths from the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Furthermore, no malformations were noted during the macroscopic examination of the inner organs and tissues of the control pups and the pups of the treatment groups.
ADULT
Cohort 1A:
Males
No observations were noted for the surviving male animals of the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
Females
No observations were noted for the female animals of the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
There were no findings were noted in the treatment groups. However, 2 females (nos. 223, 230) of the control showed a dilated uterus (filled with clear liquid) and one other control female (no. 235) exhibited a reddened thymus. Another female of the control group (no. 237) revealed an enlarged mesenteric lymph node, an enlarged spleen and intestines with a thickened mucosa.
All the macroscopic findings recorded at necropsy were lesions within the range of normal background changes or physiological alterations which maybe observed in this type of study or animals of this strain and age, or incidental gross appearances without corresponding histomorphological changes.
Cohort 1B:
Males
No observations were noted for the surviving male animals of the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
Females
No observations were noted for the female animals of the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
In the control group one female animal (no. 398) with a dilated uterus was noted. As described for the macroscopic findings of Cohort 1A, a dilated uterus belongs to the normal background changes or physiological alterations which maybe observed in this type of study or animals of this strain and age, or incidental gross appearances without corresponding histomorphological changes. - Histopathological findings:
- no effects observed
- Description (incidence and severity):
- Cohort 1A:
Males
No test item-related changes were observed for the examined male animals of the high dose group (160 mg IPDA/kg b.w./day) during the microscopic examination.
Nevertheless, one dead male of the high dose group was reported (no. 336). However, histomorphologically the direct cause(s) of animals morbidity was not established in male no. 336 of group 4 from F1 Generation Cohort 1A. The remaining microscopic findings of male no. 336 were stress-related changes or non-specific changes that are frequently observed in animals with deteriorated general conditions. Findings that could be specific to the test item treatment were not identified. However, due to the animal’s general conditions during the exposure with higher dose level, effects attributed to the test item can not be excluded.
In the previous study (OECD 422 study) in which the treatment in the high dose group was started at a dose of 300 mg/kg b.w./day (thereafter reduced to 240 mg/kg b.w./day during the treatment period), treatment-related microscopic changes were observed in the stomach and kidneys.
However, the histopathological examination of the present study revealed that the test item did not produce treatment-related microscopic changes not only in the stomach and kidneys but also in any other organs including male and female reproductive system of the animals treated with 160 mg/kg b.w./day in F1 Generation Cohort 1A.
Male reproductive organs
No histomorphological changes to be toxicologically concerned in the view of reproductive performance were found in the male reproductive organs including testes, epididymides, prostate glands, seminal vesicles and coagulating glands in cohort 1A.
For testes, the stages were checked on completeness of cell populations, completeness of stages and checked for degenerative changes, while taking also account into the interstitial cell stucture, for both generations. As a result, all testes examined showed completeness of stages and cell population.
All the other findings recorded in the male reproductive organs were within the range of normal background changes that may be observed in this type of study or animals of this strain and age, and hence, deemed not to be treatment-related.
Females
No test item-related changes were observed for the examined female animals of the high dose group (160 mg IPDA/kg b.w./day) during the microscopic examination.
The histopathological examination of the present study revealed that the test item did not produce treatment-related microscopic changes not only in the stomach and kidneys but also in any other organs including female reproductive system of the high dose animals in F1 generation Cohort 1A.
Female Reproductive Organs - including quantitative examination of ovaries
No histomorphological changes to be of toxicologically concerend in the view of reproductive performance were found in the female reproductive organs including ovaries, oviducts, uterus, uterine cervix and vagina in the F1 cohort 1A.
All findings recorded were within the range of normal background lesions or physiological alterations that may be observed in this type of study or animals of this strain and age.
For ovaries, the quantitative evaluation was performed in F1 generation Cohort 1A, in addition to qualitative histopathology examination.
No test item-related or statistically significant differences were noted in all parameters including primordial follicles, summed up primordial and growing follicles, antral follicles and corpora lutea. - Other effects:
- no effects observed
- Description (incidence and severity):
- Cohort 1A:
Monitoring of estrous cycles - 2-week period
The stages of the estrous cycle of the cohort 1A animals were monitored on 14 test days between test days 49 and 62 of the F1 Generation Study.
No test item-related differences were noted for the mean length and the mean number of estrous cycles per female animal between the females of the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
A slight, but statistically significantly elongated mean cycle length was noted at the high dose level (4.5 days compared to 4.12 days in the control group, p ≤ 0.05). However, the difference was only small and the mean cycle length of the individual females of the high dose group were in the range of the mean cycle length of the individual females of the control group. Secondly, the physiological cycle length of a rat lies between 4 and 5 days. Hence, the observed increase in the mean cycle length is still in physiological range. Therefore, this observation was considered to be spontaneous.
Examination of the sperm number, viability and morphology
No test item-related difference was noted between the rats of the control group and the rats treated with 25, 80 or 160 mg IPDA/kg b.w./day for the sperm number, the viability and morphology.
Cohort 1B: Stage of estrous cycle at necropsy
No test item-related differences were noted between the control group and the treatment groups (25, 80 or 160 mg IPDA/kg b.w./day) in the distribution of the stages of the estrous cycle evaluated from vaginal lavages that were taken at necropsy. - Key result
- Dose descriptor:
- NOAEL
- Generation:
- other: F1 pups
- Effect level:
- > 160 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: pre- and postnatal development
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- other: F1 adult (Cohort 1A & 1B)
- Effect level:
- 80 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- viability
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- The oral exposure to 25, 80, and 240/160 mg test item/kg bw (male and female Sprague-Dawley rats; OECD 443; Provivo, 2021) in the extended one generation reproductive toxicity study led to no detectable changes in fertility and reproductive performance of the parental animals.
The pre- and post-development of the pups and the postnatal development of the pups was also not affected by the test item. No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.
No test item-related influence was noted on the development of the reproductive system of eighter sex.
However, a test item-related effect was noted at 240/160 mg test item/kg b.w./day for the male and female animals in the form of 3 test item-related premature deaths from in total 48 high dose animals. Further, breathing sounds and piloerection were noted at the high dose level. The mortality rate of the high dose correlates with the observations made in the OECD 422 study (Provivo , 2021). One test item-related death was noted in the adult F1 animals at the high dose of 160 mg/kg b.w./day. The animals of the F1 Generation showed no further signs of general toxicity (body weight, food consumption thyroid hormone levels). Breathing sounds and piloerection were only noted for a few animals of the F1 Generation at 160 mg test item/kg/d.
The No Observed Adverse Effect Level (NOAEL) for reproduction was therefore considered to be above 240 mg test item/kg/day.
The NOEAL for pre- and postnatal development was above 160 mg IPDA/kg b.w./day.
The No Observed Adverse Effect Level (NOAEL) for systemic toxicity in the F0 and in the adult animals of Cohort 1A and 1B was determined to be 80 mg/kg/day. - Executive summary:
The aim of the study was to evaluate the effects of the test item IPDA at dose levels of 25, 80 or 240/160 mg/kg b.w./day on the general and reproductive toxicity of the F0 Parents and of the developmental toxicity of the F1 Generation from weaning until adulthood (OECD 443). The high dose level was reduced from 240 to 160 mg/kg b.w./day on test day 51 (after the administration of 240 mg/kg for 37 days) due to changes in behaviour and the external appearance and one test item-related premature death. For this reason, the high dose animals of the F1 Generation also only received a dose level of 160 mg/kg.
General and reproductive toxicity (F0 Generation and F1 Pups)
General toxicity
A test item-related effect was noted at 240/160 mg IPDA/kg b.w./day for the male and female animals in the form of 3 test item-related premature deaths from in total 48 high dose animals. Further, breathing sounds and piloerection were noted at the high dose level. The mortality rate of the high dose correlates with the observations made in the OECD 422 study.
No influence was noted on body weight, food consumption, the laboratory examinations, during necropsy and the histopathological examination.
Reproductive toxicity
No test item-related influence was noted on the reproductive performance of the parental animals (number and length of estrous cycles, fertility and gestation index, pre-coital time and gestation length).
The prenatal development of the pups (number of resorptions, stillbirths and live born pups) and the postnatal development of the pups (pup body weight, viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights) was also not effected by the test item. No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy.
Developmental toxicity (F1 - Cohorts 1A and 1B)
During their post-weaning development one test item-related death was noted at the high dose of 160 mg/kg b.w./day. The animals of the F1 Generation showed no further signs of general toxicity (body weight, food consumption thyroid hormone levels). Breathing sounds and piloerection that were noted as adverse and test item-related observations for several animals of the F0 Generation at 240/160 mg IPDA/kg were only noted for a few animals of the F1 Generation at 160 mg IPDA/kg. Due to the low incidences of breathing sounds and piloerection at the F1 Generation, these observations were not considered as an adverse effect for the developmental toxicity of the F1 Generation.
No test item-related influence was noted on the development of the reproductive system (time points of sexual maturations, number and length of estrous cycles, sperm parameter, detailed histopathological examination of testis and epididymides, number of primordial and growing follicles and number of corpora lutea in the ovaries).
The following no-observed-adverse-effect levels (NOAEL) were established for the parental animals of the F0 Generation and the F1 Generation:
F0 Generation:
General toxicity
NOAEL 80 mg IPDA/kg b.w./day
Based on the premature death of 3 animals (for which a test item-related cause of death cannot be excluded) and clinical signs observed in animals treated with 240/160 mg IPDA/kg b.w./day.
Reproductive toxicity
NOAEL above 240/160 mg IPDA/kg b.w./day
F1 Generation:
Developmental toxicity
Adverse effects on pre- and postnatal development
- Adverse effects on prenatal development (conceptus to birth)
NOAEL above 160 mg IPDA/kg b.w./day
- Adverse effects on postnatal development (pup)
NOAEL above 160 mg IPDA/kg b.w./day
General toxicity (Cohorts 1A and 1B)
NOAEL 80 mg IPDA/kg b.w./day
Based on one premature death observed in animals treated with 160 mg IPDA/kg b.w./day, for which a test item-related premature death cannot be excluded.
1.1.1 Findings - F0 Generation and F1 Pups
Mortality
Males
None of the male animals treated with 25 or 80 mg IPDA/kg b.w./day died prematurely.
At (240/160 mg IPDA/kg b.w./day) one premature death was noted. Male no. 153 was prematurely sacrificed on test day 73 due to poor health condition. The histopathological examination revealed no specific test item-related findings for the animal that was prematurely sacrificed due to moribund conditions. Also no signs of misgavage, accidental influx or incidental regurgitation of the test item-formulation into the respiratory tract were noted.
Females
None of the female animals treated with 25 or 80 mg IPDA/kg b.w./day died prematurely.
At (240/160 mg IPDA/kg b.w./day) two females (no. 182 and no. 178) were prematurely sacrificed due to poor health condition on test days 24 or 59, respectively.
The histopathological examination revealed no specific test item-related findings for the 2 animals that were prematurely sacrificed due to moribund conditions. No signs of misgavage or an accidental influx or incidental regurgitation of the test item-formulation into the respiratory tract were noted for these 2 animals.
Clinical signs
Males and females
Salivation was noted for several male and female animals at 80 mg IPDA/kg b.w./day.
At the high dose level (240/160 mg IPDA/kg b.w./day) test item-related changes were noted for all or several male and female animals in the form of salivation, breathing sounds and piloerection.
The other observations were considered to be spontaneous, due to their low incidence.
Start and duration:
In nearly all cases salivation was a short lasting post‑dosing symptom, whereas breathing sounds were often noted for several hours after dosing or for the whole day.
Detailed clinical observations:
No further observations in addtion to those made during the daily cage side observations were noted for the male and female animals of the control and the treatment groups.
Body weight and
body weight gain
Males and females
No test item-related differences were noted.
Food consumption
Males and females
No test item-related differences were noted.
Haematology
(test days 127 to 133,
at necropsy)
Males and females
No test item-related changes were noted.
Nevertheless, for the female animals a decrease in the number of white blood cells was observed, which was statistically significant for the 25 and 80 mg IPDA/kg b.w./day. However, these changes were considered spontaneous as no dose response-relationship was noted.
Clinical biochemistry
(test days 127 to 133,
at necropsy)
Males and females
No test item-related changes were noted.
Urinalysis
(test days 127 to 133,
at necropsy)
Males and females
No test item-related changes were noted.
Thyroid hormone levels
(test days 127 to 133,
at necropsy)
Males and females
No test item-related changes were noted for the examined thyroid hormones T4 and TSH.
Differences between the control group and all treatment groups were noted for the TSH concentration of the male and female animals. However, as the differences were not statistically significant and nearly all individual values were within the Provivo background data range and no dose-response relationship was noted, they were not considered to be test item-related.
Sperm parameter
(test days 127, 128, 129,
at necropsy)
Males
No test item-related changes were noted in sperm number, motility and morphology.
Necropsy
(test days 127, 128, 129 for the males,
test days 129 to 134 for the females)
Macroscopic post-mortem
findings
Males and females
No toxicologically relevant macroscopic changes that could be associated with the test item were noted for the surviving male and female animals that were dosed with 25, 80 or 240/160 mg IPDA/kg b.w./day.
Body weight at autopsy
Males and females
No test item-related changes were noted.
Estrous stage at necropsy
No test item-related differences were noted.
Organ weights
Males and females
No test item-related differences were noted.
Nevertheless, in the males the relative and absolute thymus weight showed a decrease. This observation was considered to be spontaneous, as no correlation with the histopathology results was noted.
The thyroid of the female animals showed a non‑dose dependent weight increase with no correlation in histopathology. Hence, the thyroid weight increase was also considered to be spontaneous and of no toxicological relevance.
Bone marrow examination
Males and females
No test item-related differences were noted.
Histopathological examinations
(Groups 1 and 4)
Males and females
No test item-related observations were noted for the examined male and female animals of group 4.
Additionally, previously (OECD 422, Provivo study no. 37482, 2021) observed microscopic changes in the kidney and stomach at the high dose animals (300/240 mg IPDA/kg b.w./day) were not observed at this study at 240/160 mg IPDA/kg b.w./day.
Examination of reproductive organs
No test item-related observations were noted for the examined reproductive organs of the male and female animals of group 4.
All testes examined showed completeness of stages and cell population.
Reproductive toxicity
Reproductive parameters of the parental females
Estrous cycle data
(test day15 until evidence of
copulation)
No test item-related influence was noted on the mean number and the mean length of the estrous cycles.
Fertility index
No test item-related influence was noted.
Gestation index
No test item-related influence was noted.
Pre-coital time
No test item-related influence was noted.
Gestation length
No test item-related influence was noted.
F1 Pups - Pre- and postnatal development
- Prenatal development (from conceptus to birth)
Reproductive parameters
No test item-related influence was noted on the birth index, the live birth index, and the percentage of post-implantation loss.
- Postnatal development (pup)
Mortality (Viability index)
Pre- and post-cull period
No test item-related influence was noted.
Pup body weight
No test item-related influence was noted.
Nevertheless, a significant reduced body weight was noted at LD14, which resolved at LD21. Therefore, these observations were considered to be spontaneous.
Ano-genital distance
No test item-related influence was noted.
However, a slight reduction was observed at both sexes, which probably resulted from the developmental delay at LD4, which had normalised until LD21. Hence, these slight differences were considered to be spontaneous and have no toxicological relevance.
Count of male nipples
(nipple retention)
No test item-related influence was noted.
F1 Pups - Examination at necropsy (surplus pups; not used for F1 generation)
External and
internal examination
No malformations or variations were noted.
T4 determination
(lactation day 4; culled pups)
No test item-related difference was noted.
T4, TSH determination
(lactation day 21/22)
No test item-related difference was noted.
Pup organ weights
No test item-related difference was noted.
1.1.1 Findings - Cohort 1A
Mortality
Males and females
One male animal (no. 336) of the high dose group (160 mg IPDA/kg b.w./day) was prematurely sacrificed on test day 73 due to poor health condition. No specific test item-related findings were noted during the histopathological examination. No signs of misgavage, accidental influx or incidental regurgitation of the test item-formulation into the respiratory tract were noted.
Clinical signs
Males and females
Salivation was noted for several male animals at 80 mg IPDA/kg b.w./day.
At the high dose level (240/160 mg IPDA/kg b.w./day) test item-related changes were noted for half the male and female animals in the form of salivation. Breathing sounds, piloerection, hunched position and laboured breathing were only noted for 1 or 2 animals per sex.
Body weight and
body weight gain
Males and females
No test item-related differences were noted.
Food consumption
Males and females
No test item-related changes were noted. A transient increase in the food consumption was noted for the high dose male animals between TD 43-57.
Haematology
(postnatal days 90 to 92,
at necropsy)
Males and females
No test item-related changes were noted.
Clinical biochemistry
(postnatal days 90 to 92,
at necropsy)
Males and females
No test item-related changes were noted.
Lymphocyte typing (spleen)
(postnatal days 90 to 92,
at necropsy)
Males and females
No test item-related changes were noted.
Urinalysis
(postnatal days 90 to 92,
at necropsy)
Males and females
No test item-related changes were noted.
Thyroid hormone levels
(postnatal days 90 to 92,
at necropsy)
Males and females
No test item-related changes were noted for the examined thyroid hormones T4 and TSH.
However, a dose dependent decrease in the TSH values in the intermediate and high dose below the detection limit were recorded for the female animals of the treatment groups. Nevertheless, the histopathology observations did not report any histopathological changes in the pituitary gland compared to the control group. Furthermore, the differences in the TSH values showed an opposite trend between the male (increased values in comparison to the control) and the female animals (decreased values in comparison to the control). Due to this opposite trend and the high variability between the individual values, the differences that were noted between the control group and the treatment groups were considered to be spontaneous and toxicological not relevant.
Sexual Maturation
Males
No test item-related influence was noted on the day of balanopreputial separation and the body weight on the day of balanopreputial separation.
Females
No test item-related influence was noted on the day of vaginal opening, the body weight on the day of vaginal opening, the day of the appearance of cornified cells in the vaginal smear and the period between the day of vaginal opening and the appearance of cornified cells in the vaginal smear.
Estrous cycle data
Females
No test item-related influence on the number and length of the estrous cycles was noted during the examination of a 2-week period between test days 50 and 63.
Sperm parameter
(PND 90 to 92; at necropsy)
Males
No test item-related changes were noted.
Necropsy
(PND 90 to 92 for the males and females)
Macroscopic post-mortem
findings
Males and females
No test item-related observations were noted.
In the female animals dilated uterus, a one lobe reddened thymus, enlarged mesenteric lymph node and a thickened intestinal mucus was observed in one or two animals of the control group.
Body weight at autopsy
Males and females
No test item-related changes were noted.
Estrous stage at necropsy
No test item-related influence was noted on the number of the different stages between the control group and the treatment groups.
Organ weights
Males and females
No test item-related differences were noted.
However, a slight decrease in the relative prostate weight was observed in the male animals. Nevertheless, observed differences were considered to be spontaneous and not test item-related.
Bone marrow examination
Males and females
No test item-related differences were noted.
Histopathology
(groups 1 and 4)
Males and females
No test item-related observation were noted for the examined male and female animals of group 4.
Additionally, previously (OECD 422, Provivo study no. 37482, 2021) observed microscopic changes in the kidney and stomach at the high dose animals (300/240 mg IPDA/kg b.w./day) were not observed at this study at 160 mg IPDA/kg b.w./day.
Examination of reproductive organs
No test item-related observation were noted for the examined reproductive organs of the male and female animals of group 4.
All testes examined showed completeness of stages and cell population.
Quantitative evaluation of primordial and small growing follicles and corpora lutea
No test item-related differences were noted between the females of the control group and the females of the high dose group in the number of follicles and corpora lutea.
1.1.2 Findings - Cohort 1B
Mortality
Males and females
No test item-related death was noted for all treatment groups (25, 80 or 160 mg IPDA/kg b.w./day).
Clinical signs
Males and females
Salivation was noted for 1 to 2 animals of the low and intermediated dose group.
At the high dose level (160 mg IPDA/kg b.w./day) test item-related changes were noted for more than half of the male and female animals in the form of salivation. Breathing sounds, haemorrhagic urine and piloerection were only noted for 1 or 2 animals per sex at the intermediate and high dose.
Detailed clinical observations:
Males and females
No further observations were noted during the weekly detailed clinical observations for the surviving male animals.
Females
Breathing sounds were noted for one female (no. 502) of the high dose group in two test weeks.
Body weight and
body weight gain
Males and females
No test item-related differences were noted.
However, marginal reductions in body weight were noted for the females of the three treatment groups compared to the control group animals from postnatal day 26 on until the end of the study on postnatal day 98. These marginal differences were considered to be non-adverse, as there was no correspondence in the body weight gain noticed.
Food consumption
Males and females
No test item-related changes were noted. Even so, a significant difference in food consumption was noted for the males of the high dose group between test days 57-64.
Sexual maturation
Males and females
No test item-related differences were noted for the time points of balanopreputial gland cleavage and vaginal opening.
Also, no test item-related differences were noted for the body weight at the time points of balanopreputial gland cleavage and vaginal opening.
Necropsy
Macroscopic post-mortem
findings
Males and females
No test item-related observations were noted.
Body weight at autopsy
Males and females
No test item-related changes were noted.
Estrous stage at necropsy
No test item-related influence was noted.
Organ weights
Males and females
No test item-related differences were noted.
However, a statistically significantly increase was noted for the relative organ weights of the left ovary at all dose levels of the females of cohort 1B. Nevertheless, no histomorphological changes were found in the female reproductive organs including ovaries in the F1 cohort 1A. Therefore, it was considered that there is no toxicological significance in the weight changes detected in these organs.
1.1.3 Analysis of test item-formulations
Test item-formulation analysis
The measured concentrations of IPDA in the test item-formulations were between 101.6 % and 109.6 % of the nominal concentration, indicating correctly prepared and homogeneous test item-formulations.
Referenceopen allclose all
Also the laboratory examinations and the examinations at necropsy (examination of haematological and biochemical parameters, determination of the T4 level of the male animals, determination of organ weights, the macroscopical and histopathological examination) revealed no test item-related changes.
However, most probably due to the properties of the test item in the drinking water a dose dependent refusal of intake of the test item-drinking water mixtures was noted for the male and female animals. This was most pronounced for the male animals of the high dose group (160/120 mg IPDA/kg b.w./day) during the first and the second treatment week and caused a reduction in body weight for the male animals. The body weight of the high dosed males recovered as the consumption of drinking water increased after the reduction of the test item concentration in the drinking water (dose level reduction from 160 to 120 mg IPDA/kg b.w./day).
The concentration of the test item in the test item-drinking water mixture was adjusted weekly to the relative drinking water consumption of the male and female animals. The actual dose levels of test item intake were calculated for the male and female animals using the total drinking water consumption and the body weight of the animals:
In most cases the actual dose levels were nearly in the range of the nominal dose levels for the male and female animals of all dose groups.
However, for the male animals of the high dose group (160/120 mg test item/kg b.w./day), the marked reduction in drinking water consumption before the reduction of the dose level revealed a decreased intake of test item via the drinking water. Hence, the calculated actual dose level was below the nominal dose level during the first and second treatment week. After the dose level reduction the actual test item-intake was in the range of the nominal dose level.
For the female animals increased actual dose levels were noted during the lactation period for all dose groups (20, 60 or 160/120 mg IPDA/kg b.w./day). This was due to increased levels of absolute and relative drinking water consumption during the lactation period in comparison to the pre-mating and gestation period that were noted for all dose groups. No influence was noted on the estrus cycles, the fertility, the gestation index, the pre-coital time and the gestation length.
During the post-natal development no influence was noted on body weight, the ano-genial distance, the number of nipples of the male pups and the T4 level.
No abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.
A reduced viability index was noted during the post-cull period for the pups of the high dose group (160/120 mg IPDA/kg b.w./day). This was considered as a secondary effect of the reduced consumption of drinking water of the high dosed females in comparison to the females of the control and the low and the intermediate dose group. This lower drinking water consumption led to a reduced milk production and hence, an inadequate feeding of some of the weaker pups. Therefore, the reduced viability index was not considered to be of toxicological relevance.
Though there were no toxicological relevant findings, the maximum dose level for possible further rat studies was considered to be 120 mg IPDA/kg b.w./day for the administration via the drinking water. At this dose level and also at the lower tested dose levels of 20 and 60 mg IPDA/kg b.w./day the test item in the drinking water already evoked a reduced drinking water consumption, but without secondary effects in the form of body weight loss or a possibly reduced milk production of the dams.
Text table 1: Groups and dose levels
Group |
IPDA dose (nominal) [mg/kg b.w./day] |
Number and sex of animals
|
Animal number |
|
1 |
0 (vehicle control) |
10 10 |
m f |
1 - 10 11 - 20 11 - 15 26 - 30 |
2 |
20 (low dose) |
10 10 |
m f |
21 - 30 31 - 40 None |
3 |
60 (low dose) |
10 10 |
m f |
41 - 50 51 - 60 None |
4 |
160 / 120 # (low dose) |
10 10 |
m f |
61 - 70 71 - 80 None |
m = male
f = female
# Dose reduction as of September 14, 2018 (TD 31) for all animals of group 4.
Text Table 2 :Reproductive outcome of the female animals per group.
Test item |
Group 1 Control |
Group 2 20 mg/kg |
Group 3 60 mg/kg |
Group 4 160/120 mg/kg |
|
Females started dosing #1 |
10 |
10 |
10 |
10 |
|
Females used for pairing |
10 |
10 |
10 |
10 |
|
Females mated |
10 |
10 |
10 |
10 |
|
Females pregnant #2 |
9 |
10 |
10 |
9 |
|
Females not pregnant |
1 |
0 |
0 |
1 |
|
Females with litters #3 |
9 |
10 |
10 |
9 |
|
Females with live born pups |
9 |
10 |
10 |
9 |
|
#1 |
Number of animals at start of the pre-mating and mating period |
||||
#2 |
Number of animals at start of the gestation period |
||||
#3 |
Number of animals at start of the lactation period |
||||
Text table 3: Fertility indices of the female animals per group.
Group / Dose level |
Fertility index % |
Pregnant females / females used for pairing |
|
Group 1 (Control) |
90 |
9 / 10 |
|
Group 2 (20 mg/kg) |
100 |
10 / 10 |
|
Group 3 (60 mg/kg) |
100 |
10 / 10 |
|
Group 4 (160/120 mg/kg) |
90 |
9 / 10 |
|
*/**: |
p ≤ 0.05 / p ≤ 0.01, Fisher test |
||
Text table 4: Gestation index per group.
Group / Dose level |
Gestation index % |
Dams with live pups / pregnant rats |
|
Group 1 (Control) |
100 |
9 / 9 |
|
Group 2 (20 mg/kg) |
100 |
10 / 10 |
|
Group 3 (60 mg/kg) |
100 |
10 / 10 |
|
Group 4 (160/120 mg/kg) |
100 |
9 / 9 |
|
*/**: |
p ≤ 0.05 / p ≤ 0.01, Fisher test |
||
Text table 5: Overview of the reproductive parameters.
|
Reproductive data |
|||||
Group 1 (control) |
Group 2 |
Group 3 |
Group 4 |
|||
Parametrical values (mean number per dam, #1) |
||||||
Implantation sites |
16.9 |
15.6 |
17.0 |
14.9 |
||
Pups (born alive and dead) |
14.7 |
14.7 |
15.7 |
13.7 |
||
Pups born alive |
14.4 |
14.6 |
15.5 |
13.7 |
||
Indices [%] |
||||||
Birth Index |
mean per dam group |
87.45 86.84 |
93.09 94.23* |
93.09 92.35 |
91.86 91.79 |
|
Live birth Index |
mean per dam group |
98.29 98.48 |
99.29 99.32 |
98.75 98.73 |
100.00 100.00 |
|
Post-implantation loss |
mean per dam group |
13.66 14.47 |
7.50 6.41* |
8.17 8.82 |
8.14 8.21 |
|
Resorptions and stillbirths |
||||||
Difference between no. of implantation sites and no. of pups born alive |
mean per dam
sum per group |
2.4
22 |
1.0
10 |
1.5
15 |
1.2
11 |
|
Number of stillbirths |
2 |
1 |
2 |
0 |
||
#1: |
Statistical calculation was performed by ANOVA / DUNNETT (*/**: p ≤ 0.05 / p ≤ 0.01). |
|||||
Text table 6:Viability indices and prematurely deceased pups during the post-cull period.
Post-cull period |
|||||
Parameter # |
Group 1 |
Group 2 |
Group 3 |
Group 4 |
|
Prematurely deceased pups between lactation days 5 and 13 / Number of pups alive on lactation day 4 after culling |
6 / 90 |
11 / 96 |
7 / 100 |
24 / 89 |
|
Number of dams with dead pups between lactation days 5 and 13 / Number of dams with live pups |
1 / 9 |
6 / 10 |
2 / 10 |
7 / 9 |
|
Between lactation days 5 and 13: Dams with 1 dead pup Dams with 2 dead pup Dams with ≥ 3 dead pup |
- - 1 |
3 1 2 |
- 1 1 |
1 3 3 |
|
Viability index (group values) |
93.33 |
88.54 |
93.00 |
73.03** |
|
|
(*/**: p ≤ 0.05 / p ≤ 0.01) Chi2test (group values) |
||||
Parental male and female animals
At 300 mg/kg b.w./day 2 females were prematurely sacrificed due to a poor general condition, one female one day before and the other female one day after dose reduction.
At 300/240 mg/kg b.w./day 2 male animals were found dead 12 or 15 test days after dose reduction.
At 300/240 mg/kg b.w./day a few of the surviving females showed salivation, a haemorrhagic nose/snout, piloerection, breathing sounds and / or reduced motility during the gestation and or lactation period. No changes in behaviour, the external appearance or the faeces were noted for the surviving male animals of the high dose group.
A marked body weight loss was noted at 300/240 mg/kg b.w./day for the prematurely deceased or sacrificed male and female animals.
The surviving female animals showed a slightly reduced body weight in comparison to control during the gestation and the lactation period. This and the observed general bad condition of the females are signs of maternal toxicity. No differences in body weight were noted for the surviving high dosed male animals.
At 300/240 mg/kg b.w./day an increased number of neutrophilic granulocytes and an increased LDH activity were noted for the female animals. Both were the secondary effects due to the corrosivity of the test item, which is manifesting in the very severe histopathological observations of erosions/ulcer in the female animals. No test item-related differences for the haematological and biochemical parameters were noted for the male animals.
The macroscopic examination at necropsy revealed kidney changes for 2 of the 8 surviving male animals of the high dose group (300/240 mg/kg b.w./day) that could possibly be test item-related.
Kidney changes that were considered to be test item-related were noted during the microscopic examination for the surviving male animals of the high dose group (300/240 mg/kg b.w./day), as well as for one of the prematurely deceased male animals and one of the prematurely sacrificed female animals, but not for the surviving female animals.
Test item-related changes for the surviving animals from both sexes were noted during the microscopic examinations of the stomachs from the male and female animals of the high dose group (300/240 mg/kg b.w./day) in form of an erosion of the stomach wall and an inflammatory response. These stomach changes were considered as local and secondary effects that were caused by the corrosive properties of the test item.
The additional microscopic examinations of the kidneys from the male animals and the stomachs from the male and female animals of the low and the intermediate dose groups revealed no test item-related changes.
No test item-related influence or adverse effects were noted for the male and female animals on food consumption, on the parameters of the neurological screening, for the organ weights and on the T4 serum levels of the male animals.
Reproductive toxicity
Parental females
No influence was noted on the number and length of the estrous cycles, the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period.
Pups
At 300/240 mg/kg b.w./day an effect was noted on the prenatal development in the form of an increased number of stillbirths. However, compared to the OECD 443 study of the test item the increased number of stillbirths in the high dose group can be regarded to be within the range of biological variability as also 9 stillbirths were noted in the low dose group of the OECD 443 study. No dose response-relationship was observed.
During the postnatal development a reduced pup body weight was noted at 300/240 mg/kg b.w./day, which was a secondary effect due to maternal toxicity.
No influence was noted on the viability of the pups, the ano-genital distance, the number of nipples per male pup and on the T4 concentration. No changes were noted for the thyroid glands from the pups during the microscopic examination.
No external gross abnormalities (malformations or variations) were noted for any of the pups.
Parental male and female animals (F0 Generation)
Three test item-related deaths were noted at the high dose level (240/160 mg IPDA/kg b.w./day). In the histopathological examination no direct cause of morbidity of these animals could be established.
Adverse test item-related changes in behaviour and the external appearance were noted for the male and female animals at 240/160 mg IPDA/kg b.w./day in the form of breathing sound and piloerection.
No test item-related influence was noted for the male and female animals on body weight, food consumption, drinking water consumption, the haematological and biochemical parameters, urinalysis, the levels of the thyroid hormones and the sperm parameters.
No test item-related changes were noted during the macroscopic examination, the examination of bone marrow and for the relative and absolute organ weights.
No test item-related observations were noted during the histopathological examination of the male and female animals of the high dose group.
Reproductive toxicity
Parental females
No test item-related influence was noted on the length and numbers of the estrous cycles during the pre-mating period, the fertility index, the gestation index, the duration of the pre-coital time interval and the gestation period.
Birth indices and post-implantation loss - F1 Pups
No test item-related differences were noted for the mean number of implantation sites per dam, the mean number of pups born (alive and dead) per dam and the mean number of live born pups per dam between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
Also the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
A slightly decreased birth index was noted between the control group (94.27 ± 8.38) and the treatment groups 25, 80 or 240/160 mg IPDA/kg b.w./day, 91.16 ± 10.31%, 93.10 ± 8.24 % and 89.46 ± 10.34%, respectively. However, as no dose response relationship was noted, these differences were considered to be spontaneous.
The decreased birth index was due to an increased number of resorptions in the treatment groups. Further on, as no dose response relationship was noted, these differences were considered to be spontaneous.
Furthermore, due to an increased number of stillbirths at the low and the intermediate dose group a slightly and statistically not significantly decreased live birth index was noted in these groups (97.25 ± 6.27 % or 97.85 ± 6.03 %) in comparison to the control group (99.68 ± 1.52 %).
In detail, 9 stillbirths (from 5 dams) were noted in the low dose group and 7 (from 4 dams) in the intermediate dose group in comparison to 1 stillbirths in the control group and 2 in the high dose group.
The increased number of resorptions in the treated groups as well as the increased number of stillbirths in the low and the intermediate dose led to an increased post-implantation loss (statistically not significant). In detail, the percentages of post-implantation loss that were noted in the treatment groups were 11.31 ± 11.91 % for low dose, 8.83 ± 10.38 % for intermediate dose and 11.20 ± 10.65 % for high dose in comparison to 6.05 ± 8.28 % for the control group. Additionally, the percentages of post-implantation loss per group in the treatment groups were within the Provivo background range.
Furthermore, as no dose response relationship was noted for the number of resorptions and the number of stillbirths, these differences were considered to be spontaneous
Number of live pups - F1 Pups
No test item-related or statistically significant differences were noted for the mean number of live pups per dam between the control group and the treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day) during the lactation period
Male to female ratio of the pups - F1 Pups
No test item-related influence on the male to female ratio was noted for all treatment groups (25, 80 or 240/160 mg IPDA/kg b.w./day).
F1 ADULTS
Cohort 1A
This part of the study reports the effects of the test item IPDA on the development of the F1 male and female animals of cohort 1A after weaning on lactation day 21 until necropsy. Necropsy was carried out between postnatal days 90 to 92 (males and females).
One male animal of the high dose group (160 mg IPDA/kg b.w./day) had to be prematurely sacrificed due to poor health conditions. The death is considered to be test item-related.
Adverse test item-related observations in the form of breathing sounds that were noted for several high dosed animals of the F0 Generation were markedly reduced for the animals of Cohort 1A.
No influence was noted for the male and female animals on body weight, food consumption, the haematological and biochemical parameters, lymphocyte typing in the spleen, urinalysis, the levels of the thyroid hormones, the sperm parameter and sexual maturation.
Nevertheless, a dose dependent decrease in the TSH values in the females of the intermediate and high dose below the detection limit were recorded. However, the histopathology observations did not report any histopathological changes in the pituitary gland compared to the control group. Furthermore, the observed differences in the TSH concentration were considered to be spontaneous, as they showed an opposite trend for the male and female animals.
Therefore, it was considered that the decreased TSH values were not toxicological relevant.
No test item-related changes were noted during the macroscopic examination, the examination of bone marrow and for the relative and absolute organ weights.
The histopathological examination of all organs did not reveal any test item-related changes or differences. Moreover, no histomorphological changes to be toxicologically concerned in the view of reproductive performance were found in the male reproductive organs (testes, epididymides, prostate glands, seminal vesicles and coagulating glands) and female reproductive organs (ovaries, oviducts, uterus, uterine cervix and vagina).
Cohort 1B
This part of the Study reports the effects of the test item IPDA on the development of the F1 male and female animals of cohort 1B after weaning on lactation day 21 until necropsy. Necropsy was carried out between postnatal days 98 to 102 (males and females).
No test item-related premature death was noted for the animals of Cohort 1B. Even so, that one male animal of the intermediate dose group was found dead. But this death was considered to result from an accident during the process of dosing.
Adverse test item-related observations in the form of breathing sounds and piloerection were noted for one or two male and female animals of the intermediate and high dose group of Cohort 1B.
No influence was noted for the male and female animals on food consumption and sexual maturation. For the female animals a slightly but statistically significantly reduced body weight was noted. Due to no correspondence with the body weight gain of the females of the treated groups this was considered to be spontaneous.
No test item-related changes were noted during the macroscopic examination and for the relative and absolute organ weight for the male and female animals. Nevertheless, an increase of the left ovary weight in the female animals of Cohort 1B was observed. However, no statistically significant changes were noted for the relative and absolute organ weights of the right ovary of the females from Cohort 1B and the left and the right ovary weights from the females of Cohort 1A. Additionally, no histomorphological changes of toxicologically concerned were found in the female reproductive organs, including ovaries of the F1 Cohort 1A. Hence, the increased weights of the left ovary that were noted for the females of Cohort 1B were considered to be spontaneous.
Text Table 7‑1: Reproductive outcome of the female animals.
IPDA | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | |
Females started dosing | 24 | 24 | 24 | 24 | |
Females deceased during pre-mating period | 0 | 0 | 0 | 3 (nos. 182, 174, 178) | |
Females used for pairing (placed together with a male at the beginning of the mating period) | 24 | 24 | 24 | 21 | |
Females with no verified copulation during the mating period of 14 days | 0 | 1 (no. 95) | 0 | 2 (nos. 177, 189) | |
| - thereof non-pregnant | - | 1 (no. 95) | - | 2 (nos. 177, 189 |
| - thereof pregnant | - | - | - | - |
Females with verified copulation during the mating period of 14 days, | 24 | 23 | 24 | 19 | |
| - thereof non-pregnant | 1 (no. 28) | - | 2 (nos. 137, 140) | - |
| - thereof pregnant | 23 | 23 | 22 | 19 |
Fertility index (Pregnant females with verified copulation related to females with verified copulation) | 96 % (23 of 24) | 100 % (23 of 23) | 92 % (22 of 24) | 100 % (19 of 19) | |
Females deceased during gestation period | 1 (no. 40) | 0 | 1 (no.126) | 1 (no. 187) | |
Females with total resorption of implants | 0 | 0 | 0 | 0 | |
Females with live born pups | 22 | 23 | 21 | 18 | |
Text Table 7‑2: Prematurely sacrificed animals for which no direct cause of death could be established.
Prematurely sacrificed animals due to poor health condition (most likely test item-related) | ||||||
No. | Sex | Group | Test day #1 | Number of doses | Type of fate | |
182 | Female | 4 | 24 | 7 #2 | Premature sacrifice
| |
178 | Female | 4 | 59 | 45 | ||
153 | Male | 4 | 73 | 59 | ||
#1: | Dose reduction on test day 51. | |||||
#2: | Dosing for animal no. 182 was stopped on test day 21 (see 2.13 ‘Study Plan deviations). | |||||
Text Table 7-3: Observations that were noted for the prematurely sacrificed animal nos. 153, 178 and 182 during the daily cage side inspection on the days before sacrifice (common observations as salivation are not considered).
Observations during the daily cage side inspections for the prematurely sacrificed animal nos. 153, 178 and 182 #1 | |||||
Gr. | No, | Sex | Symptom | Observed on test day (day of death #2) | |
4 | 153 | M | Gasping Reduced motility Breathing sounds Laboured breathing | 72, 73 73 Consistently from test day 60 until 73 72, 73 (day of death: 73) | |
178 | F | Breathing sounds | 58, 59 (59) | ||
182 | F | Breathing sounds | 21 - 23 | ||
Laboured breathing | 24 | ||||
Haemorrhagic nose/snout | 24 (day of death: 24) | ||||
| Gr.: Group; M; F: Male; Female | ||||
#1: | See tables 3-1B ‘Clinical Signs – Prematurely Deceased Males - Individual Data’ and 3-2B ‘Clinical Signs – Prematurely Deceased Females – Premating/Mating - Individual Data - Females’ | ||||
#2: | Day of death is given in brackets. | ||||
Text Table 7‑4A: Prematurely deceased or sacrificed animals due to an accident during the process of dosing.
Prematurely deceased or sacrificed animals - death caused by accident | |||||||
No. | Sex | Group | Test day #1 | Number of doses | Type of fate
| Cause of death | |
164 #2 | Male | 4 | 16 | 1 | Found dead | Accidental death | |
151 | Male | 4 | 49 | 35 | Found dead | ||
174 | Female | 4 | 49 | 35 | Premature sacrifice | ||
126 #3 | Female | 3 | 95 (GD6) | 81 | Found dead
| ||
187 #4 | Female | 4 | 106 (GD20) | 92 | Premature sacrifice | ||
40 #5 | Female | 1 | 107 (GD19) | 93 | Found dead | ||
#1: | Dose reduction on test day 51. | ||||||
#2: | No. 164 was replaced by spare animal no. 193 (Amendment no. 2). | ||||||
#3: | Female no. 126 was pregnant. During necropsy (including laparotomy) on gestation day 6, 15 implantation sites were noted. | ||||||
#4: | Female no. 187 was pregnant. During necropsy (including laparotomy) on gestation day 20, 13 implantation sites were noted. | ||||||
#5: | Female no. 40 was pregnant. During necropsy (including laparotomy) on gestation day 19, 14 implantation sites were noted. | ||||||
Text Table 7-4B: Observations that were noted during the daily cage side inspection on the days before sacrifice or death (common observations as salivation are not considered).
Observations during the daily cage side inspections #1 Prematurely deceased or sacrificed animals - death caused by accident | ||||||
Gr. | No, | Sex | Symptom | Observed on test day (day of death #2) | ||
1 | 40 | F | No observations | - (GD 19) | ||
3 | 126 | F | No observations | - (GD 6) | ||
4 | 151 | M | Haemorrhagic nose/snout | 47 | ||
Haemorrhagic canthus | 47 (49) | |||||
164 | M | No observations | - (16) | |||
174 | F | Breathing sounds | 46 - 49 (174) | |||
187 | F | Breathing sounds | GD 18 - 20 | |||
Piloerection | GD 18 | |||||
Haemorrhagic nose/snout | 18 – 20 (GD 20) | |||||
| Gr.: Group; M; F: Male; Female, GD = gestation day. | |||||
#1: | See tables 3-1B ‘Clinical Signs – Prematurely Deceased Males – Individual Data’, 3-2B ‘Clinical Signs - Prematurely Deceased Females – Pre-mating/Mating – Individual Data’ and 3-3B ‘Clinical Signs - Prematurely Deceased Females – Gestation – Individual Data’ | |||||
#2: | Day of death is given in brackets. | |||||
Text Table 7‑5: Observations that were noted during the daily cage-side observations for the male animals of the low, the intermediate and the high dose level.
Observations in group 2 (25 mg IPDA/kg b.w./day) #1 | ||||
Observation (males) | Affected animals | First to last seen (test days) #2 | Number of times recorded #3 | |
Haemorrhagic nose / snout | 1 of 24 | 98 (no. 71) | 1 | |
Water consumption increased | 1 of 24 | 69 - 75 (no. 52) | 7 | |
Pultaceous faeces | 1 of 24 | 75 (no. 64) | 1 | |
Observations in group 3 (80 mg IPDA/kg b.w./day) | ||||
Salivation | 17 of 24 | 24 - 125 | 65 | |
Water consumption increased | 1 of 24 | 72 - 75 (no. 120) | 4 | |
Observations in group 4 (240/160 mg IPDA/kg b.w./day) | ||||
Salivation | 22 of 22 #4 | 16 - 127 | 704 | |
Breathing sounds | 10 of 22 | 16 - 117 | 67 | |
Piloerection | 5 of 22 | 22 - 73 | 14 | |
Haemorrhagic urine | 1 of 22 | 1 (no. 159) | 1 | |
Water consumption increased | 2 of 22 | 72 – 79 (nos. 146, 158) | 11 | |
Pultaceous faeces | 1 of 22 | 103 -104 (no. 145) | 2 | |
#1: | See table 3-1A ‘Clinical Signs - Summary - Males - F0 Generation’ and table 3-5 ‘Clinical Signs - Individual Data - Males - F0 Generation’ for the number of the individual animals. | |||
#2: | If only 1 or 2 animals were affected, the animals are listed in brackets. | |||
#3: | Number of times the symptom was observed for the affected animals. | |||
#4: | Group 4: The prematurely deceased animals nos. 151, 164 were excluded from the summary. | |||
Text Table 7‑6: Observations that were noted during the daily cage-side observations for the female animals during the pre-mating/mating period, the gestation period and the lactation period until necropsy.
Observations in group 2 (25 mg IPDA/kg b.w./day) | |||||||
| Affected females per period | Number of times recorded | |||||
Observation (females) | Pre-mating/ Mating #1 | Gestation #1 | Lactation #1 | Pre-mating/ Mating | Gestation | Lactation | |
Salivation | n.d. | n.d. | 1 of 23 #2 | n.d. | n.d. | 3 | |
Observations in group 3 (80 mg IPDA/kg b.w./day) | |||||||
Salivation | 9 of 24 | 2 of 21 #3 | 2 of 21 #3 | 18 | 2 (no. 125, 134) | 2 (no. 125, 135) | |
Observations in group 4 (240/160 mg IPDA/kg b.w./day) | |||||||
Salivation | 21 of 21 #4 | 12 of 18 #5 | 12 of 18 #5 | 440 | 26 | 32 | |
Breathing sounds | 10 of 21 | 4 of 18 | 1 of 18 | 159 | 19 | 7 (no. 186) | |
Piloerection | 5 of 21 | n.d. | 1 of 18 | 31 | n.d. | 2 (no. 184) | |
Gasping | 1 of 21 | n.d. | n.d. | 1 (no. 187) | n.d. | n.d. | |
Haemorrhagic nose-snout | 1 of 21 | n.d. | n.d. | 3 (no. 187) | n.d. | n.d. | |
Increased water consumption | 1 of 21 | n.d. | n.d. | 8 (no. 188) | n.d. | n.d. | |
Respiratory rate increased | 1 of 21 | n.d. | n.d. | 1 (no. 181) | n.d. | n.d. | |
Laboured breathing | 1 of 21 | n.d. | n.d. | 6 (no. 187) | n.d. | n.d. | |
Thickened Abdomen | 1 of 21 | n.d. | n.d. | 1 (no. 187) | n.d. | n.d. | |
n.d.: | Not detected | ||||||
#1: | See table 3-2A ‘Clinical Signs - Summary - Females - Pre-mating/Mating - F0 Generation’, table 3-3A ‘Clinical Signs - Summary - Females - Gestation - F0 Generation’ and table 3-4 ‘Clinical Signs - Summary - Females - Lactation - F0 Generation’. | ||||||
#2: | Group 2: The non-mated and non-pregnant female no. 95 is not considered for the gestation and the lactation period. | ||||||
#3: | Group 3: The non-pregnant female nos. 137, 140 and the prematurely deceased female no. 126 are missing in the gestation period and the lactation period. | ||||||
#4: | Group 4: The prematurely deceased animals no. 174, 178, 182 (died during pre-mating period) are excluded from the summary. | ||||||
#5: | Group 4: The prematurely deceased animal no. 187 (died during gestation period) and the non-mated and non-pregnant female nos. 177, 189 are excluded from the summaries in the gestation and the lactation periods. | ||||||
Text Table 7‑7: Start and duration of observations that were noted for the male animals.
Start and duration of symptoms - Males #1 | |||||
Symptom | Time frames in relation to application | Number of times recorded (in all groups) | |||
Appearing of the symptom | Disappearing of the symptom | ||||
Salivation | 0 - 5 min | 5 - 20 min | 4 | ||
0 - 5 min | 20 - 60 min | 100 | |||
5 - 20 min | 5 - 20 min | 11 | |||
5 - 20 min | 20 - 60 min | 651 | |||
5 - 20 min | 6 - 24 h | 2 | |||
Consistently during the day | 1 | ||||
Breathing sounds | 0 - 5 min | - | 1 #2 | ||
0 - 5 min | 2 - 6 h | 1 | |||
0 - 5 min | 6 - 24 h | 2 | |||
5 - 20 min | 20 - 60 min | 1 | |||
Consistently during the day | 62 | ||||
#1: | See table 3-9-1 ‘Start and Duration of Symptoms - Summary - Males - F0 Generation’. | ||||
#2: | No endpoint was determined in one case. | ||||
Text Table 7‑8: Start and duration of observations that were noted for the female animals.
Start and duration of symptoms - Females #1 | ||||
Symptom | Time frames in relation to application | Number of times recorded (in all groups and periods) | ||
Appearing of the symptom | Disappearing of the symptom | |||
Salivation | 0 - 5 min | 5 - 20 min | 2 | |
0 - 5 min | 20 - 60 min | 39 | ||
5 - 20 min | - | 1 #2 | ||
5 - 20 min | 5 - 20 min | 10 | ||
5 - 20 min | 20 - 60 min | 471 | ||
Breathing sounds | - | - | 1 #3 | |
0 - 5 min | - | 2 #2 | ||
0 - 5 min | 6 -24 h | 3 | ||
5 - 20 min | 1- 2 h | 1 | ||
20 - 60 min | 2 - 6 h | 1 | ||
Consistently during the day | 177 | |||
Laboured breathing | Consistently during the day | 6 | ||
Gasping | Consistently during the day | 1 | ||
Increased respiratory rate | Consistently during the day | 1 | ||
#1: | See table 3-9-2 ‘ Start and Duration of Symptoms - Summary - Females - Pre-mating / Mating - F0 Generation’, table 3-9-3 ‘Start and Duration of Symptoms - Summary - Females - Gestation - F0 Generation’ and table 3-9-4 ‘Start and Duration of Symptoms - Summary - Females - Lactation - F0 Generation’. | |||
#2: | No endpoint was determined during the pre-mating / mating period in one case each for salivation and breathing sounds. | |||
#3: | No start and no endpoint was determined in one case of breathing sounds during the pre-mating / mating period. | |||
Text Table 7‑9: Body weight gain of the male animals from test day 15 to test day 126.
Males | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | |
Body weight gain # (test day 15 to test day 126) | +44.6% | +47.3% | +43.1% | +43.6% | |
#: | Values taken from table 6-1 'Body Weight Gain - Summary - Males - F0 Generation' | ||||
Text Table 7‑10: Body weight gain of the female animals during the pre-mating period, the gestation period and the lactation period.
Females | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg |
Body weight gain (%) (pre-mating period) (test days 15 to 85) #1 | +21.3% | +20.6% | +20.3% | +19.0% |
Body weight gain (%) (gestation period) #2 | +50.3% | +47.7% | +47.0% | +49.1% |
Body weight gain (%) (lactation period) #3 | +1.1% | +2.5% | +3.9% | +6.7% |
Text Table 7-11: Comparison of haematological parameters with the Provivo background data.
F0 Females Parameter | Values from this study #2 Mean value per group ± SD (range of the individual values (n = 10)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
WBC [x10E3 cells/µL] | Group 1 | 9.292 ± 1.488 (6.20 – 11.75) [n=1] |
5 to 95 % Percentile
4.65 - 11.38
| |
Group 2 | 8.161 ± 1.965 (5.80 – 12.11) [n=1] | |||
Group 3 | 6.761 ± 1.506* (4.70 - 9.91) | |||
Group 4 | 9.852 ± 3.564 (5.06 – 12.24) [n=2] | |||
EOS [x10E3 cells/µL] | Group 1 | 0.241 ± 0.136 (0.09 - 0.54) [n=1] |
5 to 95 % Percentile
0.05 - 0.42
| |
Group 2 | 0.115 ± 0.050** (0.06 - 0.23) | |||
Group 3 | 0.130 ± 0.062* [n=1] (0.04 - 0.22) | |||
Group 4 | 0.117 ± 0.036* (0.05 - 0.16) | |||
Cohort 1A WBC [x10E3 cells/µL]
| Group 1 | 14.031 ± 22.068 (5.07 - 76.60) [n=1] | 5 to 95 % Percentile
4.33 - 11.52 | |
Group 2 | 7.167 ± 1.766 (4.47 - 8.93) | |||
Group 3 | 8.677 ± 2.281 (6.05 - 12.96) [n=1] | |||
Group 4 | 8.937 ± 2.091 (6.58 - 12.38) [n=2] | |||
Baso [x10E3 cells/µL] | Group 1 | 0.042 ± 0.018 (0.02 - 0.07) |
5 to 95 % Percentile
0.00 - 0.07
| |
Group 2 | 0.042 ± 0.020 (0.02 - 0.07) | |||
Group 3 | 0.021 ± 0.009* (0.01 - 0.04) | |||
Group 4 | 0.037 ± 0.017 (0.01 - 0.07) | |||
#1: | Data not audited by QAU | |||
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test) | |||
#2: | Taken from table 8-2 'Haematological Parameters - Summary - Females' and table 8-4 'Haematological Parameters - Individual Data - Females'. | |||
Text Table 7-12: Statistically significant changes of the haematological parameters that were not considered to be test item-related.
|
| Changes in comparison to control #1 [%] | Reason | ||||
Parameter # (F0 Generation) | Sex | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | ||
| |||||||
WBC [x10E3/µL] | Females #2 | n.a. (9.292) | -12.2 (8.161) | -27.2* (6.761) | +6.0 (9.852) | A | |
WBC [x10E3/µL] | Males #3 | n.a. (9.609) | +16.4 (11.187) | - 9.0 (8.745) | - 2.4 (9.380) | D | |
WBC [x10E3/µL] | Males #4 (Co1A) | n.a. (9.340) | +1.1 (9.440) | +18.1 (11.033) | +27.6 (11.917) | D | |
WBC [x10E3/µL] | Females #5 (Co1A) | n.a. (14.031) | - 48.9 (7.167) | - 38.2 (8.677) | - 36.3 (8.937) | D | |
WBC [x10E3/µL] | Females #6 (Co1A) | n.a. (7.079) | +1.2 (7.167) | +22.6 (8.677) | +26.2 (8.937) | D | |
EOS [x10E3/µL] | Females #1 | n.a. (0.241) | -52.3** (0.115) | -46.1* (0.130) | -51.5* (0.117) | B | |
Baso [x10E3/µL] | Females #1 | n.a. (0.042) | 0.0 (0.042) | -50.0* (0.021) | -11.9 (0.037) | C | |
n.a.: | Not applicable | ||||||
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) | ||||||
A: | The statistically significantly decreased value at the intermediate dose level was considered to be spontaneous, as no dose response-relationship was noted. Furthermore, a comparison with the statistically significant changes that were noted for the F0 male animals and the statistically not significant changes that were noted for the F0 females and the male and female animals of Cohort 1A revealed no consistent tendencies. | ||||||
B: | The similar reductions in the treatment groups were considered to be due to an increased value of the control group and considered as not relevant. | ||||||
C; | No dose response-relationship was noted and all values were within the Provivo background range. | ||||||
D: | Only for comparison. | ||||||
#1: | The group mean values are given in the brackets. | ||||||
#2: | Values taken from table 8-2 'Haematological Parameters - Summary - Females'. | ||||||
#3: | Values taken from table 8-1 'Haematological Parameters - Summary - Males'. | ||||||
#4: | Values taken from table 6-1-Co1A 'Haematological Parameters - Summary - Males'. | ||||||
#5: | Values taken from table 6-2-Co1A 'Haematological Parameters - Summary - Females'. | ||||||
#6: | Mean of the control group without animal no. 237 that had extremely increased numbers of white blood cells. | ||||||
Text Table 7-12: Comparison of haematological parameters with the Provivo background data.
F0 Females Parameter | Values from this study #2 Mean value per group ± SD (range of the individual values (n = 10)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
WBC [x10E3 cells/µL] | Group 1 | 9.292 ± 1.488 (6.20 – 11.75) [n=1] |
5 to 95 % Percentile
4.65 - 11.38
| |
Group 2 | 8.161 ± 1.965 (5.80 – 12.11) [n=1] | |||
Group 3 | 6.761 ± 1.506* (4.70 - 9.91) | |||
Group 4 | 9.852 ± 3.564 (5.06 – 12.24) [n=2] | |||
Cohort 1A WBC [x10E3 cells/µL]
| Group 1 | 14.031 ± 22.068 (5.07 - 76.60) [n=1 | 5 to 95 % Percentile
4.33 - 11.52
| |
Group 2 | 7.167 ± 1.766 (4.47 - 8.93) | |||
Group 3 | 8.677 ± 2.281 (6.05 - 12.96) [n=1] | |||
Group 4 | 8.937 ± 2.091 (6.58 - 12.38) [n=2] | |||
EOS [x10E3 cells/µL] | Group 1 | 0.241 ± 0.136 (0.09 - 0.54) [n=1] |
5 to 95 % Percentile
0.05 - 0.42
| |
Group 2 | 0.115 ± 0.050** (0.06 - 0.23) | |||
Group 3 | 0.130 ± 0.062* [n=1] (0.04 - 0.22) | |||
Group 4 | 0.117 ± 0.036* (0.05 - 0.16) | |||
Baso [x10E3 cells/µL] | Group 1 | 0.042 ± 0.018 (0.02 - 0.07) |
5 to 95 % Percentile
0.00 - 0.07
| |
Group 2 | 0.042 ± 0.020 (0.02 - 0.07) | |||
Group 3 | 0.021 ± 0.009* (0.01 - 0.04) | |||
Group 4 | 0.037 ± 0.017 (0.01 - 0.07) | |||
#1: | Data not audited by QAU | |||
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test) | |||
#2: | Taken from table 8-2 'Haematological Parameters - Summary - Females' and table 8-4 'Haematological Parameters - Individual Data - Females'. | |||
Text Table 7‑13: Statistically significant changes of the biochemical parameters that were not considered to be test item-related.
Parameter # F0 Gemeration |
| Changes in comparison to control [%] | Reason | |||
Sex | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | |||
|
| |||||
Bilirubin (total) [µmol/L] | Females | +15.1* | +0.3 | -3.4 | A | |
Glucose [mmol/L] | Females | +0.2 | -8.0 | -18.7* | B | |
Glucose [mmol/L] | Males | - 1.2 | - 4.0 | - 2.2 | C | |
Glucose [mmol/L] | Males Co1A | +5.3 | +8.6 | +6.8 | C | |
Glucose [mmol/L] | Females Co1A | - 12.2 | +5.5 | - 5.0 | C | |
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) | |||||
A: | No dose response relationship was noted and all values of the low dose group were within the Provivo background range. | |||||
B: | No changes were noted for the F0 males and the males and females of Cohort 1A. | |||||
C: | Only for comparison. | |||||
#: | Values taken from table 9-1 'Biochemical Parameters - Summary - Males', table 9-2 'Biochemical Parameters - Summary - Females', table 7-1-Co1A 'Biochemical Parameters - Summary - Females' and from table 7-2-Co1A 'Biochemical Parameters - Summary - Females'. | |||||
Text Table 7-14: Comparison of biochemical parameters with the Provivo background data.
F0 Females Parameter | Values from this study #2 Mean value per group ± SD (range of the individual values (n = 10)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
Bilirubin (total) [µmol/L] | Group 1 | 2.98 ± 0.31 (2.5 – 3.4) |
5 to 95 % Percentile 2.4 – 4.5 | |
Group 2 | 3.43 ± 0.53* (2.5 – 4.4) | |||
Group 3 | 2.99 ± 0.31 (2.5 – 3.6) | |||
Group 4 | 2.88 ± 0.37 [n=1] (2.2 – 3.4) | |||
Glucose [mmol/L] | Group 1 | 7.651 ± 0.878 (6.69 – 9.60) | 5 to 95 % Percentile 5.88 – 9.72 | |
Group 2 | 7.666 ± 1.737 (5.94 –10.89) [n=2] | |||
Group 3 | 7.036 ± 0.943 [n=1] (5.82 – 8.10) | |||
Group 4 | 6.223 ± 0.845* [n=5] (5.43 – 7.90) | |||
#1: | Data not audited by QAU | |||
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test) | |||
#2: | Taken from table 9-2 'Biochemical Parameters - Summary - Females' and table 9-4 'Biochemical Parameters - Individual Data - Females'. | |||
Text Table 7‑15: Changes of the TSH concentration that were not considered to be test item-related.
|
| Changes in comparison to control [%] | Reason | |||
Parameter #1 | Sex | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | ||
|
| |||||
TSH [ng/mL] | males | -51.5 | -38.7 | -14.2 | A | |
TSH [ng/mL] | females | -7.0 | +39.4 | +30.4 | A | |
#1: | Values taken from table 11-1 ' Thyroid Hormone Level Analysis - Summary - Males ' and table 11-2 ' Thyroid Hormone Level Analysis - Summary - Females' | |||||
A: | The observed differences in the TSH concentration were not considered to be test item-related, as they were without statistical significance and no dose-response relationship was noted. | |||||
Text Table 7-16A: Comparison of TSH concentration of the male and female animals with the Provivo background data.
Parameter (F0)
| Values from this study #2 Mean value per group ± SD (Range of the individual values (n = 10)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
TSH [ng/mL] | Group 1 (males) | 3.3264 ± 1.6885 (1.545 - 7.507) [n=1] |
5% to 95% Percentile
0.081 – 6.964
25% to 95% Percentile
1.506 #3 – 6.964 | |
Group 2 (males) | 1.6145 ± 0.7953 [n=3 #3] (0.081 - 2.667) | |||
Group 3 (males) | 2.0380 ± 1.9448 [n=4] (0.081 - 6.157) | |||
Group 4 (males) | 2.8549 ± 3.4244 [n=5] (0.373 - 9.500) [n=2] | |||
Group 1 (females) | 2.1299 ± 1.5930 [n=1] (0.081 - 6.017) [n=1] | 5% to 95% Percentile
0.081 – 4.123
25% to 95% Percentile
0.238 #3 – 4.123 | ||
Group 2 (females) | 1.9813 ± 1.0509 (0.917 - 4.029) | |||
Group 3 (females) | 2.9693 ± 1.5341 (1.472 - 6.391) [n=2] | |||
Group 4 (females) | 2.7774 ± 1.4983 (1.241 - 6.161) [n=1] | |||
#1: | Data not audited by QAU | |||
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (Dunnett test) | |||
#2: | See tables 11-1 and 11-3 'Thyroid Hormone Level Analysis - Summary and Individual Data - Males', 11-2 and 11-4 'Thyroid Hormone Level Analysis - Summary and Individual Data - Females'. | |||
#3 | The 5 % percentile is at the lower level of detection (LOD = 0.081). Hence, for the comparison with the study values the 25 % percentile was used. | |||
Text Table 7-16B: Comparison of T4 concentration of the male and female animals with the Provivo background data.
Parameter (F0)
| Values from this study #2 Mean value per group ± SD (Range of the individual values (n = 10)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
T4 [nmol/L] | Group 1 (males) | 53.6940 ± 8.0417 (44.155 – 67.412) [n=1] |
5% to 95% Percentile
33.618 – 66.057 | |
Group 2 (males) | 53.1960 ± 6.5840 (46.028 – 64.072) | |||
Group 3 (males) | 53.6609 ± 7.0554 (41.450 – 64.077) | |||
Group 4 (males) | 53.2547 ± 5.2753 (45.532 – 60.183) | |||
Group 1 (females) | 38.1577 ± 9.351 (24.134 – 51.513) [n=1] | 5% to 95% Percentile
18.464 – 49.518 | ||
Group 2 (females) | 38.3400 ± 6.9228 (27.011 – 49.735) [n=1] | |||
Group 3 (females) | 40.4866 ± 7.4368 (30.140 – 50.574) [n=1] | |||
Group 4 (females) | 38.3674 ± 8.1366 (19.662 – 47.310) | |||
#1: | Data not audited by QAU | |||
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (Dunnett test) | |||
#2: | See tables 11-1 and 11-3 'Thyroid Hormone Level Analysis - Summary and Individual Data - Males', 11-2 and 11-4 'Thyroid Hormone Level Analysis - Summary and Individual Data - Females'. | |||
Text Table 7-17: Changes of the sperm motility that were not considered to be test item-related.
Percentage of motile spermatozoa in the cauda epididymis (%) | |||||
Parameter #1 | Group 1 control | Group 2 25mg/kg | Group 3 80mg/kg | Group 4 240/160mg/kg | |
Percentage of motile spermatozoa | 71.3 | 71.5 | 75.8* #2 | 68.5 | |
#1: | Taken from table 12-1 'Sperm Count and Motility - Summary - F0 Generation'. | ||||
#2: | Not considered to be test item-related, as the difference between group 1 and group 3 was only small, no dose-response relationship was noted and an increased number of motile spermatozoa is not an adverse effect. | ||||
Text Table 7-18: Comparison of sperm motility with the Provivo background data.
F0 Males Parameter | Values from this study #2 Mean value per group ± SD (range of the individual values (n = 24/22)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
Sperm motility [%] | Group 1 | 71.3 ± 6.9 (61 – 79) |
5 to 95 % Percentile
51 – 86 | |
Group 2 | 71.5 ± 8.0 (62 – 85) | |||
Group 3 | 75.8 ± 5.5 (66 – 87) [n=1] | |||
Group 4 | 68.5 ± 9.0 (62 – 80) | |||
#1: | Data not audited by QAU | |||
#2: | Taken from table 12-1 'Sperm Count and Motility - Summary - F0 Generation' and table 12-3 'Sperm Motility – Individual Data - F0 Generation' | |||
Text Table 7-19: Pairs without verified copulation.
Pairs without verified copulation | |||
Group | Male partner | Female partner | |
2 | 72 | 95 | |
4 | 152 | 177 | |
4 | 163 | 189 | |
#1: | See table 1 ‘Pairing List - F0 Generation’. | ||
Text Table 7-20A: ------------------------------------------------------------------------------------------- Macroscopic findings that were noted for the male animals at necropsy (only the surviving animals were considered).
Observations at necropsy for the female animals | ||||||
Organ | Observation | Group 1 Control (n = 24 #1) | Group 2 25 mg/kg (n = 24) | Group 3 80 mg/kg (n = 24) | Group 4 240/160 mg/kg (n = 22) | |
Liver | petechial bleeding | 1 / 24 (22) | - | - | - | |
Trachea | tissue enlargement | - | 1 / 24 (57) | - | - | |
Lymph node, mesenteric | dark-red discoloured | - | 1 / 24 (62) | - | - | |
#1: | Number of animals (only the surviving female animals are included). For the macroscopic post-mortem findings of the prematurely deceased animals refer to Appendix 5 section 3.1 'Mortality' of the histopathology phase report. | |||||
…/…: | Affected animals / Animals examined | |||||
(…): | The number of the affected animal is written in brackets. | |||||
Text Table 7-20B: -- Macroscopic findings that were noted for the female animals at necropsy (only surviving animals were considered).
Observations at necropsy for the female animals # | ||||||
Organ | Observation | Group 1 Control (n = 23) | Group 2 25 mg/kg (n = 24) | Group 3 80 mg/kg (n = 23) | Group 4 240/160 mg/kg (n = 20] | |
Duodenum | mucosa reddened | 2 / 23 (29, 33) | 4 / 24 (82, 83, 91, 93) | 7 / 23 (129, 131, 134-136, 143-144) | 4 / 20 (170, 171, 175, 180) | |
dilated | 1 / 23 (33) | - | - | - | ||
thickened | - | - | 1 / 23 (135) | 1 / 20 (180) | ||
Stomach | haemorrhagic foci | 1 / 23 (35) | 1 / 24 (87) | 1 / 23 (133) | 2 / 20 (176, 190) | |
mucosa black discoloured | - | 1 / 24 (92) | - | - | ||
Uterus | uterus horn: thickened | 1 / 23 (34) | 1 / 24 (93) | - | 1 / 20 (186) | |
dilated, filled with liquid | - | 1 / 24 (91) | 2 / 23 (137, 138) | - | ||
Lymph node (cervical) | enlarged and indurated | 1 / 23 (32, right)) | - | - | - | |
Axilla | tissue enlargement | 1 / 23 (32, right) | - | - | - | |
abscess | - | - | 1 / 23 (128, right) | - | ||
Thymus | reduced in size | 1 / 23 (45) | - | - | - | |
Reddened (extremely) |
|
|
| 1 / 24 (175) | ||
Pituitary gland | enlarged (slightly) | - | - | 1 / 23 (131) | - | |
Heart, lungs | adhered with thoracic wall | - | - | 1 / 23 (134) | - | |
Lungs | petechial bleeding | - | - | - | 1 / 24 (175) | |
Kidney | Tissue enlargement | - | - | - | 1 / 24 (172, left) | |
#: | Number of animals (only the surviving female animals are included). For the macroscopic post-mortem findings of the prematurely deceased animals refer to Appendix 5 section 3.1 'Mortality' of the histopathology phase report. | |||||
…/…: | Affected animals / Animals examined | |||||
(…): | The number of the affected animal is written in brackets. | |||||
Text Table 7-21: Stage of the estrous cycle at necropsy. The stage of the estrous cycle was evaluated from vaginal lavages that were taken at necropsy and during the microscopic examination of the vagina (only group 1 and 4).
Stage of estrous cycle at necropsy (F0 Generation) #1 | Group 1 Control #2 | Group 2 25 mg/kg #3 | Group 3 80 mg/kg #4 | Group 4 240/160 mg/kg | |||
N | H | N | N | N | H | ||
Proestrus | 2 of 22 | 6 of 20 | 2 of 23 | 1 of 22 | 2 of 24 | 5 of 20 | |
Estrus | 4 of 22 | 2 of 20 | 2 of 23 | 3 of 22 | 6 of 24 | 2 of 20 | |
Metestrus | 6 of 22 | - | 4 of 23 | 10 of 22 | 5 of 24 | 1 of 20 | |
Diestrus | 10 of 22 | 12 of 20 #5 | 15 of 23 | 8 of 22 | 11 of 24 | 12 of 20 #5 | |
-: | not detected. | ||||||
#1: | The values are taken from table 15 ‘Stage of Estrous Cycle at Necropsy - Individual Data - F0 Generation’. | ||||||
N: | Stage of the estrous cycle was determined from the vaginal lavage at necropsy. | ||||||
H: | Stage of the estrous cycle was determined during the microscopic examination of the vagina (see Appendix 5 'Histopathological Phase Report'). | ||||||
#2: | The vaginal lavages of female nos. 36 and 40 were not evaluable. | ||||||
#3: | The vaginal lavage of female no. 92 was not evaluable. | ||||||
#4: | The vaginal lavages of female nos. 126 and 143 were not evaluable. | ||||||
#5: | Termed as lactational diestrus in the histopathology report. | ||||||
Text Table 7-22: Statistically significant changes in organ weights, not considered to be test item-related.
F0 Generation |
| Changes in comparison to control [%] | Reason | |||
Parameter | Sex | Group 2 | Group 3 | Group 4 | ||
|
| |||||
Thymus (absolute) | Male #1 | - 9.4 | - 14.1 | - 19.1** | A | |
Thymus (relative) | Male #2 | - 11.5 | - 12.9 | - 18.5** | A | |
Thyroid / Parathyroid (left, absolute) | Female #3 | +30.1* | +6.1 | +12.8 | B | |
Thyroid / Parathyroid (left, relative) | Female #4 | +30.8 | +6.1 | +16.8 | B | |
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) | |||||
#1: | Taken from table 18-1 ‘Absolute Organ Weights - Summary - Males - F0’. | |||||
#2: | Taken from table 17-1 ‘Relative Organ Weights - Summary - Males - F0’. | |||||
#3: | Taken from table 18-2 ‘Absolute Organ Weights - Summary - Females - F0’. | |||||
#4: | Taken from table 17-2 ‘Relative Organ Weights - Summary - Females - F0’. | |||||
A: | The observation was considered to be spontaneous, as no correlation with the histopathology results was noted. | |||||
B: | The observation was considered to be spontaneous, as no dose response relationship and no correlation with the histopathology results were noted. | |||||
Text Table 7-23: Comparison of organ weights with the Provivo background data.
Parameter | Values from this study #2 Mean value per group ± SD (range of the individual values (n= up to 24)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
males | ||||
Thymus [g] | Group 1 | 0.349 ± 0.079 [n=1] (0.20 – 0.48) |
5 to 95 % Percentile 0.22 – 0.48 | |
Group 2 | 0.316 ± 0.070 (0.22 – 0.53) [n=1] | |||
Group 3 | 0.300 ± 0.086 [n=4] (0.16 – 0.49) [n=1] | |||
Group 4 | 0.282 ± 0.054** [n=2] (0.19 – 0.41) | |||
Thymus [g/kg b.w.] | Group 1 | 0.580 ± 0.125 [n=2] (0.35 – 0.87) [n=1] | 5 to 95 % Percentile 0.41 – 0.85 | |
Group 2 | 0.513 ± 0.094 [n=1] (0.36 – 0.75) | |||
Group 3 | 0.505 ± 0.133 [n=6] (0.27 – 0.83) | |||
Group 4 | 0.473 ± 0.080** [n=3] (0.34 – 0.68) | |||
females | ||||
Thyr./Parathyr. [g] | Group 1 | 0.0106 ± 0.0024 (0.007 – 0.017) | 5 to 95 % Percentile 0.007 – 0.017 | |
Group 2 | 0.0138 ± 0.0045* (0.007 – 0.027) [n=4] | |||
Group 3 | 0.0112 ± 0.0037 (0.007 – 0.019) [n=1] | |||
Group 4 | 0.0119 ± 0.0033 [n=1] (0.006 – 0.017) | |||
Thyr./Parathyr. [g/kg b.w.] | Group 1 | 0.0341 ± 0.0084 [n=3] (0.029 – 0.049) | 5 to 95 % Percentile 0.023 – 0.056 | |
Group 2 | 0.0446 ± 0.0170 [n=1] (0.027 – 0.055) [n=4] | |||
Group 3 | 0.0361 ± 0.0134 [n=1] (0.023 – 0.051) [n=2] | |||
Group 4 | 0.0398 ± 0.0120 [n=1] (0.025 – 0.052) [n=2] | |||
#1: | Data not audited by QAU | |||
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test) | |||
#2: | Taken from table 17-1 to table 18-4. | |||
Text Table 7-24: Mean length and mean number of estrous cycles.
Parameter F0 | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | |
Pre-mating: Test day 15 (start of treatment) until pairing (test day 85) #1 | |||||
Mean cycle length (days) | 4.50 | 4.72 | 4.52 | 5.05* | |
Number of cycles | 15.0 | 14.3 | 14.8 | 13.3 | |
Pre-mating: Test day 85 (start of pairing) until verification of copulation #1 | |||||
Mean cycle length (days) | #3 | #3 | #3 | 4.00 #2 | |
Number of cycles | #3 | #3 | #3 | 3 | |
#1: | Values taken from table 19-1 'Estrous Cycle Data – Start of Dosing until Mating - Summary - F0 Generation'. | ||||
#2: | Female no. 189 revealed 3 complete estrous cycles during her mating period of 15 test days. No positive mating sign was noted during the mating period and the non-pregnancy status was confirmed by Salewski Staining at laparotomy at the end of the pseudo-gestation period. | ||||
#3: | No rats with complete estrous cycles were noted. | ||||
Text Table 7-25A: Range of mean cycle length.
Parameter #1 | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | |
Pre-mating: Test day 15 (start of treatment) until pairing (test day 85) # | |||||
Range of mean cycle length of the individual females (days) | 4.0 - 6.8 | 4.0 - 6.6 | 3.9 - 6.1 | 4.1 - 6.7 + 7.7 #2 | |
#1: | Values taken from table 19-2 'Estrous Cycle Data – Start of Dosing until Mating - Individual Data - F0 Generation'. | ||||
#2: | No. 187 with a mean cycle length of 7.7 days revealed 2 largely elongated estrous cycles of 16 and 14 test days. However, such elongated estrous cycles were also noted for females of the control group (nos. 39 and 48 with estrous cycles of 19 or 28 test days; mean cycle length 4.0 or 5.4). | ||||
Text Table 7-25B: Comparison of mean cycle length with the Provivo background data.
F0 Females Parameter | Values from this study #2 Mean value per group ± SD (range of the individual values (n = 24)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
Mean cycle length (pre-mating period) [days] | Group 1 | 4.50 ± 0.78 (4.0 – 6.8) [n=1] |
5 to 95 % Percentile
3.8 – 6.4 | |
Group 2 | 4.72 ± 0.86 (4.0 – 6.6) [n=1] | |||
Group 3 | 4.52 ± 0.67 (3.9 – 6.1) | |||
Group 4 | 5.05 ± 0.98* (4.1 – 7.7) [n=2] | |||
#1: | Data not audited by QAU | |||
#2: | Taken from table 19-1 'Estrous Cycle Data – Start of Dosing until Mating - Summary - F0 Generation' and table 19-2 'Estrous Cycle Data – Start of Dosing until Mating – Individual Data - F0 Generation' | |||
Text Table 7-26: Non-pregnant females.
Parameter #1 | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | |
Non-pregnant females | |||||
Non-pregnant female with verified copulation (sperm detection) | no. 28 (no. 3 #2) |
| nos. 137, 140 (nos. 114, 115) |
| |
Non-pregnant female without verified copulation (no sperm detection) |
| no. 95 (no. 72) |
| nos. 177, 189 (nos. 152, 163) | |
#1: | See table 21-2 ‘Reproductive Outcomes - Individual Data - Females - Fertility and Gestation Indices - F0 Generation’. | ||||
#2: | The numbers of the male partners are given in brackets (see table 1 ‘Pairing List’. No changes were noted for the sperm parameters of their male partners (see section 8.12 ‘Examination of the sperm number, viability and morphology). | ||||
Text Table 7-27: Fertility indices per group.
Group / Dose level | Fertility index % #1 | Pregnant females with verified copulation / Females with verified copulation | |
Group 1 (Control) | 96 | 23 / 24 #2 | |
Group 2 (25 mg/kg) | 100 | 23 / 23 #3 | |
Group 3 (80 mg/kg) | 92 | 22 / 24 #4 | |
Group 4 (240/160 mg/kg) | 100 | 21 / 21 #5 | |
#1: | See table 21-1 ‘Reproductive Outcomes and Indices per Group - Females - Fertility and Gestation Indices - F0 Generation’. | ||
#2: | Group 1: One non-pregnant animal with a verified copulation (no. 28) was noted. | ||
#3: | Group 2: One non pregnant female with no verified copulation was noted (no. 95). | ||
#4: | Group 3: Two non-pregnant animals with verified copulation were noted (nos. 137. 140). | ||
#5: | Group 4: Two non-pregnant animals with no verified copulation were noted (nos. 177, 189). Three animals deceased during the pre-mating period, hence only 21 animals were left for pairing. | ||
Text Table 7-28: Gestation indices per group.
Group / Dose level | Gestation index % #1 | Dams with live pups / Pregnant rats | |
Group 1 (Control) | 100 | 22 / 22 #2 | |
Group 2 (25 mg/kg) | 100 | 23 / 23 | |
Group 3 (80 mg/kg) | 100 | 21 / 21 #3 | |
Group 4 (240/160 mg/kg) | 100 | 18 / 18 #4 | |
#1: | See table 21-1 ‘Reproductive Outcomes and Indices per Group - Females - Fertility and Gestation Indices - F0 Generation’. | ||
#2: | Group 1: Female animal no. 40 which died during the gestation period by accident was excluded. | ||
#3: | Group 3: Female animal no. 126 which died during the gestation period by accident was excluded. | ||
#4: | Group 4: Female animal no. 187 was prematurely sacrificed during the gestation period and excluded. | ||
Text Table 7-29A: Comparison of the percentages of post-implantation loss per group with the Provivo background data.
F0 Females Parameter | Values from this study #2 Percent of post-implantation loss per group | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
Post-implantation loss (percent per group) | Group 1 | 6.29 % |
Range of the post-implantation loss from 6 control groups
6.14 – 14.86 % | |
Group 2 | 11.90 % | |||
Group 3 | 9.01 % | |||
Group 4 | 11.45 % | |||
#1: | Data not audited by QAU | |||
#2: | Taken from table 22 'Number of Pups and Indices Overview per Group'. | |||
Text Table 7-29B: Overview of the reproductive data.
Parental females (F0 Generation) | Reproductive data | ||||
Group 1 (Control) | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | ||
Number of implantation sites and pups | |||||
Implantation sites #1 | 15.0 ± 2.7 | 15.1 ± 3.0 | 15.9 ± 2.0 | 16.2 ± 2.5 | |
Pups #1 (born alive and dead) | 14.3 ± 2.7 | 13.9 ± 2.7 | 15.2 ± 1.9 | 14.7 ± 2.7 | |
Pups born alive #1 | 14.2 ± 2.7 | 13.5 ± 2.7 | 14.9 ± 2.1 | 14.6 ± 2.7 | |
Reproductive indices [%] | |||||
Birth index | |||||
per dam (group mean) #1 per group #2 | 94.27 ± 27 94.01 | 91.16 ± 10.31 90.65 | 93.10 ± 8.24 93.02 | 89.46 ± 10.34 89.23 | |
Live birth index | |||||
per dam (group mean) #1 per group #2 | 99.68 ± 1.52 99.68 | 97.25 ± 6.27 97.19 | 97.85 ± 6.03 97.81 | 99.25 ± 2.25 99.25 | |
Post-implantation loss | |||||
per dam (group mean) #1 per group #2 | 6.05 ± 8.28 6.29 | 11.31 ± 11.91 11.90 | 8.83 ± 10.38 9.01 | 11.20 ± 10.65 11.45 | |
Twins (number of shared placentae) | |||||
per dam (group mean) per group | 1.0 ± 0.0 3 | 1.7 ± 1.2 5 | 2.5 ± 1.3 10 | 2.0 ± 1.0 6 | |
Resorptions and stillbirths | |||||
Sum of resorptions and stillbirths (difference between number of implantation sites and stillbirths) | |||||
per dam (group mean) per group | 1.0 ± 1.3 21 | 1.8 ± 2.0 42 | 1.5 ± 1.8 31 | 1.9 ± 1.8 34 | |
Number of stillbirths | 1 | 9 | 7 | 2 | |
Number of resorptions | 20 | 33 | 24 | 32 | |
#1: | Statistical calculation was performed by ANOVA / DUNNETT (*/**: p ≤ 0.05 / p ≤ 0.01). | ||||
#2: | No statistical evaluation was performed for the group values. | ||||
#3: | See table 23-1 ‘Birth Indices and Post-implantation Loss - Values per Dam - Summary’. | ||||
#4: | See table 22 ‘Number of Pups and Indices - Overview per Group’. | ||||
Text Table 7‑30: Viability indices and prematurely deceased pups during the pre-cull period.
Pre-cull period | |||||
Parameter # | Group 1 (Control) | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | |
Prematurely deceased pups between lactation days 0/1 to 4 / Total number of live born pups | 2 / 313 | 6 / 311 | 8 / 313 | 3 / 263 | |
Viability index (group values) [%] | 99.36 | 98.07 | 97.44 | 98.86 | |
#: | See table 22 ‘Number of Pups and Indices - Overview per Group’. | ||||
Text Table 7‑31: Dams with prematurely deceased pups during the pre-cull period.
Number of prematurely deceased pups per dam (pre-cull period) # | ||||||||
Group 1 (Control) | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | |||||
dam no. | deceased pups | dam no. | deceased pups | dam no. | deceased pups | dam no. | deceased pups | |
29 | 1 | 77 | 1 | 123 | 1 | 171 | 1 | |
35 | 1 | 79 | 1 | 124 | 1 | 172 | 1 | |
| 93 | 4 | 127 | 1 | 192 | 1 | ||
| 134 | 1 |
| |||||
135 | 3 | |||||||
144 | 1 | |||||||
#: | See table 24-2 ‘Dead Pups per Dam and Viability Index - Individual Data - F1 Pups’. | |||||||
Text Table 7‑32: Viability indices and prematurely deceased pups during the post-cull period.
Post-cull period | |||||
Parameter #1 | Group 1 (Control) | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | |
Prematurely deceased pups between lactation days 5 and 21 / Number of pups alive on lactation day 4 after culling | 0 / 217 | 0 / 226 | 0 / 210 (1 / 210) #2 | 1 / 180 | |
Viability index (group values) [%] | 100.00 | 100.00 | 100.00 (99.52) #2 | 99.44 | |
#1: | See table 22 ‘Number of Pups and Indices - Overview per Group’. | ||||
#2: | Pup 133-13 died on lactation day 20 but due to technical problems, it could not be written as dead to the databank (see Table A ‘Individual Pup Data - Dam no. 133’). | ||||
|
| ||||
Text Table 7‑33: Male to female ratios on lactation days 1 and 4.
Male / Female ratio of the pups # |
| ||||||
Time point | Group 1 (Control) | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg |
| ||
Lactation day 1 | 0.80 | 0.98 | 1.00 | 0.74 |
| ||
Lactation day 4 | 0.81 | 0.98 | 1.02 | 0.73 |
| ||
| #: | See table 22 ‘Number of Pups and Indices - Overview per Group’. | |||||
Text Table 7-34: Changes of pup body weight in comparison to the control group for the male and female pups combined. Not considered to be test item-related.
Pup body weight | Changes in comparison to the control group [%] # | ||||
Group 2 (25 mg/kg) | Group 3 (80 mg/kg) | Group 4 (240/160 mg/kg) | |||
| |||||
Lactation day 1 | males + females combined | - 0.2 | - 2.6 | +1.2 | |
Lactation day 4 | males + females combined | - 0.7 | - 2.4 | - 0.1 | |
Lactation day 7 | males + females combined | - 3.1 | - 3.2 | - 1.8 | |
Lactation day 14 | males + females combined | - 4.1 | - 3.7 | - 4.5 | |
Lactation day 21 | males + females combined | - 3.2 | - 1.5 | - 2.2 | |
#: | Values are taken from table 26-1 'Mean Body Weight of the Pups per Dam - Summary'. | ||||
Text Table 7-35: Changes in litter weight that were not considered to be test item-related.
| Changes in comparison to control [%] # | Reason | ||||
Parameter Sex | Control | Group 2 | Group 3 | Group 4 | ||
|
| |||||
Litter weight (male pups on lactation day 14) | n.c. | 4.6 | 10.7 | - 1.2 | - | |
Litter weight (female pups on lactation day 14) | n.c. | - 12.2* | - 13.3* | - 5.5 | A | |
Litter weight (male and female pups combined on lactation day 14) | n.c. | - 4.4 | - 2.2 | - 3.5 | - | |
Pup body weight (female pups on lactation day 14) | n.c. | - 4.8* | - 4.4 | - 5.1* | - | |
Mean number of female pups per dam on lactation day 14 | 5.4 | 4.9 | 4.9 | 5.3 | - | |
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) | |||||
A: | Not considered to be test item-related as the differences in litter weight were due to small and spontaneous differences in pup body weight and pup number on lactation day 14. | |||||
n.c.: | Not calculable | |||||
-: | Only for clarification. | |||||
#: | Values taken from table 27-1 'Liter Weight per Dam - Summary' | |||||
Text Table 7-36: Ano-genital distance of the pups. Changes in comparison to the control value. Not considered to be test item-related.
Ano-genital Distance # |
| Changes in comparison to control [%] | |||
Sex | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | ||
| |||||
Ocular units (absolute) | Male | -1.5 | -4.8* | -3.6 | |
Ocular units (absolute) | Female | -0.4 | -4.8 | -5.6 | |
Ocular units / g b.w. (relative) | Male | -1,4 | -4.2** | -3.9* | |
Ocular units / g b.w. (relative) | Female | +0.3 | -3.8 | -5.3* | |
*/**:
| Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) Ocular units/g b.w.: Ano-genital distance was normalized to cube root of pup body weight. | ||||
#: | Values taken from table 28-1 ‘Ano-genital Distance of the Pups - Mean Values per Dam - Summary’. | ||||
Text Table 7‑37: Overview of the pups with nipple retention.
Pups with nipples #1 | ||||||||
Group 1 (Control) | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | |||||
Dam no. | Pup no. (number of nipples) #2 | dam no. | Pup no. (number of nipples) | dam no. | Pup no. (number of nipples) | dam no. | Pup no. (number of nipples) | |
25 | 25-03 (1) | 77 | 77-1 (4) | 123 | 123-3 (1) | 169 | 169-4 (1) | |
26 | 26-05 (1) |
| 77-2 (1) | 127 | 127-5 (1) |
| 169-5 (1) | |
29 | 29-06 (1) |
| 77-3 (2) | 130 | 130-1 (1) | 171 | 171-1 (2) | |
32 | 32-06 (1) |
| 77-4 (4) | 132 | 132-7 (1) | 172 | 172-2 (2) | |
34 | 34-01 (1) |
| 77-5 (4) |
| 132-8 (2) |
| 172-4 (3) | |
42 | 42-07 (3) | 79 | 79-2 (1) | 133 | 133-3 (1) |
| 172-5 (1) | |
46 | 46-07 (1) |
| 79-3 (1) | 134 | 134-2 (1) | 173 | 173-7 (1) | |
48 | 48-01 (1) | 81 | 81-2 (1) | 135 | 135-2 (1) |
| 173-9 (1) | |
| 48-02 (1) |
| 81-3 (1) | 141 | 141-3 (1) | 179 | 179-1 (3) | |
|
| 82 | 82-3 (4) | 144 | 144-5 (2) | 183 | 183-3 (1) | |
|
| 84 | 84-7 (1) |
|
|
| 183-5 (1) | |
|
| 96 | 96-3 (2) |
|
| 184 | 184-4 (4) | |
|
|
| 96-4 (1) |
|
| 188 | 188-5 (1) | |
Summary | ||||||||
8 | 9 Pups | 6 | 13 Pups | 9 | 10 Pups | 8 | 13 Pups | |
Number of nipples per pup | ||||||||
1 Nip | 8 Pups | 1 Nip | 7 Pups | 1 Nip | 8 Pups | 1 Nip | 8 pups | |
2 Nip | - | 2 Nip | 2 Pups | 2 Nip | 2 Pups | 2 Nip | 2 Pups | |
3 Nip | 1 Pup | 3 Nip | - | 3 Nip | - | 3 Nip | 2 Pup | |
|
| 4 Nip | 4 Pups | 4 Nip | - | 4 Nip | 1 Pup | |
#1: | See table A ‘Individual Pup Data’ from the listed dams. | |||||||
#2: | The number of nipples that were noted for the listed pup are given in brackets. | |||||||
Text Table 7-38A: Changes of T4 level in comparison to the control group for the male and female pups alone or combined.
T4 level (nmol/L) | Changes in comparison to the control group [%] # | ||||
Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | |||
| |||||
Lactation day (PND) 4 | males | +27.1** | +17.3 | +31.6* | |
females | +15.3 | +12.1 | +20.9* | ||
males + females combined | +18.6* | +16.5 | +26.0** | ||
Lactation day (PND) 21/22 | males | - 4.0 | - 15.4** | - 10.0 | |
females | - 11.5* | - 16.7** | - 9.7 | ||
males + females combined | - 7.4 | - 15.7** | - 9.6* | ||
#: | Values are taken from table 30-1 'Thyroid Levels of the Pups on Lactation Day 4 - Mean Values per Dam - Summary' and table 30-2 ‘Thyroid Levels of the Pups on Lactation Days 21/22 - Mean Values per Dam - Summary’. | ||||
Text Table 7-38B: Comparison of thyroid hormone levels with the Provivo background data.
Parameter | Values from this study #2 Mean value per group ± SD (Range of the individual values) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
T4 Males + Females combined PND 4 (nmol/L) | Group 1 | 20.5283 ± 4.5130 (14.115 – 30.476) [n=2] | 5% to 95% Percentile
11.437 – 26.191 | |
Group 2 | 24.3513 ± 3.6228* (17.623 – 33.460) [n=6] | |||
Group 3 | 23.9188 ± 11.4526 (10.953 – 51.619) [n=4] | |||
Group 4 | 25.8694 ± 5.2237** (18.943 – 37.331) [n=8] | |||
T4 Males + Females combined PND 21/22 (nmol/L) | Group 1 | 52.0715 ± 6.8407 (36.019 – 64.788) [n=4] | 5% to 95% Percentile
29.035 – 59.099 | |
Group 2 | 48.2146 ± 6.4779 (35.380 – 57.225) | |||
Group 3 | 43.8786 ± 6.7427** (32.621 – 55.705) | |||
Group 4 | 47.0810 ± 4.6636* (37.765 – 56.833) | |||
#1: | Data not audited by QAU | |||
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test) | |||
#2: | Taken from tables 30-3 ‘Thyroid Levels of the Pups on Lactation Day 4 – Mean Values per Dam – Individual Data’ and 30-4 'Thyroid Levels of the Pups on Lactation Days 21/22 – Mean Values per Dam – Individual Data'. | |||
Text Table 7-39A: Changes of TSH levels in comparison to the control group for the male and female pups alone or combined.
TSH level (ng/mL) | Changes in comparison to the control group [%] # | ||||
Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 240/160 mg/kg | |||
| |||||
Lactation day (PND) 21/22 | males | - 11.8 | +10.1 | - 27.9 | |
females | - 9.5 | +50.6 | - 2.6 | ||
males + females combined | - 11.2 | +29.9 | - 16.2 | ||
#: | Values are taken from table 30-2 ‘Thyroid Levels of the Pups on Lactation Days 21/22 - Mean Values per Dam - Summary’. | ||||
Text Table 7-39B: Comparison of thyroid hormone levels with the Provivo background data.
Parameter | Values from this study #2 Mean value per group ± SD (Range of the individual values) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
TSH Males + Females combined PND 21/22 (ng/mL) | Group 1 | 0.7054 ± 0.2389 [n=4 #3] (0.406 – 1.255) | 5% to 95% Percentile
0.081 #3 – 2.576
25% to 95% Percentile
0.460 – 2.576 | |
Group 2 | 0.6267 ± 0.3018 [n=8] (0.235 – 1.356) | |||
Group 3 | 0.9162 ± 0.4584 [n=3] (0.245 – 2.120) | |||
Group 4 | 0.5912 ± 0.5466 [n=8] (0.081 – 1.748) | |||
#1: | Data not audited by QAU | |||
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test) | |||
#2: | Taken from tables 30-3 ‘Thyroid Levels of the Pups on Lactation Day 4 – Mean Values per Dam – Individual Data’ and 30-4 'Thyroid Levels of the Pups on Lactation Days 21/22 – Mean Values per Dam – Individual Data'. | |||
#3: | The 5 % percentile is at the lower level of detection (LOD = 0.081). Hence, for the comparison with the study values the 25 % percentile was used. | |||
Text Table 8-2: Observations that were noted for the prematurely deceased animal no. 336 during the daily cage side inspection on the days before death (common observations as salivation are not considered).
Observations during the daily cage side inspections for the prematurely deceased animal no. 336 # | ||||
Gr. | No. | Sex | Symptom | Observed on PND (day of death) |
4 | 336 | M | Piloerection Respiratory rate decreased Breathing sounds Laboured breathing | 50 - 53 52, 53 51 53 (54) |
Text Table 8-3: Observations that were noted during the daily cage-side observations for the male animals of the low, the intermediate and the high dose level.
Observations in group 3 #1 (80 mg IPDA/kg b.w./day) | |||
Observation (males) | Affected animals | First to last seen (postnatal days) #2 | Number of times recorded |
Salivation | 3 of 20 | 42 - 77 | 5 |
Observations in group 4 (160 mg IPDA/kg b.w./day) | |||
Salivation | 11 of 19 #3 | 30 - 77 | 26 |
Breathing sounds | 2 of 19 | 54 -77 (325, 326) | 23 |
Hunched | 1 of 19 | 63 - 74 (326) | 12 |
Piloerection | 1 of 19 | 54 - 74 (326) | 18 |
Laboured breathing | 1 of 19 | 63 - 73 (326) | 11
|
Text Table 8-4: Observations that were noted during the daily cage-side observations for the female animals of the low, the intermediate and the high dose level.
Observations in group 4 #1 (160 mg IPDA/kg b.w./day) | |||
Observation (females) | Affected animals | First to last seen (postnatal days) #2 | Number of times recorded |
Salivation | 15 of 20 | 29 - 76 | 27 |
Breathing sounds | 2 of 20 | 33 - 58 (351, 342) | 5 |
Text Table 8‑7: Differences in body weight in comparison to control on postnatal day 26 and on postnatal day 88 / 89.
Cohort 1A Body Weight
| Changes in comparison to control [%] | ||
Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg | |
Males #1 | |||
PND 26 #3 | - 1.5 | - 2.5 | - 1.3 |
PND 88 #4 | +0.5 | +0.1 | +0.4 |
Females #2 | |||
PND 26 #3 | - 1.6 | - 1.4 | - 4.3 |
PND 89 #4 | - 0.2 | - 3.7 | - 4.5 |
Text Table 8‑8: Body weight gain of the male and female animals from postnatal day 26 to postnatal day 88.
Body weight gain | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg |
Males #1 (postnatal day 26 to 88) | +495.2% | +508.1% | +510.3% | +504.4% |
Females 2 (postnatal day 26 to 89) | +259.6% | +266.4% | +250.7% | +258.4% |
Text Table 8-10A: Statistically significant changes of the haematological parameters that were not considered to be test item-related.
Parameter # (Co1A) | Sex | Changes in comparison to control [%] | Reason | |||
Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg | ||||
|
| |||||
Platelets (PLT) [x103/µl] | Males #1 | +9.5 | +13.7 | +28.3** | A | |
Platelets (PLT) [x103/µl] | Females #2 | - 9.3 | +8.6 | +7.6 | C | |
MCV [fL] | Females #2 | - 0.7 | 0.0 | - 2.6* | B | |
MCV [fL] | Males #1 | +0.5 | +1.5 | - 0.6 | C | |
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) | |||||
#1: | Values taken from table 6-1-Co1A 'Haematological Parameters - Summary - Males'. | |||||
#2: | Values taken from table 6-2-Co1A 'Haematological Parameters - Summary - Females'. | |||||
A: | The moderate increase is considered to be spontaneous, as no statistically significant changes were noted for the female animals and only 2 individual values were above the Provivo background data. | |||||
B: | The decreased MCV value was considered to be spontaneous. Only one individual value of the high dosed females was below the Provivo background range and no changes were noted for the male animals. | |||||
C: | Only for comparison. | |||||
Text Table 8-10B: Comparison of haematological parameters with the Provivo background data.
Parameter Co1A | Values from this study #2 Mean value per group ± SD (range of the individual values (n = 10)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
Platelets (PLT) [x103/µl] (males) | Group 1 | 913.0 ± 119.4 (744 – 1049) |
5 to 95 % Percentile
562 - 1268
| |
Group 2 | 999.6 ± 137.3 (810 – 1166) | |||
Group 3 | 1038.2 ± 108.1 (906 - 1191) | |||
Group 4 | 1171.6 ± 237.2** (876 – 1727) [n=2] | |||
MCV [fL] (females) | Group 1 | 53.21 ± 1.74 [n=1] (50.4 – 55.5) |
5 to 95 % Percentile
50.7 – 55.8
| |
Group 2 | 52.85 ± 0.56 (52.0 – 53.9) | |||
Group 3 | 53.20 ± 1.07 (52.0 – 55.0) | |||
Group 4 | 51.83 ± 1.33* [n=1] (49.9 – 54.3) | |||
#1: | Data not audited by QAU | |||
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / Dunnett test) | |||
#2: | Taken from table 6-3-Co1A 'Haematological Parameters – Individual Data - Males' and table 6-4-Co1A 'Haematological Parameters - Individual Data - Females'. | |||
Text Table 8-11A: Statistically significant changes of the biochemical parameters that were not considered to be test item-related.
Parameter Co1A | Sex | Changes in comparison to control [%] | Reason | |||
Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg | ||||
|
| |||||
Sodium (mmol/L) | Females #1 | - 0.2 | - 1.1* | - 0.7 | A | |
Sodium (mmol/L) | Males #2 | - 0.4 | - 0.7 | - 0.7 | B | |
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) | |||||
#1: | Values taken from table 7-2-Co1A ‘Biochemical Parameters - Summary - Females'. | |||||
#2: | Values taken from table 7-1-Co1A 'Biochemical Parameters - Summary - Males'. | |||||
A: | The decrease at the intermediate dose level is considered to be spontaneous. No dose response-relationship was noted and no decrease was noted for the male animals. | |||||
B: | Only for comparison | |||||
Text Table 8-11B: Comparison of biochemical parameters with the Provivo background data.
F1 Females Parameter | Values from this study #2 Mean value per group ± SD (range of the individual values (n = 10)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | |
Sodium [mmol/L] | Group 1 | 137.3 ± 1.3 [n=1] (135 – 139) |
5 to 95 % Percentile 136 – 141 |
Group 2 | 137.0 ± 0.9* [n=1] (135 – 138) | ||
Group 3 | 135.8 ± 1.0 [n=4] (134 – 137) | ||
Group 4 | 136.3 ± 1.6 [n=2] (134 – 139) | ||
Text Table 8-12A: Statistically significant changes of the thyroid hormone TSH (not considered to be test item-related).
Parameter #1 (Cohort 1A) | Sex | Changes in comparison to control [%] | Reason | |||
Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg | ||||
|
| |||||
TSH [ng/mL] | males | +4.7 | +50.0 | +80.8 | A | |
TSH [ng/mL] | females | - 31.5 | - 66.6 | - 79.5* | B | |
#1: | Values taken from table 10-1-Co1A 'Thyroid Hormone Level Analysis - Summary - Males' and from table 10-2-Co1A ‘Thyroid Hormone Level Analysis - Summary - Females'. | |||||
A: | The increased TSH concentrations that were noted at the intermediate and the high dose group were considered to be spontaneous. No individual value from the treatment groups was above the upper range of the background data and the individual values were highly variable. Finally an opposite trend was noted for the female animals. | |||||
B: | The decreased TSH concentrations were not accompanied by histomorphological changes of the pituitary gland. | |||||
Text Table 8-12B: Comparison of TSH concentration of the male and female animals with the Provivo background data.
Parameter (Co1A)
| Values from this study #2 Mean value per group ± SD (Range of the individual values (n = 10)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | ||
TSH [ng/mL] | Group 1 (males) | 0.8242 ± 0.6144 [n=3 #3] (0.081 – 1.530) |
5% to 95% Percentile
0.081 #3 – 5.699
25% to 95% Percentile
0.142 – 5.699 | |
Group 2 (males) | 0.8627 ± 0.8861 [n=4] (0.081 - 2.589) | |||
Group 3 (males) | 1.2363 ± 1.4994 [n=2] (0.081 – 5.126) | |||
Group 4 (males) | 1.4902 ± 1.2938 [n=1] (0.081 – 4.043) | |||
Group 1 (females) | 1.0719 ± 0.9819 (0.081 – 2.765) [n=2] | 5% to 95% Percentile
0.081 #3 – 2.523
25% to 95% Percentile
0.081 #4 – 2.523 | ||
Group 2 (females) | 0.7345 ± 0.5231 (0.081 – 1.620) | |||
Group 3 (females) | 0.3585 ± 0.4497 (0.081 – 1.128) | |||
Group 4 (females) | 0.2197 ± 0.2818* (0.081 – 0.962) | |||
#1: | Data not audited by QAU | |||
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (Dunnett test) | |||
#2: | See tables 10-1-Co1A and 10-3-Co1A 'Thyroid Hormone Level Analysis - Summary and Individual Data - Males', 10-2-Co1A and 10-4-Co1A 'Thyroid Hormone Level Analysis - Summary and Individual Data - Females'. | |||
#3: | The 5 % percentile is at the lower level of detection (LOD = 0.081). Hence, for the comparison with the study values the 25 % percentile was used. | |||
#4: | Also the 25 % percentile is at the lower level of detection. | |||
Text Table 8-12C: Comparison of T4 concentration of the male and female animals with the Provivo background data.
Parameter (Co1A)
| Values from this study #2 Mean value per group ± SD (Range of the individual values (n = 10)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | |
T4 [nmol/L] | Group 1 (males) | 64.6330 ± 5.9232 (55.943 – 73.694) |
5% to 95% Percentile
42.528 – 76.219 |
Group 2 (males) | 66.1330 ± 18.4558 (32.569 – 93.925) [n=4) | ||
Group 3 (males) | 66.3058 ± 9.4665 (46.540 – 78.571) [n=1] | ||
Group 4 (males) | 64.5040 ± 7.4090 (51.423 – 76.457) [n=1] | ||
Group 1 (females) | 38.1076 ± 12.4532 [n=1] (19.673 – 60.465) [n=1] | 5% to 95% Percentile
21.861 – 59.502 | |
Group 2 (females) | 40.3537 ± 11.5878 (24.899 – 65.859) [n=1] | ||
Group 3 (females) | 31.8250 ± 6.4195 [n=1] (19.395 – 41.300) | ||
Group 4 (females) | 29.8639 ± 5.2173 (25.313 – 42.504) | ||
Text Table 8-13: Time points (postnatal day) of balano preputial separation
Parameter (Co1A and Co1B combined) | Values from this study #2 Mean value per group ± SD (range of the individual values (n= up to 40) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | |
Day balano preputial separation (postnatal day) | Group 1 | 22.6 ± 0.7 (22 – 24) [n=4] |
5 to 95 % Percentile 22.0 – 23.0 |
Group 2 | 22.5 ± 0.6 (22 – 24) [n=2] | ||
Group 3 | 22.2 ± 0.5* (22 – 24) [n=1] | ||
Group 4 | 22.2 ± 0.5** (22 – 24) [n=2] | ||
Text Table 8-14A: Time points (postnatal day) of vaginal opening.
Parameter | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg |
Cohort 1A and 1B combined | ||||
Day of vaginal opening (PND) #1 | 33.1 ± 2.5 | 34.2 ± 2.8 | 33.1 ± 1.9 | 34.6 ± 2.3** |
Day of vaginal opening (PND) (Range of individual animals) #2 | 30 - 43 | 30 - 44 | 30 - 37 | 30 - 43 |
Body weight on the day of vaginal opening (% changes in comparison to control) #1 | - | +4.3 | - 0.7 | +3.9 |
Text Table 8-14B: Day of vaginal opening - comparison with the Provivo background data.
Parameter (Co1A and Co1B combined) | Values from this study #2 Mean value per group ± SD (range of the individual values (n= up to 40) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | |
Day of vaginal opening (postnatal day) | Group 1 | 33.1 ± 2.5 (30 – 43) [n=1] |
5 to 95 % Percentile 30 – 37 |
Group 2 | 34.2 ± 2.8 (30 – 44) [n=2] | ||
Group 3 | 33.1 ± 1.9 (30 – 37) | ||
Group 4 | 34.6 ± 2.3** (30 – 43) [n=1] | ||
Text Table 8-15: Sexual maturation of female animals.
Parameters # (Co1A) | Parameters of sexual maturation Mean values per group | ||||
Group 1 (Control) | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg | ||
- | day of vaginal opening (PND) (Co1A only) | 33.0 ± 3.0 | 34.5 ± 3.4 | 33.1 ± 1.7 | 34.9 ± 2.7* |
- | day of appearance of cornified cells (PND) | 34.9 ± 3.5 | 36.3 ± 4.0 | 34.8 ± 3.1 | 35.6 ± 3.7 |
- | period between day of vaginal opening and day of appearance of cornified cells (test days) | 1.9 ± 1.7 | 1.8 ± 2.0 | 1.7 ± 2.3 | 0.7 ± 1.3 |
Text Table 8-16: Range of mean cycle length.
Parameter (Cohort 1A) | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg |
From test day 49 until test day 62 | ||||
Number of cycles #1 | 1.9 ± 1.0 | 1.7 ± 0.8 | 2.0 ± 0.6 | 1.9 ± 0.6 |
Mean cycle length per group #1 | 4.12 ± 0.33 | 4.29 ± 0.56 | 4.08 ± 0.30 | 4.50 ± 0.58* |
Range of mean cycle length of the individual females (days) #2 | 4.0 - 5.0 | 4.0 - 6.0 | 3.5 - 5.0 | 4.0 - 6.0 |
Number of females with no complete estrous cycle #2 | 3 (nos. 229, 231, 236) | 3 (nos. 264, 270, 271) | 1 (no. 303) | 1 (no. 360) |
Text Table 8-17: Macroscopic findings noted for the females of the control group from Cohort 1A.
Sex | Gr. | No. | Macroscopic findings # | |
Affected Organ | Finding | |||
F | 1 | 223 | Uterus | Dilated and filled with clear liquid. |
230 | Uterus | Dilated and filled with clear liquid. | ||
235 | Thymus | Reddened left lobe. | ||
237 | Lymph node | Enlarged mesenteric lymph node | ||
Spleen | Enlarged | |||
Intestines | Thickened mucosa | |||
Text Table 8-18: Stage of the estrous cycle at necropsy. The stage of the estrous cycle was evaluated from vaginal lavages that were taken at necropsy and during the microscopic examination of the vagina (only group 1 and 4).
Stage of estrus at necropsy (Cohort 1A females) | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg #3 | Group 4 160 mg/kg | |||
N #1 | H #2 | N | N | N | H | ||
Proestrus | - | 5 of 20 | 3 of 20 | - | - | 5 of 20 | |
Estrus | 6 of 20 | 6 of 20 | 3 of 20 | 4 of 20 | 4 of 20 | 5 of 20 | |
Metestrus | 5 of 20 | 1 of 20 | 8 of 20 | 5 of 20 | 9 of 20 | 3 of 20 | |
Diestrus | 9 of 20 | 8 of 20 | 6 of 20 | 10 of 20 | 7 of 20 | 7 of 20 | |
- : | not detected | ||||||
| N = Necropsy; H = Histopathological examination of the vagina | ||||||
Text Table 8-19A: Changes in organ weights of the male animals of Cohort 1A, unrelated to the test item.
Parameter Male (Co1A) | Cohort | Changes in comparison to control [%] | Reason | ||
Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg | |||
|
| ||||
Prostate #1 (relative) | Cohort 1A | - 0.5 | - 9.2 | - 13.9* | A |
Prostate #2 (relative) | Cohort 1B | - 9.4 | - 9.6 | - 5.5 | B |
Prostate #5 (relative) | Cohort 1A + 1B | - 5.2 | - 9.5* | - 9.2* | B |
Prostate #3 (absolute) | Cohort 1A | - 4.4 | - 8.9 | - 8.4 | A |
Prostate #4 (absolute) | Cohort 1B | - 7.1 | - 9.1 | - 4.3 | B |
Prostate #6 (absolute) | Cohort 1A + 1B | - 4.4 | - 8.9 | - 8.4 | B |
Epididymis, r. #5 (relative) | Cohort 1A + 1B | - 7.7* | - 4.5 | - 4.1 | C |
Epididymis, r. #6 (absolute) | Cohort 1A + 1B | - 6.5 | - 4.3 | - 3.0 | C |
Text Table 8-19B: Comparison of organ weights with the Provivo background data.
Parameter Males (Co1A) | Values from this study #2 Mean value per group ± SD (range of the individual values (n= up to 20)) [number of individual values below or above the stated range] | Provivo Background Data #1 obtained from the control groups of 6 OECD 443 studies performed at Provivo from 2019 – 2021 | |
Prostate [g/kg b.w.] | Group 1 | 2.7998 ± 0.4325 [n=2] (1.851 – 3.472) |
5 to 95 % Percentile 2.337 – 4.294 |
Group 2 | 2.7851 ± 0.5185 [n=5] (2.230 – 4.173) | ||
Group 3 | 2.5428 ± 0.3962 [n=6] (1.901 – 3.159) | ||
Group 4 | 2.4107 ± 0.4434* [n=11] (1.814 – 3.659) | ||
Text Table 9-2: Observations that were noted for the prematurely deceased animal no. 443 during the daily cage side inspection on the days before death (common observations as salivation are not considered).
Observations during the daily cage side inspections for the prematurely deceased animal no. 443 # | |||||
Gr. | No. | Sex | Symptom | Observed on postnatal day (day of death) | |
3 | 443 | M | Piloerection Pale, whole body Pale ears Exopthalmus, eyes | 75, 76; 78 - 83 75 - 83 61 61 (day of death: 84) | |
| Gr.: Group; M; F: Male; Female | ||||
Text Table 9-3: Observations that were noted during the daily cage-side observations for the male animals of the low, the intermediate and the high dose level.
Observations in group 2 (25 mg IPDA/kg b.w./day) | ||||
Observation (males) #1 (Cohort 1B) | Affected animals | First to last seen (postnatal days) #2 | Number of times recorded | |
Salivation | 1 of 20 | 63 (406) | 1 | |
Observations in group 3 (80 mg IPDA/kg b.w./day) #3 | ||||
Salivation | 1 of 19 | 45 (452) | 1 | |
Piloerection | 1 of 19 | 100 (456) | 1 | |
Haemorrhagic urine | 1 of 19 | 100 (460) | 1 | |
Observations in group 4 (160 mg IPDA/kg b.w./day) | ||||
Salivation | 16 of 20 | 28 - 100 | 38 | |
Breathing sounds | 2 of 20 | 48 - 75 (492, 493) | 2 | |
Haemorrhagic urine | 1 of 20 | 96 (488) | 1 | |
#1: | Taken from table 2-1A-Co1B ' Clinical Signs - Summary - Males'. | |||
#2: | If only 1 or 2 animals were affected, the animals are listed in brackets. | |||
#3: | Group 3: The prematurely deceased male no. 443 was excluded from the summary. | |||
Text Table 9-4: Observations that were noted during the daily cage-side observations for the female animals of the low, the intermediate and the high dose level.
Observations in group 3 (80 mg IPDA/kg b.w./day) | |||
Observation (females) #1 (Cohort 1B) | Affected animals | First to last seen (postnatal days) #2 | Number of times recorded |
Salivation | 2 of 20 | 45 - 76 (463, 466) | 2 |
Observations in group 4 (160 mg IPDA/kg b.w./day) | |||
Salivation | 20 of 20 | 26 - 99 | 62 |
Breathing sounds | 1 of 20 | 46 - 70 (502) | 16 |
Piloerection | 1 of 20 | 28 (506) | 1 |
Text Table 9‑6: Start and duration of observations that were noted for the female animals.
Start and duration of symptoms - Females | |||
Symptom (Cohort 1B) # | Time frames in relation to application | Frequency observed (in all groups) | |
Appearing of the symptom | Disappearing of the symptom | ||
Salivation | 0 - 5 min | 5 - 20 min | 17 |
| 20 - 60 min | 47 | |
Breathing sounds | 0 - 5 min | 6 - 24 h | 16 |
Text Table 9‑7A: Differences in body weight in comparison to control on postnatal day 26 and on postnatal day 98.
Cohort 1B Body Weight
| Changes in comparison to control [%] | |||
Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg | ||
Males #1 | ||||
PND 26 #2 | - 1.9 | +1.0 | - 0.5 | |
PND 98 #3 | +1.5 | +0.7 | +0.9 | |
#1: | See table 4-1-Co1B ‘Body Weight - Summary - Males’. | |||
#2: | At start of the F1 Study, the transferred pups were between 22 and 26 days old. Hence, the first day of age, for which a body weight was available for all transferred pups was postnatal day 26. | |||
#3: | Before removal of the male animals of cohort 1A. | |||
Text Table 9‑7B: Body weight gain of the male animals of Cohort 1B from postnatal day 26 to postnatal day 98.
Body weight gain | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg |
Males (postnatal day 26 to 98) | +543.5% | +564.3% | +543.1% | +552.8% |
Text Table 9-8A: Differences in body weight in comparison to control between postnatal days 26 and 98.
Cohort 1B Body Weight
| Changes in comparison to control [%] | ||
Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg | |
Females #1 | |||
PND 26 #2 | - 4.1 | - 2.3 | - 4.5 |
PND 40 | - 3.6 | - 5.9** | - 8.2** |
PND 47 | - 4.6* | - 8.2** | - 8.4** |
PND 54 | - 5.4* | - 6.0* | - 7.2** |
PND 61 | - 5.4* | - 5.9* | - 6.8** |
PND 68 | - 5.2 | - 6.2* | - 6.4* |
PND 96 | - 4.4 | - 5.8* | - 5.3 |
PND 98 | - 3.2 | - 5.0 | - 5.3 |
Text Table 9‑8B: Body weight gain of the female animals of Cohort 1B from postnatal day 26 to postnatal day 98.
Body weight gain | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg |
Females (postnatal day 26 to 98) | +285.2% | +289.0% | +274.5% | +282.3% |
Text Table 9‑9: Statistically significant differences in food consumption that were considered to be spontaneous.
Parameter (male) | Ref. Table no. | Difference to control [%] | Group / sex | Test days | Statistical significance | Reason |
Relative food consumption | 6-1-Co1B | - 5.0 | 4 m | 57 - 64 | p £ 0.05 | A |
A: The reduction was only slight and temporary
Text Table 9-10: Time points (postnatal day) of balano-preputial separation.
Parameter | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg | |
Cohort 1A | |||||
Day of balano-preputial separation (PND) #1 | 22.6 ± 0.7 | 22.5 ± 0.7 | 22.2 ± 0.4 | 22.3 ± 0.7 | |
Cohort 1B | |||||
Day of balano-preputial separation (PND) #2 | 22.6 ± 0.7 | 22.4 ± 0.5 | 22.3 ± 0.6 | 22.1 ± 0.2* | |
Cohort 1A and 1B combined | |||||
Day of balano-preputial separation (PND) #3 | 22.6 ± 0.7 | 22.5 ± 0.6 | 22.2 ± 0.5* | 22.2 ± 0.5** | |
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (DUNNETT test) | ||||
Text Table 9-11: Stage of the estrous cycle at necropsy. The stage of the estrous cycle was evaluated from vaginal lavages that were taken at necropsy.
Stage of estrus at necropsy (Cohort 1B females) # | Group 1 Control | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg | |
Proestrus | - | 2 of 20 | 1 of 20 | 2 of 20 | |
Estrus | 7 of 20 | 9 of 20 | 4 of 20 | 8 of 20 | |
Metestrus | 2 of 20 | 3 of 20 | 6 of 20 | 3 of 20 | |
Diestrus | 11 of 20 | 6 of 20 | 9 of 20 | 7 of 20 | |
- : | not detected | ||||
Text Table 9-12: Statistically significant changes in organ weights unrelated to the test item for the male animals of Cohort 1B (the values of Cohort 1A are given for comparison).
Parameter Cohort 1B (males) | Cohort | Changes in comparison to control [%] | Reason | |||
Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg | ||||
|
| |||||
Pituitary #1 (relative) | Cohort 1B | +5.6 | - 2.4 | +21.9** | A | |
Cohort 1A | - 11.1 | - 4.1 | - 4.2 | B | ||
Pituitary #2 (absolute) | Cohort 1B | +8.0 | - 1.3 | +22.6* | A | |
Cohort 1A | - 1.5 | - 2.3 | +9.4 | B | ||
*/**: | Statistically significant from control at p ≤ 0.05 (ANOVA / DUNNETT test) | |||||
#1: | Values taken from table 11-1-Co1B 'Relative Organ Weights - Summary - Males'. | |||||
#2: | Values taken from table 12-1-Co1B 'Absolute Organ Weights - Summary - Males' | |||||
A: | The increased weights that were noted at the low dose level and the high dose level for the weight of the pituitary gland were considered to be spontaneous, as no dose response relationship and no such changes were noted in Cohort 1A. | |||||
B: | Only for comparison. | |||||
Text Table 9-13: Changes in the relative and absolute weight of the right adrenal gland and the relative and absolute weight of the left ovary (not considered to be test item-related). The values form the left adrenal gland, the right ovary and the respective values from Cohort 1A are given for comparison.
Cohort 1B (females) | Cohort | Changes in comparison to control [%] | Reason | |||
Parameter | Group 2 25 mg/kg | Group 3 80 mg/kg | Group 4 160 mg/kg | |||
|
| |||||
Adrenal gland; left (relative) #1 | Cohort 1B | - 5.0 | - 0.2 | - 5.8 | C | |
Cohort 1A | - 1.4 | - 5.7 | - 0.7 | C | ||
Adrenal gland, right (relative) #1 | Cohort 1B | - 12.4 | - 1.9 | - 11.5 | A | |
Cohort 1A | - 0.4 | - 2.9 | - 2.2 | C | ||
Adrenal gland; left (absolute) #2 | Cohort 1B | - 6.8 | - 3.4 | - 9.7 | C | |
Cohort 1A | - 0.7 | - 8.0 | - 3.5 | C | ||
Adrenal gland; right (absolute) #2 | Cohort 1B | - 14.5* | - 5.0 | - 15.4** | A | |
Cohort 1A | - 0.3 | - 5.6 | - 5.3 | C | ||
Ovary; left (relative) #1 | Cohort 1B | +16.6* | +20.1** | +20.8* | B | |
Cohort 1A | - 0.4 | +5.9 | +10.6 | C | ||
Ovary; right (relative) #1 | Cohort 1B | +5.5 | +14.8 | +14.3 | C | |
Cohort 1A | +2.5 | - 2.0 | +8.7 | C | ||
Ovary; left (absolute) #2 | Cohort 1B | +13.8 | +15.2 | +15.0 | B | |
Cohort 1A | +1.4 | +3.6 | +8.2 | C | ||
Ovary; right (absolute) #2 | Cohort 1B | +2.7 | +9.6 | +8.7 | C | |
Cohort 1A | +4.2 | - 4.2 | +5.8 | C | ||
*/**: | Statistically significant from control at p ≤ 0.05/0.01 (ANOVA / DUNNETT test) | |||||
#1: | Values taken from table 18-Co1A 'Relative Organ Weights – Cohort 1A' and table 11-Co1B 'Relative Organ Weights – Cohort 1B' | |||||
#2: | Values taken from table 19-Co1A 'Absolute Organ Weights – Cohort 1A' and table 12-Co1B 'Absolute Organ Weights – Cohort 1B' | |||||
A: | The reduced absolute and relative organ weights of the right adrenal gland were considered to be spontaneous. No statistically significant changes were noted for the left adrenal gland from the females of Cohort 1B and the left and right adrenal gland from the females of Cohort 1A. | |||||
B: | The increased absolute and relative organ weights of the left ovary were considered to be spontaneous. No statistically significant changes were noted for the right ovary from the animals of Cohort 1B and the left and right ovaries from the females of Cohort 1A. In addition, the histopathological examination of the reproductive organs from the high dosed females of Cohort 1A revealed no relevant toxicological changes. | |||||
C: | For comparison only. | |||||
Text Table 10-1: Test item formulation analysis.
Parameter | Sampling / Dealing | Percent of nominal concentration [%] | |
Concentration | F0 Generation: At the end of the pre-mating period, before administration to the last animals (test day 84 of the F0 Study) | 105.7 % - 107.8 % | |
F0 Generation: At termination of the F0 Generation, before administration of the last animals (test day 128 of the F0 Study). | 104.7 % - 108.4 % | ||
F1 Generation: At termination of the F1 Generation, before administration of the last animal (test day 66 of the F1 Study | 101.6 % - 103.1 % | ||
Homogeneity | F0 Generation (at start of dosing on test day 15): At start of administration, during administration and before administration to the last animal per group. | 102.0 % - 105.7 % | |
F0 Generation (at dose reduction of the high dose level on test day 51): At start of administration, during administration and before administration to the last animal per group. | 103.4 % - 104.7 % | ||
F1 Generation (test day 1 of the F1 Study): At start of administration, during administration and before administration to the last animal per group. | 106.0 % - 109.6 % | ||
| Taken from table 1-F0/F1 ‘Test Item Formulation Analysis'. | ||
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 160 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- Klimisch 1
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Studies in animals from public sources
Partly cited from SIAR for SIAM 18 (Paris, April 2004): "No studies have been performed to explicitly address the question of reproductive effects in animals caused by 3-aminomethyl-3,5,5-trimethylcyclohexylamine. Histopathological results of a subchronic 90-day investigation on rats according to OECD TG 408 showed no effects regarding the reproductive organs (epididymides, mammary gland, ovaries, seminal vesicles, testes and uterus) in concentrations up to 160 mg/kg bw/day. Testes weights were also not affected (RCC Research and Consulting Company, 1986)."
The toxicological information regarding effects on Developmental toxicity (no abortion or total resorption, no treatment releated effects on the pre- or post-implantation loss, no treatment related effects on sex-ratio and on the fetal weight; CIT, 2002; see section 7.8.3 of IUCLID) and the fact that Isophorone diamine do not cause adverse effects on the examined reproductive organs in the 90 day subchronic study (RCC, 1986; see section 7.5 of IUCLID) leading to the conclusion that that effects on fertility of the substance Isophorone diamine at doses, which do not cause parental toxicity, are rather unlikely.
EOGRTS study according to OECD 443
However, in the Final decision on a compliance check (from 5thof April 2017) ECHA requested to perform the OECD 443 study in rats, oral route with the registered substance specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation.
Before the start of the EOGRTS study according to OECD 443, two dose range finding studies were performed to determine suitable dose levels (OECD 421, LPT, 2019 and OECD 422, Provivo, 2021).
The aim of the studies was to obtain preliminary information on possible effects of the test item on reproduction and/or development.
OECD 421 study
In the first DRF study according to OECD guideline 421, the test item was administered orally to rats via the drinking water at dose levels of 20, 60 or 160/120 mg test item /kg b.w./day (LPT, 2019). No premature death and no changes in behaviour, the external appearance and the appearance of the faeces were noted.
Also, the laboratory examinations and the examinations at necropsy (examination of haematological and biochemical parameters, determination of the T4 level of the male animals, determination of organ weights, the macroscopical and histopathological examination) revealed no test item-related changes.
However, most probably due to the properties of the test item in the drinking water a dose dependent refusal of intake of the test item-drinking water mixtures was noted for the male and female animals. This was most pronounced for the male animals of the high dose group (160/120 mg test item /kg b.w./day) during the first and the second treatment week and caused a reduction in body weight for the male animals. The body weight of the high dosed males recovered as the consumption of drinking water increased after the reduction of the test item concentration in the drinking water (dose level reduction from 160 to 120 mg test item /kg b.w./day).
However, for the male animals of the high dose group (160/120 mg test item/kg b.w./day), the marked reduction in drinking water consumption before the reduction of the dose level revealed a decreased intake of test item via the drinking water. Hence, the calculated actual dose level was below the nominal dose level during the first and second treatment week. After the dose level reduction, the actual test item-intake was in the range of the nominal dose level.
For the female animals increased actual dose levels were noted during the lactation period for all dose groups (20, 60 or 160/120 mg test item /kg b.w./day). This was due to increased levels of absolute and relative drinking water consumption during the lactation period in comparison to the pre-mating and gestation period that were noted for all dose groups.
In the parental no influence was noted on the estrus cycles, the fertility, the gestation index, the pre-coital time and the gestation length.
A reduced viability index was noted during the post-cull period for the pups of the high dose group (160/120 mg test item/kg b.w./day). This was considered as a secondary effect of the reduced consumption of drinking water of the high dosed females in comparison to the females of the control and the low and the intermediate dose group. This lower drinking water consumption led to a reduced milk production and hence, an inadequate feeding of some of the weaker pups. Therefore, the reduced viability index was not considered to be of toxicological relevance. No other effects could be observed.
Unfortunately, it was not possible to determine adequate dose levels for the OECD 443 study. The animals refused to consummate the drinking water because of the bad smell of the test substance). As a consequence, the reproductive screening study was performed with another form of administration.
It was chosen to perform an OECD 422 study with rats and gavage as administration route at the dose levels of 0, 30, 100 and 300 (240) mg/kg bw/d.
OECD 422 study
The test item was administered orally to rats at dose levels of 30, 100 or 300 mg test item/kg b.w./day. Due to mortality, the high dose level was decreased to 240 mg test item /kg b.w./day, starting on test day 28.
A marked body weight loss was noted at 300/240 mg test item/kg b.w./day for the prematurely deceased or sacrificed male and female animals.
The surviving female animals showed a slightly reduced body weight in comparison to control during the gestation and the lactation period. This and the observed general bad condition of the females are signs of maternal toxicity. No differences in body weight were noted for the surviving high dosed male animals.
Test item-related changes for the surviving animals from both sexes were noted during the histopathological examinations of the stomachs from the male and female animals of the high dose group (300/240 mg test item/kg b.w./day) in form of an erosion of the stomach wall and an inflammatory response. These stomach changes were considered as local and secondary effects that were caused by the corrosive properties of the test item.
For the female animals at 300/240 mg test item/kg b.w./day the manifesting in the very severe erosions/ulcer in the stomach was accompanied by an increased number of neutrophilic granulocytes and an increased LDH activity. However, these observations were not noted in eighter sex for the treatment doses 30 or 100 mg test item/kg b.w./day.
The macroscopic examination at necropsy revealed kidney changes for 2 of the 8 surviving male animals of the high dose group (300/240 mg test item/kg b.w./day) that could possibly be test item-related.
No test item-related influence or adverse effects were noted for the male and female animals on food consumption, on the parameters of the neurological screening, for the organ weights and on the T4 serum levels of the male animals.
OECD 443 study
The OECD 443 did not reveal changes in fertility. However, a test item-related effect was noted at 240/160 mg test item/kg b.w./day for the male and female animals in the form of 3 test item-related premature deaths from in total 48 high dose animals. Further, breathing sounds and piloerection were noted at the high dose level. The mortality rate of the high dose correlates with the observations made in the OECD 422 study.
For the F0 generation no influence was noted on body weight, food consumption, the laboratory examinations, during necropsy and the histopathological examination.
One test item-related death was noted in the adult F1 animals at the high dose of 160 mg test item/kg b.w./day. The animals of the F1 Generation showed no further signs of general toxicity (body weight, food consumption thyroid hormone levels). Breathing sounds and piloerection were only noted for a few animals of the F1 Generation at 160 mg test item/kg/d.
In the previous study (an OECD 422 study in Provivo, 2021) in which the treatment in the high-dose group was started at a dose of 300 mg/kg b.w./day (thereafter it was reduced to 240 mg/kg during the treatment period), the treatment-related microscopic changes were observed in the stomach and kidneys. However, the histopathological examination of the OECD 443 study revealed that the test item did not produce treatment-related microscopic changes not only in the stomach and kidneys but also in any other organs including male and female reproductive system of the high dose animals that were treated at 240/160 mg/kg b.w./day in F0 generation and with 160 mg/kg b.w./day in F1 Generation Cohort 1A.
Effects on developmental toxicity
Description of key information
OECD 414 study, rats
Partly cited from SIAR for SIAM 18 (Paris, April 2004): "3-Aminomethyl-3,5,5-trimethylcyclohexylamine did not show any teratogenic or embryofetotoxic effects in the gavage study with rats performed in accordance with OECD TG 414 (2001) up to and including the highest tested dose level of 250 mg/kg bw/day." The NOEL for maternal toxicity was 50 mg/kg bw/day, effects at 250 mg/kg bw/day were reduced food consumption and reduced body weight gain. The NOAEL for developmental toxicity is >=250 mg/kg bw/day (CIT, 2002).
OECD 414 study, rabbits
To determine suitable dose levels for the OECD 414 study with the second species, a dose range finding study was performed. In this prenatal developmental toxicity study, the test item was administered orally to female rabbits at dose levels of 5, 25 or 50 mg/kg b.w./day from the 6th to 28th day of pregnancy. Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 50 mg test item/kg b.w./day for the dams as no effects could be observed during the study (LPT, 2018).
In a supporting 14 days tolerance study with rabbits the test item was administered orally to two groups with 3 female rabbits once daily at dose levels of 75 mg/kg bw test item and 150 mg/kg bw. test item. Group 1 dosing was extended to 21 test day, group 2 dosing was terminated on test day 7 because of humane sacrifice. A reddened gastric mucosa was seen all dosed animals and a pale liver was noted in 2 of 3 animals of the high dose group. Furthermore, a reduction in food consumption was noted for the low dose animals. No or only minimal food intake was noted for the high dose animals from start of dosing onwards until premature sacrifice on test day 8 due to animal welfare reasons (LPT, 2018).
In the following main OECD 414 study with the second species rabbit, the test substance was administered orally to female rabbits at dose levels of 10, 25 or 75 mg/kg b.w./day from the 6th to 28th day of pregnancy. Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 25 mg test item /kg b.w./day for the dams. The no-observed-adverse effect level (NOAEL) was above 75 mg test item/kg b.w./day for the fetuses and 25 mg test item /kg b.w./day for the rate of early resorptions/post implantation loss (LPT, 2020).
OECD 422 study
In the OECD 422 study (Provivo, 2021) an effect was noted on the prenatal development in the form of an increased number of stillbirths at 300/240 mg test item/kg b.w./day. However, compared to the OECD 443 study of the test item (Provivo, 2021) the increased number of stillbirths in the high dose group can be regarded to be within the range of biological variability as also 9 stillbirths were noted in the low dose group of the OEDC 443 study (Provivo, 2021). No dose response-relationship was observed.
Additionally, during the postnatal development a reduced pup body weight was noted at 300/240 mg test item/kg b.w./day, which was a secondary effect due to maternal toxicity.
No influence was noted on the viability of the pups, the ano-genital distance, the number of nipples per male pup and on the T4 concentration. No changes were noted for the thyroid glands from the pups during the microscopic examination.
No external gross abnormalities (malformations or variations) were noted for any of the pups.
Therefore, the NOAEL for pre- and postnatal development was above 300/240 mg test item/kg b.w./day.
OECD 443 study
In the OECD 443 study no adverse effects in the prenatal development of the pups (number of resorptions, stillbirths and live born pups) and the postnatal development of the pups (pup body weight, viability index, ano-genital distance, nipple retention, thyroid hormone levels, pup organ weights) were detected after the treatment with the test item. No malformations or variations were noted during the macroscopic external and internal examinations of the pups at necropsy
No test item-related influence was noted on the development of the reproductive system (time points of sexual maturations, number and length of estrous cycles, sperm parameter, detailed histopathological examination of testis and epididymides, number of primordial and growing follicles and number of corpora lutea in the ovaries).
Summarising, the NOAEL for pre- and postnatal development was above 160 mg test item/kg b.w./day.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2018-04-12 to 2018-06-19
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- This OECD 414 study is used as dose range finding study for the OECD 414 main study with rabbits (LPT, 2020).
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- adopted January 22, 2001
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.31 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- May 30, 2008
- GLP compliance:
- no
- Remarks:
- The study was performed based on Good Laboratory Practice' Regulations of the EC enacted in Germany in the 'Chemikaliengesetz' [Chemicals Act], current edition; and -OECD Principles of Good Laboratory Practice' Document Nos. 1, 8 and 13
- Limit test:
- no
- Specific details on test material used for the study:
- The test item was diluted in the vehicle to the appropriate concentration.
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or test system and environmental conditions:
- TEST ORGANISMS:
- Species: Rabbit
- Source: Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
- Strain: New Zealand White
- Age: 5 months
- body weight: 3.55 - 4.30 kg
- Diet: ad libitum, Commercial ssniff K-Z V2323 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany,
- Water: ad libitum
- Adaptation period: 20 days
-Housing: Except during the mating period, the dams were kept separately in breeding cages with wire floors
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 °C +/- 3° C
- Humidity (%): 55% +/- 15 %
- Illumination: 12 hours artifical fluorescent light and 12 hours dark
- Ventilation rate: between fifteen to twenty air changes per hour. - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- Purified water (aqua ad injectabilia)
- Details on exposure:
- ADMINISTRATION:
- Frequency: once daily, day 6 to 28 of gestation
- Dose volume: 2 ml/kg b.w.
- Dose: 0, 5, 25, 50 mg/kg/bw
- Animals: 16 females in groups 1 to 4
Test item preparation:
The test item formulations were freshly prepared every day.
The test item was diluted in the vehicle to the appropriate concentration and was administered orally at a constant volume (2 mL/kg b.w.) once daily from the 6th to the 28th day of gestation.
The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume daily in the same way. - Analytical verification of doses or concentrations:
- no
- Details on analytical verification of doses or concentrations:
- For each test or reference item that is mixed with a vehicle, test by appropriate analytical methods will be carried out for the main study to determine concentration, homogeneity and, if needed, stability of the test item in the formulations.
- Details on mating procedure:
- Sexually mature ('proved') male rabbits of the same breed served as partners. The female breeding partners were randomly chosen.
Mating was monogamous; 1 male and 1 female animal were placed in one cage in the forenoon. Successful copulation was ascertained by observation.The day of copulation was considered as day 0 of pregnancy. Only animals with positive signs of copulation were included in the study. - Duration of treatment / exposure:
- 23 administration days from gestation day 6 to 28
- Frequency of treatment:
- Once daily
- Duration of test:
- Laparotomy on gestation day 29
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Vehicle control
- Dose / conc.:
- 5 mg/kg bw/day (actual dose received)
- Remarks:
- Low dose
- Dose / conc.:
- 25 mg/kg bw/day (actual dose received)
- Remarks:
- Intermediate dose
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Remarks:
- High dose
- No. of animals per sex per dose:
- 4 female rabbits/dose
Evaluated litters: at least 12 pregnant females, 3 litters per dose - Control animals:
- yes, concurrent vehicle
- Details on study design:
- Principle
Pregnant rabbits were treated with the test item starting from the 6th and lasting until the 28th day of pregnancy (the 'critical' phase of organogenesis and the fetal development). The development of the pregnant rabbits was observed during the gestation period (day of mating is day 0 of pregnancy). One day before the calculated date of birth (29 days after mating) the dams were laparotomised and examined for corpora lutea, implantation sites, resorptions in the uterus and for the condition of the fetuses.
Dose selection rationale:
The dose levels were selected in agreement with the Sponsor based on available toxicological data. - Maternal examinations:
- Dated and signed records of all activities relating to the day to day running and maintenance of the study within the animal units, as well as to the group observations and examinations outlined in the Study Plan, were recorded in the appropriate documentation. In addition, observations relating to the individual animals made throughout the study were recorded.
The following observations were made during the course of the study:
Clinical signs
Individual animals were observed daily for any signs of behavioural changes, reaction to treatment, or illness.
Immediately after administration, any signs of illness or reaction to treatment were rec-orded. In case of changes, the animals were observed until thesymptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately
3.30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
Viability
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Fetuses obtained this way were examined for abnormal development, whenever possible. No abortion occurred in the study.
No abortion or premature delivery occurred during the study.
Body weight
The weight of each rabbit was recorded on the day of delivery (used for randomization), followed by daily weighings starting on gestation day 6 - always at the same time of the day. These measurements were also used for calculating the daily amount of test item to be administered.
The body weight gain was calculated in intervals (i.e. gestation day 6-9, 9 12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29) and for the period after the start of treatment until necropsy (gestation day 6-29).
Furthermore the carcass weight and the net weight change from day 6 are given.
Carcass weight: Carcass weight = Terminal body weight minus uterine weight
Net weight change from day 6 = Carcass weight minus body weight on day 6
These values are stated in the report.
Food and drinking water consumption
The quantity of food consumed by each rabbit was recorded daily. Food intake per rabbit (g/rabbit/day) was calculated using the total amount of food given to and left by each rabbit in each group on completion of a treatment day.
The relative food consumption (g/kg b.w./day) was calculated using the following formula:
Daily food consumption [g/kg b.w./day]= Total food intake in g / Body weight in kg
Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study.
EXAMINATIONS (NECROPSY), Examination of the dams
Dissection technique and evaluation of the animals:
One day before the calculated parturition, i.e. on gestation day 29, all surviving rabbits were sacrificed by lethal intravenous injection of 300 mg Pentobarbital/kg b.w. and exsanguinated.
In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of sacrifice or on the day on which the animals were found dead.
After ventral midline incision and skin reflection, the ovaries and uteri were removed; the gravid uteri (in toto) were weighed. A macroscopic examination of all subcutaneous tissues and internal organs of the dams was carried out. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intraabdominal lymph nodes and accessory reproductive organs were recorded.
In case of macroscopical findings, the affected maternal tissues were preserved in 10% buffered formalin for possible future histopathological examinations. - Ovaries and uterine content:
- Corpora lutea
- number per dam
- absolute number per group
- mean per group
Implantations
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group
Resorptions
- number per dam, % per litter
- distributions in the uterine horns
- absolute number per group
- mean per group
- mean % per group
- early resorptions < 2 g
- late resorptions > 2 g
Weight of placentae
- individual weight per fetus (alive and dead)
- mean placental weight per dam (placentae from male and female fetuses alone or combined)
- mean placental weight per dam and group (litter mean per group)
Weight of fetuses
- individual weight per fetus (alive and dead)
- mean fetal weight per dam (male and female fetuses alone or combined)
- mean fetal weight per dam and group (litter mean per group)
Fetuses
- absolute number per dam and group (alive and dead, male and female fetuses alone or combined)
- mean number of fetuses per dam and group (alive and dead, male and female fetuses alone or combined)
- sex ratio per litter
- distribution in the uterine horns
Runts
- number per dam
- mean per group
Malformed fetuses
- individual data per fetus
- type of malformation
- total number and incidence (%) of affected fetuses and litters per group
Total malformation rate [%] = malformed fetuses per group / fetuses per group x 100
Fetuses with variations
- individual data per fetus
- type of variation
- total number and incidence (%) of affected fetuses and litters per group8
Total variation rate [%] = fetuses per group with variations / fetuses per group x 100
Indices of pre-implantation loss and post-implantation loss:
Calculation of group indices
Pre-implantation loss [%] = corpora lutea (per group) - implantations (per group) / corpora lutea (per group) x 100
Post-implantation loss [%] = implantations (per group) - living fetuses (per group) / implantations (per group) x 100
Calculation of mean indices per litter
Pre implantation loss [%] = Sum of pre-implantation losses per litter in a group [%] / Number of litters in a group
Post implantation loss [%] = Sum of post-implantation losses per litter in a group [%] / Number of litters in a group - Fetal examinations:
- The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
(b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) The gravid uterus weight was determined.
(g) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(h) All fetuses (dead and alive) were inspected externally for damages, especially for malformations .
(i) The fetuses were sacrificed by a lethal intraperitoneal injection of 60 mg Pentobarbi-tal/kg b.w..
Each fetus was dissected:
The thorax and peritoneal cavity (without damage to ribs and sternum) were opened. Location, size and condition of the internal organs were determined and examined for abnormalities (e.g. liver, discolouration, situs inversus, alterations of urinary bladder, brain, lungs, cleft palate) of soft tissue.
The sex was determined.
The kidneys were removed and incised to check for damages (e.g. dilatation of the renal pelvis).
The abdominal organs were removed.
The diaphragm was carefully removed to check the position of the heart (left - right).
The thoracic organs were removed using surgical forceps; the heart was incised to check for damages. - Statistics:
- The statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), inter-group comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
Non-parametrical data
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01)
The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external macro-scopic examination of the fetuses).
Note:
The statistical evaluation of the pre- and post-implantation index (per group) using the number of corpora lutea, implantation sites and/ or fetuses per group was done using StatXact 4.0.1 software. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No changes in behaviour, the external appearance or faeces were noted for the control group or the test item-treated groups (5, 25 or 50 mg test item/kg b.w./day).
- Mortality:
- no mortality observed
- Description (incidence):
- No premature death was noted for the control group and the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related changes in body weight or body weight gain were noted between the dams of the control group and the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
Body weight gain:
The changes in body weight gain after the start of treatment on gestation day 6 until necropsy on gestation day 29 are given in the table below:
Body weight gain (mean %) # Group 1 Control Group2, 25 mg/kg Group 3, 25 mg/kg Group 4, 50 mg/kg
Gestation day 6 to gestation day 29 4.5% 7.8% 7.4% 7.4% - Food consumption and compound intake (if feeding study):
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related changes in food consumption were noted between the dams of the control group and the dams treated with 5, 25 or 50 mg test item/kg b.w./day.
However, a statistically significantly increased food consumption in comparison to the control group was noted on several test days between gestation day 19 and gestation day 24 for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day; at maximum 241.5% above the value of the control group at the intermediate dose level between gestation days 23 and 24). This was due to a low food intake of the control dams on several test days. Hence, the statistically significantly increased food consumption that was noted for the treatment groups was considered to be spontaneous. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- No test item-related changes in the consumption of drinking water were noted for the dams treated with 5, 25 or 50 mg test item/kg b.w./day by visual appraisal.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- Gravid uterus and carcass weight
No test item-related changes in the gravid uterus weight and the carcass weight in comparison to the control group were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
Net body weight gain from day 6
No test item-related changes between the control group and the treatment groups (5, 25 or 50 mg test item/kg b.w./day) were noted for the net body weight gain (without gravid uterus) between gestation day 6 and gestation day 29. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related pathological findings were noted during macroscopic inspection of the dams of the control group and dams treated with 5, 25 or 50 mg test item/kg b.w./day.
One animal of the high dose group revealed changes in the liver and the stomach. However, as only one animal was affected, these changes were considered to be spontaneous and not test item-related - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Details on results:
- General toxicity maternal animals: no effects
- Number of abortions:
- no effects observed
- Description (incidence and severity):
- No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
The values of pre- and post-implantation loss were in the range of normal variability. - Total litter losses by resorption:
- no effects observed
- Description (incidence and severity):
- No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
- Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- No dead fetus was noted in the control group and in the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- No effects observed
- Changes in number of pregnant:
- no effects observed
- Details on maternal toxic effects:
- Maternal toxic effects:no effects
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- > 50 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- other: no maternal toxicity
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related influence was noted on the fetal weight after administration of 5, 25 or 50 mg test item/kg b.w./day.
One runt (no. 15-7) was noted in the high dose group (50 mg/kg b.w./day). This was in the normal range of variability and considered to be spontaneous. - Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- No dead fetus was noted in the control group and in the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- The sex distribution of the fetuses in the control group and in the treatment groups (5, 25 or 50 mg test item/kg b.w./day) was inside the range of normal biological variability.
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- No test item-related influences on the number of resorptions and the number of live fetuses were noted for the dams of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
- Changes in postnatal survival:
- not examined
- External malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No external malformation was noted for the fetuses of the control group and the treat-ment groups (5, 25 or 50 mg test item/kg b.w./day).
External variations in the form of a hyperflexion of the extremities were noted for 3 fetuses from 2 dams of the low dose group (5 mg test item/kg b.w./day). As no extremities with a hyperflexion were noted for the fetuses of the intermediate and the high dose group (25 or 50 mg test itemkg b.w./day) the observations at the low dose level were considered to be spontaneous. - Skeletal malformations:
- not examined
- Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Internal gross alterations
A macroscopic internal examination was performed to detect gross alterations of the internal organs. No internal malformation or variation was noted for the fetuses of the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
At the control group a spontaneous variation in the form of a malpositioned left kidney was noted for fetus no. 4-8. - Other effects:
- no effects observed
- Description (incidence and severity):
- One runt each was noted in the intermediate dose group (300 mg test item/kg b.w./day) and in the control group.
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
No dead fetuses were noted in the litters of the control group and the treatment groups (treated orally with 5, 25 or 50 mg /kg b.w./day).
No test item-related malformations or variations were noted during the macroscopic external examination and the macroscopic gross inspection of the internal organs at laparotomy for the treatment groups (5, 25 or 50 mg test item/kg b.w./day). - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 50 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: no developmental toxicity
- Key result
- Developmental effects observed:
- no
- Conclusions:
- Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 50 mg test item/kg b.w./day for the dams. The no-observed-adverse effect level (NOAEL) for the fetal organism was also above 50 mg test item/kg b.w./day.
Based on the data obtained in this dose-range-finding study, it is not possible to derive dose levels for main study. Therefore, an additionally tolerance test was conducted (7.5.1 LPT_DRF_21 days_Rabbits_2018, LPT Study No. 36299).
Based on data obtained in both dose range finding studies, the following dose levels are suggested for LPT Study No. 35503 (Prenatal developmental toxicity study in rabbits):
Group 1: Control
Group 2: 10 mg test item/kg b.w./day, p.o
Group 3: 25 mg test item/kg b.w./day, p.o
Group 4: 75 mg test item/kg b.w./day, p.o - Executive summary:
The aim of this dose-range-finding study was to determine the dose levels for a prenatal developmental toxicity study of the test item in pregnant rabbits when administered orally during the critical period of organogenesis and the fetal development (6th to 28th day of gestation).
In this prenatal developmental toxicity study, the test item was administered orally to female rabbits at dose levels of 5, 25 or 50 mg/kg b.w./day from the 6th to 28th day of pregnancy.
Findings:
Examination of the dams
Mortality
No premature death was noted for the control group and the treatment groups (5, 25 or 50 mg test item/kg b.w./day).
Clinical signs
No changes in behavior, the external appearence and the consistency of the faeces were noted in the control group and the test item-treated groups.
Body weight and
body weight gain
No test item-related difference was noted for the body weight and the body weight gain.
Food consumption
No test item-related difference was noted for the food consumption.
Drinking water consumption
No test item-related influence was noted for the drinking water consumption at any of the tested dose levels (visual assessment).
Necropsy findings
No test item-related observations were noted for the treatment groups during necropsy.
Uterus and carcass weights
No test item-related influence on the uterus and carcass weight was noted for the treatment groups.
Examination of the fetuses
No test item-related influence was detected on the prenatal fetal development at 5, 25 or 50 mg test item/kg b.w./day with respect to the incidence of resorptions, number of live fetuses and the values calculated for the pre- and post-implantation loss.
Nodead fetuswas noted in the test item groups or the control group.
Sex distribution
No test item-related differences were noted.
Fetal weights
No test item-related influence on the fetal weights was noted for the treatment groups.
Placental weights
No test item-related influence was noted.
Fetal alterations
Malformations
No malformation was noted for the fetuses treated with 5, 25 or 50 mg test item/kg b.w./day during the inspection at laparotomy, including a macroscopic external examination and a macroscopic gross inspection of the internal organs.
Variations
The macroscopic inspection at laparotomy revealed no test item-related external variation for the fetuses treated with 5, 25 or 50 mg test item/kg b.w./day. No variation was noted during the gross inspection of the internal organs.
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was above 50 mg test item/kg b.w./day for the dams.
None of the animals died prematurely.
No changes were noted in behaviour, the external appearance and the consistency of the faeces.
No test item-related effect was noted for the body weight, body weight gain or food consumption.
Necropsy revealed no test item-related pathological findings.
The no-observed-adverse effect level (NOAEL) for the fetal organism was also above 50 mg test item/kg b.w./day.
No test item-related effects were noted for the reproduction parameters (number of implantation sites, number of fetuses, number of resorptions) in any of the treatment groups.
No test item-related influence was noted on the body weight of the fetuses in the treatment groups.
No dead fetuses, no malformation and no test item-related variations were noted.
Based on the data obtained in this dose-range-finding study, it is not possible to derive dose levels for main study. Therefore, an additionally tolerance test was conducted (LPTStudy No. 36299). Based on data obtained in both dose range finding studies, the following dose levels are suggested forLPTStudy No.35503 (Prenatal developmental toxicity study in rabbits):
Group 1:
Control
Group 2:
10 mg test item/kg b.w./day, p.o
Group 3:
25 mg test item/kg b.w./day, p.o
Group 4:
75 mg test item/kg b.w./day, p.o
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- June 2018 - October 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 2018
The 2018 update is to include rat-specific requirements in the TG 414 only; thus applies to rats and not to rabbits (for details see overall remarks/attachments below) - Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.31 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 2008
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or test system and environmental conditions:
- Test species / Strain Rabbit / New Zealand White
Breeder Manfred Bauer Kaninchen, Lohe 7/1, 74632 Neuenstein, Germany
Age (on day 6 of gestation) 5 months
Body weight (on day 6 of gestation) 3.75 kg to 4.68 kg
Number of animals 128 treated females in groups 1 to 4:
Mated and treated animals:
Groups 1 + 2: 30 females per group
Groups 3 + 4: 34 females per group
Evaluated females with viable fetuses:
Group 1: 20 dams
Group 2: 20 dams
Group 3: 20 dams
Group 4: 20 dams
ENVIRONMENTAL CONDITIONS
DIET: ad libitum, Commercial diet ssniff® K-Z V2323 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
WATER: ad libitum, tap water in bottles, Samples of drinking water are taken periodically by the Wasserwerk Wankendorf (24601 Wankendorf, Germany)
ENVIRONMENTAL CONDITIONS
Room temperature: 20°C ± 3°C (maximum range)
Relative humidity: 55% ± 10% (maximum range)
Illumination: 12-hour light/12-hour dark cycle
Ventilation rate: 15 - 20 air changes/hour - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- purified water (aqua ad injectabilia)
- Details on exposure:
- ADMINISTRATION
Frequency of administration Once daily, via gavage
Treatment period Day 6 to 28 of gestation
Vehicle Purified water
Administration volume 2 mL/kg b.w./day
Selection of route of administration According to OECD guideline 414
DOSAGE PREPARATION
The test item formulations were freshly prepared every day.
The test item was diluted in the vehicle to the appropriate concentrations and was administered orally at a constant volume (2 mL/kg b.w.) once daily from the 6th to the 28th day of gestation.
The amount of the test item was daily adjusted to the current body weight of the animal.
The control animals received the vehicle at the same administration volume daily in the same way.
In addition, the stability, homogeneity, and concentration of the test item mixture was monitored - Analytical verification of doses or concentrations:
- yes
- Remarks:
- For the analysis of the application formulations of groups 2 to 4, samples of approximately 2 x 5 mL each were taken at the following times and stored at 20°C ± 10% until analysis
- Details on analytical verification of doses or concentrations:
For the analysis of the application formulations of groups 2 to 4, samples of approximately 2 x 5 mL each were taken at the following times and stored at 20°C ± 10% until analysis:
At start of dosing
Analysis of stability and concentration
Immediately after preparation of the formula-tion as well as after 8 and 24 hours storage of the test item preparations at room temperature.
(3 samples / test item group; groups 2 - 4).
Number of samples: 3 x 3 = 9
(Date of sampling: 26 June 2018)
Homogeneity
At start of administration, during (middle) administration and before administration to the last animal of the dose group.
(3 samples / test item group; groups 2 - 4).
Number of samples: 3 x 3 = 9
(Date of sampling: 26 June 2018)
At end of the dosing period (at a time when the majority of animals was dosed)
Analysis of concentration
During treatment with the test item always before administration to the last animal of the group.
(1 sample / test item group; groups 2 - 4).
Number of samples: 1 x 3 = 3
(Date of sampling: 23 July 2018)
The samples were labelled with the study number, species, type of sample, concentration, sampling time and date.
The method of analysis was validated in LPT Study No. 36470. The following parameters were determined:
- Linearity
- Accuracy
- Precision
- Sensitivity
- Specificity
- Stability
The validation of the method revealed that the method employed was suitable for the determination and quantification of IPDA in the test item formulation samples.- Details on mating procedure:
- For this experiment, sexually mature, purebred female artificially inseminated rabbits (New Zealand White) were used.
- Duration of treatment / exposure:
- Day 6 to 28 of gestation
- Frequency of treatment:
- Once daily
- Duration of test:
- On gestation day 29 (one day before the calculated date of parturition) the dams were laparotomised.
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- control group
- Dose / conc.:
- 10 mg/kg bw/day (nominal)
- Remarks:
- low dose group
- Dose / conc.:
- 25 mg/kg bw/day (nominal)
- Remarks:
- intermediate dose group
- Dose / conc.:
- 75 mg/kg bw/day (nominal)
- Remarks:
- high dose group
- No. of animals per sex per dose:
- 20 pregnant females per dose group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The dose levels were selected in agreement with the Sponsor based on the results of two dose-range-finding studies which were conducted in pregnant rabbits with IPDA dosed at 5, 25 and 50 mg/kg b.w./day (LPT Study No. 35502) and in non-pregnant rabbits with IPDA dosed at 75 and 150 mg/kg b.w./day (LPT Study No. 36299).
LPT Report No. 35502 revealed no test item-related influences on any of the treatment groups.
In LPT Study No. 36299 the high dose group (150 mg IPDA/kg b.w./day) was noted with a distinctly reduced food consumption and body weight and therefore the animals were prematurely sacrificed on TD8. Necropsy revealed a severely reddened gastric mucosa in 3 of 3 examined animals.
The animals of the low dose group (75 mg IPDA/kg b.w./day) were noted with a reduced food consumption but no test item-related change for the body weight. A reddened gastric mucosa and ulcers were noted for 2 of 3 low dose animals during necropsy.
Based on complete lethality at 150 mg IPDA/kg b.w./day, 75 mg IPDA/kg b.w./day
were considered as the maximal tolerated dose (MTD).
The oral route was selected since this has been proven to be suitable for the detection of a toxicological hazard. - Maternal examinations:
- Dated and signed records of all activities relating to the day to day running and maintenance of the study within the animal units as well as to the group observations and examinations outlined in the Study Plan were recorded in the appropriate documentation. In addition, observations relating to the individual animals made throughout the study were recorded.
Clinical signs
Animals were individually observed at least once daily for any signs of behavioural changes, reaction to treatment or illness.
Immediately after administration any signs of illness or reaction to treatment were recorded. In case of changes the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m.
On Saturdays and Sundays, animals were checked regularly from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
Viability
Further checks were made early in each working day and again in the afternoon to look for dead or moribund animals. This would have allowed post mortem exami-nations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.
Animals showing signs of abortion or premature delivery were sacrificed on the same day. Fetuses obtained this way were examined for abnormal development whenever possible.
Body weight
The weight of each rabbit was recorded on the day of delivery (used for randomiza-tion), followed by daily weighings starting on GD 6 - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. day 6-9, 9 12, 12-15, 15-18, 18-21, 21-24, 24-27, 27-29). Furthermore, the carcass weight and the net weight gain from day 6 are given.
Food and drinking water consumption
The quantity of food consumed by each rabbit was recorded. Food intake per rabbit (g/rabbit/day) was calculated using the total amount of food given to and left by each rabbit in each group on completion of a treatment day.
Necropsy
One day before the calculated parturition, i.e. on gestation day 29, all surviving rabbits were sacrificed by lethal intravenous injection of 300 mg Pentobarbi-tal/kg b.w. and exsanguinated.
In order to check for possible drug effects, a dissection with macroscopic examina-tion of the internal organs and placentae of the dams was carried out on the day of sacrifice.
After ventral midline incision and skin reflection, the ovaries and uteri were removed; the gravid uteri (in toto) were weighed. A macroscopic examination of all subcutaneous tissues and internal organs of the dams was carried out. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumi-nation. The liver and the kidneys were examined. Any abnormalities in the appear-ance and size of the gonads, adrenal glands, uterus, intraabdominal lymph nodes and accessory reproductive organs were recorded.
In case of macroscopical findings, the affected maternal tissues were preserved in 10% buffered formalin for possible future histopathological examinations. - Ovaries and uterine content:
- Corpora lutea
- number per dam
- absolute number per group
- mean per group
Implantations
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group
Resorptions
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group
- mean % per group
- number of early resorptions (< 2 g)
- number of late resorptions (> 2 g)
Weight of placentae
- individual data per fetus
- mean per litter
- mean per sex and litter
- litter mean per group
- litter mean per sex and group
Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per sex and litter
- litter mean per group
- litter mean per sex and group
Fetuses
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive and dead) per sex and dam
- sex ratio per litter/group
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive/dead per group
Runts
- number per dam/group
Malformed fetuses
- individual data per fetus
- type of malformation: number and incidence (%) per group and litter
- number of affected fetuses per group
Total malformation rate [%] = malformed fetuses per group x 100
fetuses per group
Fetuses with variations7
- individual data per fetus
- type of variation: number and incidence (%) per group and litter
- number of affected fetuses per group8
Total variation rate [%] = fetuses per group with variations x 100
fetuses per group
Fetuses with retardations7
- individual data per fetus
- type of retardation: number and incidence (%) per group and litter
- number of affected fetuses per group8
Total retardation rate [%] = fetuses per group with retardations x 100
fetuses per group
Indices of pre-implantation loss and post-implantation loss:
Calculation of group indices (see table 7-1)
Pre implantation
loss [%] = Corpora lutea (per group) - implantations (per group) x 100
Corpora lutea (per group)
Post implantation
loss [%] = Implantations (per group) - living fetuses (per group) x 100
Implantations (per group)
Calculation of mean indices per litter (see table 7-2 and A7)
Pre implantation
loss [%] = Sum of pre-implantation losses per litter in a group [%]
Number of litters in a group
Post implantation
loss [%] = Sum of post-implantation losses per litter in a group [%]
Number of litters in a group
- Fetal examinations:
- The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
(b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) The gravid uterus weight was determined.
(g) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(h) All fetuses (dead and alive) were inspected externally for damages, especially for malformations .
(i) The fetuses were sacrificed with pentobarbital (60 mg/fetus, i.p.).
Each fetus was dissected:
The thorax and peritoneal cavity (without damage to ribs and sternum) were opened. Location, size and condition of the internal organs were determined and examined for abnormalities (e.g. liver, discolouration, situs inversus, alterations of urinary bladder, brain, lungs, cleft palate) of soft tissue.
The sex was determined.
The kidneys were removed and incised to check for damages (e.g. dilatation of the renal pelvis).
The abdominal organs were removed.
The diaphragm was carefully removed to check the position of the heart (left - right).
The thoracic organs were removed using surgical forceps; the heart was incised to check for damages.
The head was removed from 50% of the fetuses and fixed in BOUIN'S solution. An examination according to WILSON was carried out, inspecting the internal head structures (e.g. eyes).
The cranium was opened for the remaining 50% of the fetuses and the brain was removed for external inspection in toto.
The eviscerated fetuses (intact and headless bodies) were dehydrated in ethanol and cleared in potassium hydroxide solution. The skeleton was stained with Alizarin red (according to DAWSON). The skeletal system was examined (determi-nation of the number and type of retardations, variations as well as malfor-mations). - Statistics:
- The statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT and SHAPIRO-WILK test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT test (p ≤ 0.01 and p ≤ 0.05).
Non-parametrical data:
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test:
FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01)
or
Chi2 test, n ≥ 100 (p ≤ 0.05 and p ≤ 0.01)
The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy or findings during the external macroscopic examination of the fetuses) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
Note:
The statistical evaluation of the pre- and post-implantation index (per group) using the number of corpora lutea, implantation sites and/ or fetuses per group (see table 7-1 'Reproduction Data - Summary - Values per Group') was done using StatXact 4.0.1 software, as such a calculation is not possible in Provantis. - Indices:
- Malformation rate, total variation rate, pre implantation loss index, post implantation loss index, viability index, retardations index
- Historical control data:
- LPT Background Data
Values of control groups (n = 18) and test groups (n = 55) not significantly influenced by any test compound; data taken from the years 2013 to September 2018 - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No changes in behaviour, the external appearance or faeces were noted for the control group or the test item-treated groups (10, 25 or 75 mg IPDA/kg b.w./day).
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- No premature deaths were noted for the control group and the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
However, one dam of the control group, two dams of the intermediate dose group and two dams of the high dose group were prematurely sacrificed after abortion. Macroscopic post mortem examination revealed no test item-related findings. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- BODY WEIGHT
No test item-related changes in body weight were noted in the dams after oral treatment with 10 mg IPDA/kg b.w./day.
In the intermediate dose group (25 mg IPDA/kg b.w./day), a not statistically significant but constantly lower body weight compared to the control group was noted from GD 9 until study termination on GD 29 (at maximum 2.4% below the value of the control group). Due to this slight reduction, the effect was considered to be not test item-related and within the normal range of variation.
At 75 mg IPDA/kg b.w./day, a more pronounced and constant reduction in body weight was noted from GD 9 until study termination on GD 29 (statistically signifi-cant between GD 9 and GD 23 at p ≤ 0.05 or 0.01) in comparison to the control group (at maximum 5.5% below the value of the control group on GD 16, p ≤ 0.05). The effect is considered a combination of two statistically non-significant effects, namely the reduced gravid uterus weight and the slightly reduced carcass weight. However, this distinctly and constantly lower body weight was dose dependent and therefore considered to be test item-related.
BODY WEIGHT GAIN
No test item-related differences between the control group and the low dose group (10 mg IPDA/kg b.w./day) was noted for the body weight gain.
A slightly but not statistically significantly lower body weight gain was noted for the intermediate dose group (25 mg IPDA/kg b.w./day). However, as the body weight, this small reduction was considered to be within the normal range of variation.
According to the lower body weight of the high dose group (75 mg IPDA/kg b.w./day), also the body weight gain was slightly lower but not statistically significantly different from the control group.
However, the distinctly and constantly lower body weight gain was dose dependent and therefore considered to be test item-related and adverse.
NET BODY WEIGHT GAIN FROM DAY 6
No differences of toxicological relevance between the control group and the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day) were noted for the net body weight gain (without gravid uterus) between GD6 and GD29. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- No test item-related changes in food consumption were noted between the dams of the control group and the dams treated with 10 or 25 mg IPDA/kg b.w./day.
At 75 mg IPDA/kg b.w./day, a decreased food consumption was noted between GD8 and GD17 (at maximum 22.9% below the value of the control group on GD14 to GD15, p ≤ 0.05). This distinctly lower food consumption was considered to be test item-related and adverse (results see figure below). - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Description (incidence and severity):
- No test item-related changes in the consumption of drinking water were noted for the dams treated with 10, 25 or 75 mg IPDA/kg b.w./day by visual appraisal.
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- Behaviour, external appearance, faeces
No changes in behaviour, the external appearance or faeces were noted for the control group or the test item-treated groups (10, 25 or 75 mg IPDA/kg b.w./day). - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Gravid uterus weight
No test item-related changes in the gravid uterus weight in comparison to the control group were noted for the dams of the low and the intermediate dose group (10 or 25 mg IPDA/kg b.w./day).
A slightly decreased mean value of the gravid uterus weight was noted at the high dose level (75 mg IPDA/kg b.w./day) (8.7% below the control value, statistically not significant). This finding was mainly due to the low uterine weight of animal no. 82 with a total implantation loss and was considered to be test item-related.
Carcass weight
No test item-related changes in the carcass weight in comparison to the control group were noted for the dams of all treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
A slightly reduced carcass weight was noted at the high dose level (3.3% below the value of the control group). The slight reduction was related to the reduction in body weight and considered to be test item-related. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related pathological findings were noted during macroscopic inspection of the dams treated with 10, 25 or 75 mg IPDA/kg b.w./day at necropsy.
In the low dose group there was one female with blck foci in the placenta and in the high dose group marked red discoloration in the stomach of one female was observed.
These single findings were considered to be spontaneous and not test item-related. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- no effects observed
- Number of abortions:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related abortions were noted for the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
However, one control dam, two intermediate dose dams and two high dose dams were noted with spontaneous abortions. As abortions are known to occur spontaneously in rabbits of this strain and age and an abortion was also noted in the control group, the abortions noted in the intermediate and high dose group were considered to be not test item-related. Furthermore, the number of abortions is still in the range of LPT background data (see table below). - Pre- and post-implantation loss:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Animal no. 82 of the high dose group showed a total post-implantation loss.
Control 8.9 % showed pre-implantation loss & 2.9 % animals showed post implantation loss
Low dose 2.0% showed pre-implantation loss & 1% showed post implantation loss
Medium dose 4.6 % showed pre-implantation loss & 3.8% showed post implantation loss
High dose 2.6% showed pre-implantation loss & 13.8% showed post implantation loss
More details in attached table. - Total litter losses by resorption:
- effects observed, treatment-related
- Description (incidence and severity):
- The treatment with the test substance at 75 mg IPDA/kg b.w./day revealed increased incidences of early and total resorptions, and in consequence an increased percentage of post-implantation loss, which were statistically significantly different from the control.
A treatment related effect for the statistically significantly increased rate of early resorptions and the statistically significantly increased percentage of post-implantation loss, cannot totally be excluded, since both values were at the upper end of the LPT historical control data.
Although treatment related and adverse effects cannot totally be excluded, the main reason for those increases is animal no. 82 that showed a total post-implantation loss. When assessing the number of live fetuses the high dose group showed no remarkable difference to the control group (199 vs. 194 out of 20 animals with viable fetuses each). A fetotoxic effect of the test substance can be excluded, since no test item-related malformations, variations or retardations were evident. - Early or late resorptions:
- effects observed, treatment-related
- Description (incidence and severity):
- No test item-related influence was noted on the number of resorptions for the dams of the low and intermediate dose groups (10 or 25 mg IPDA/kg b.w./day).
The treatment with the test substance at 75 mg IPDA/kg b.w./day revealed increased incidences of early and total resorptions, and in consequence an increased percentage of post-implantation loss, which were statistically significantly different from the control.
A treatment related effect for the statistically significantly increased rate of early resorptions and the statistically significantly increased percentage of post-implantation loss, cannot totally be excluded, since both values were at the upper end of the LPT historical control data.
Although treatment related and adverse effects cannot totally be excluded, the main reason for those increases is animal no. 82 that showed a total post-implantation loss. When assessing the number of live fetuses the high dose group showed no remarkable difference to the control group (199 vs. 194 out of 20 animals with viable fetuses each). A fetotoxic effect of the test substance can be excluded, since no test item-related malformations, variations or retardations were evident. - Dead fetuses:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No dead fetus was noted in the control group and in the intermediate and high dose groups (25 or 75 mg IPDA/kg b.w./day).
At 10 mg IPDA/kg b.w./day, dam no. 30 was noted with two dead fetuses (nos. 30-8 and 30-9). As the two dead fetuses were only noted for one dam and no dose response-relationship was present, the two dead fetuses were considered to be spontaneous and not test item-related. Furthermore, the incidence of 2 dead fetuses in one of the treatment groups is in the range of the LPT background data (see table below). - Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Control: From 30 mated and treated females in the control group 6 animals became not pregnant and 21 animals were pregnant. 3 animals were excluded and not examined.
Low dose group. From 30 animals 5 became not not pregnant and 20 animals were pregnant, 5 Animals were excluded from further examinations.
Medium dose group. From 34 mated and treated animals, 8 animals not became pregnatn and 22 animals were pregnant.
High dose group. From 34 mated and treated naimals 7 animals became not pregnant and 23 animals were pregnant.
Altogether in all dose groups including the control group, a poor pregnancy rate was observed for seasonal reasons. For details see also attached table. - Other effects:
- no effects observed
- Description (incidence and severity):
- No test item-related differences for the placental weights were noted for any of the dosing groups (10, 25 or 75 mg IPDA/kg b.w./day) in comparison to the control group. The placental weights were within the normal variability.
- Details on maternal toxic effects:
- Mortality: No test item-related premature death was noted for the control group and the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
Abortions: No test item-related abortions were noted for the treatment groups.
Clinical signs: No changes in behaviour and the external appearance were noted in the control group and the test item-treated groups.
Body weight and body weight gain: A test item-related reduced body weight was noted for the high dose dams treated with 75 mg IPDA/kg b.w./day from GD9 until study termination on GD29 (at maximum 5.5% below the value of the control group on GD16, p ≤ 0.05). Accordingly a lower body weight gain was noted for the high dose group (12.6% body weight gain for the high dose group compared to 14.1% in the control group).
Food and drinking water consumption: A test item-related decrease in food consumption was noted for the high dose group (75 mg IPDA/kg b.w./day) from GD8 to GD17 (at maximum 22.9% below the value of the control group on GD14 to GD15, p ≤ 0.05).
Necropsy findings: No test item-related observations were noted for the treatment groups during necropsy.
Uterus weight, carcass weight and net body weight gain:
At 75 mg IPDA/kg b.w./day a slight reduction in the gravid uterus weight was noted (8.7% below the control value, statistically not significant).
A slight reduction was noted for the carcass weight at the high dose level (3.3% below the control value, statistically not significant). - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 25 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity see below
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 75 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- early or late resorptions
- food consumption and compound intake
- organ weights and organ / body weight ratios
- pre and post implantation loss
- Fetal body weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No test item-related influence was noted on the mean fetal weights after administration of 10, 25 or 75 mg IPDA/kg b.w./day.
No test item-related effect was noted for the number of runts. In total, 12 runts were noted at laparotomy: 5 runts were noted in the control group, 4 runts in the low dose group (10 mg IPDA/kg b.w./day), one runt in the intermediate dose group (25 mg IPDA/kg b.w./day) and two runts were noted in the high dose group (75 mg IPDA/kg b.w./day). - Reduction in number of live offspring:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No dead fetus was noted in the control group and in the intermediate and high dose groups (25 or 75 mg IPDA/kg b.w./day).
At 10 mg IPDA/kg b.w./day, dam no. 30 was noted with two dead fetuses (nos. 30-8 and 30-9). As the two dead fetuses were only noted for one dam and no dose response-relationship was present, the two dead fetuses were considered to be spontaneous and not test item-related. Furthermore, the incidence of 2 dead fetuses in one of the treatment groups is in the range of the LPT background data. - Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- The sex distribution of the fetuses in the dose groups (10, 25 or 75 mg IPDA/kg b.w./day) was comparable to the control fetuses. Slight differences observed were within the biological variability.
Sex ratio in study groups(male/female):
Control 0.88
Low dose 0.76
Medium dose 1.06
High dose 1.06 - Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- no effects observed
- External malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No visible gross alteration (malformation or variation) was noted for fetuses of the low and intermediate dose groups (10 or 25 mg IPDA/kg b.w./day).
In the high dose group (75 mg IPDA/kg b.w./day), the fetus no. 84-9 was noted with multiple malformations in form of an absent upper jaw and cranial roof as well as a small brain and a raschisis in the neck region. This malformation complex confirmed the external observation and was considered to be not test item-related but spontaneous according to type and incidence. - Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No alterations in the form of malformations were noted during skeletal examina-tions of the fetuses according to DAWSON at the low and intermediate dose level (10 or 25 mg IPDA/kg b.w./day).
At the high dose level (75 mg IPDA/kg b.w./day), the fetus no. 84-9 was noted with a malformation of the skull in form of an absent cranium and maxilla as well as a partly ossified temporal bone and cervical vertebral arches being partly reduced in size and misaligned. However this single occurrence was considered to be sponta-neous and not test item-related.
The skeletal variations observed during examination according to DAWSON were related to the caudal vertebral bodies (fused), to the lumbar vertebral bodies (misaligned), to the thoracic vertebral arches (fused), to the sternum (sternebra(e) bipartite, fused, misaligned, misshapen) or the rib(s) (accessory 13th ribs (uni- or bilateral), short, less than 12 ribs ossified).
No test item-related influences were noted on the incidence of the above men-tioned skeletal variations in the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
The retardations observed during skeletal examination (according to DAWSON) were related to the skull (incomplete ossification), the lumbar vertebral arches (reduced in size), the lumbar vertebral bodies (reduced in size), the sternum (sternebra(e) unossified, incompletely ossified or reduced in size), the talus (unossified), the thoracic vertebral arches (unossified) and the thoracic vertebral bodies (reduced in size or unossified).
No test item-related influences were noted on the incidence of the above men-tioned skeletal retardations in the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
However, statistically significant increases were noted for the incidences of the incomplete ossification of the skull in the intermediate dose group (p ≤ 0.05) as well as for the incidences of unossified sternebra(e) and the total retardations in the low dose group (for both p ≤ 0.01).
As no dose response relationship was noted and the values were within the range of the LPT background data ; these 3 statistically significantly increased incidences were considered to be not test item-related.
For details see attached table. - Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A macroscopic internal examination was performed to detect gross alterations of the internal organs. No malformations or variations were noted during the internal examination of the fetuses of the control group and the dose groups (10, 25 or 75 mg IPDA/kg b.w./day). The gross inspection of the brain in 50% of the fetuses revealed no changes for any of the fetuses after opening of the cranium and removal of the brain.
No alterations in the form of malformations were noted during soft tissue examina-tions of the fetal head according to WILSON at any tested dose level (10, 25 or 75 mg IPDA/kg b.w./day).
No test item-related influence was noted in the number of soft tissue variations of the fetal head compared to the control at any tested dose level (10, 25 or 75 mg IPDA/kg b.w./day).
The observed and classified soft tissue variations that were noted during the examination of the fetal head according to WILSON were in the form of dilatations of the 4th cerebral ventricle, subdural haemorrhages in the meninges and haemor-rhages in the cerebrum as well as cystic or semicircular cystic areas in the cerebral hemisphere. No test item-related differences in the incidences of the observed variations of the fetal head were noted between the control group and the test groups.
The observed cystic or semicircular cystic areas in the cerebrum noted in altogeth-er 6 fetuses of the test item groups are known to be fixation-induced artefacts . - Other effects:
- no effects observed
- Details on embryotoxic / teratogenic effects:
- One abortion was noted for the control group (no. 1) and two abortions each were noted for the intermediate dose group (nos. 58 and 63) and the high dose group (nos. 76 and 90) treated with 25 or 75 mg IPDA/kg b.w./day). As abortions are known to occur spontaneously in rabbits of this strain and age, these single incidences were considered to be an incidental finding and hence, are judged to be not test item-related.
Two dead fetuses were noted for dam no. 30 (nos. 30-8 and 30-9) treated with 10 mg IPDA/kg b.w./day. As no dose dependence-relationship was noted and because of the low incidence, the occurrence of one dam with two dead fetuses was considered to be not test item-related.
No test item-related increase was noted for the incidence of runts at any tested dose level.
No test item-related malformations or variations were noted during the macro-scopic external examination and the macroscopic gross inspection of the internal organs at laparotomy and the skeletal examination according to DAWSON or the soft tissue examination of the fetal heads according to WILSON for the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
Furthermore, the skeletal examination according to DAWSON revealed no test item-related retardations. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- > 75 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no prenatal developmental toxicity effects observed up to the highest tested dose
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
- Conclusions:
- Under the conditions of the study, 3-Aminomethyl-3,5,5-trimethylcyclo-hexanamine did not show any teratogenic potential.
- Executive summary:
Under the conditions of the study, IPDA did not show any teratogenic potential.In this prenatal developmental toxicity study according to OECD 414 (version 2001), the test item 3-Aminomethyl-3,5,5-trimethylcyclo-hexanamine was administered orally to 128 inseminated female rabbits at dose levels of 0, 10, 25 or 75 mg/kg b.w./day from the 6th to 28th day of pregnancy. One group served as control group, in which the animals received the vehicle (purified water) without test substance. The body weight of the 5-month-old animals at day 6 of gestation was 3.75 kg to 4.68 kg. 20 dams per dose group were examined and evaluated at the end of the study.
Examination of the dams
Mortality: No test item-related premature death was noted for the control group and the treatment groups (10, 25 or 75 mg IPDA/kg b.w./day).
Abortions: No test item-related abortions were noted for the treatment groups.
Clinical signs: No changes in behaviour and the external appearance were noted in the control group and the test item-treated groups.
Body weight: A test item-related reduced body weight was noted for the high dose dams treated with 75 mg IPDA/kg b.w./day from GD9 until study termination on GD29 (at maximum 5.5% below the value of the control group on GD16, p ≤ 0.05). Accordingly, a lower body weight gain was noted for the high dose group (12.6% body weight gain for the high dose group compared to 14.1% in the control group).
Food and DW
Consumption: A test item-related decrease in food consumption was noted for the high dose group (75 mg IPDA/kg b.w./day) from GD8 to GD17 (at maximum 22.9% below the value of the control group on GD14 to GD15, p ≤ 0.05).
Necropsy findings: No test item-related observations were noted for the treatment groups during necropsy.
Uterus weight, carcass weight and net body weight gain:
At 75 mg IPDA/kg b.w./day a slight reduction in the gravid uterus weight was noted (8.7% below the control value, statistically not significant).
A slight reduction was noted for the carcass weight at the high dose level (3.3% below the control value, statistically not significant).
Summary table:Overview of the animals (total numbers)
IPDA
Group 1
Control
Group 2
10 mg/kg
Group 3
25 mg/kg
Group 4
75 mg/kg
1
Mated and treated
females
30 #
30 #
34 #
34 #
2
Not examined females (excluded/spare animals)
3
5
4
4
3
Non-pregnant females
6
5
8
7
4
Deceased animals
none
none
none
none
5
Evaluated pregnant
females
21
20
22
23
6
Evaluated dams without viable fetuses (total post implantation loss)
none
none
none
1
7
Evaluated dams with abortion
1
none
2
2
8
Evaluated litters with
viable fetuses
20
20
20
20
# Further animals were included to yield 20 evaluable litters with viable fetuses.
A poor pregnancy rate was observed for seasonal reasons.
Examination of the fetuses
In the high dose group (75 mg IPDA/kg b.w./day), a test item-related increase was noted for the number of early resorptions (22 early resorptions in the high dose group in comparison to 1 early resorption in the control group). Accordingly, also the post-implantation loss was increased in the high dose group (13.8% in the high dose group compared to 2.9% in the control group).
No test item-related deaths of fetuses were noted.
No test item-related increase was noted for the incidence of runts in the test item groups in comparison to the control group.
Sex distribution: No test item-related differences were noted.
Fetal weights: No test item-related influence on the fetal weights was noted for the treatment groups.
Placental weights: No test item-related differences were noted.
Fetal alterations
Malformations: No test item-related malformation was noted during the external examination at laparotomy, the gross inspection of the internal organs, the skeletal examination according to DAWSON and the soft tissue examination of the fetal head according to WILSON.
Variations: The external examination at laparotomy, the gross inspection of the internal organs, the skeletal examination according to DAWSON and the soft tissue examination of the fetal head according to WILSON revealed no test item-related variations.
Retardations: Examination according to DAWSON revealed no test item-related delays in the ossification.
Analysis of test item formulations
The measured actual concentrations of the test item in the test item vehicle-mixtures were between 102.7% and 106.5% of the nominal concentrations, indicating correctly prepared, stable and homogeneous formulations.
Conclusion
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 25 mg IPDA/kg b.w./day for the dams:
No test item-related premature death was noted for any of the dose groups.
For the high dose group (75 mg IPDA/kg b.w./day), a reduction was noted for the body weight or the body weight gain. Also a decrease was noted in food consumption at 75 mg IPDA/kg b.w./day.
No changes in behaviour, external appearance or faeces were noted for the treatment groups.
No test item-related pathological findings were noted during necropsy for the treatment groups.
At 75 mg IPDA/kg b.w./day, a slight decrease was noted for the gravid uterus and the carcass weight.
The no-observed-adverse effect level (NOAEL) was above 75 mg IPDA/kg b.w./day for the fetuses and 25 mg IPDA/kg b.w./day for the rate of early resorptions/postimplantation loss:
At the maternotoxic dose level of 75 mg IPDA/kg b.w./day (reduced body weight, reduced body weight gain, reduced carcass weight and reduced food consumption), an increased number of early resorptions and accordingly an increased post-implantation loss were noted. Due to the increased number of early resorptions, the total resorption rate was slightly above the LPT historical control data (up to 1.3). The early resorption rate and the percentages of post-implantation loss were at the upper range but still within the LPT historical control data.
No test item-related influence was noted on the mortality or body weight of the fetuses in the treatment groups.
No test item-related deaths of the fetuses and no test item-related malformations, variations or retardations were noted.
Under the conditions of the study, IPDA did not show any teratogenic potential.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2001-06-05 - 2001-06-28
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 1981
- Deviations:
- no
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Remarks:
- Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free, (COBS-VAF®).
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, L'Arbresle (France)
- Strain and sanitary status: Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free, (COBS-VAF®).
- Age: 10-11 weeks
- Weight at study initiation: 206-301, mean 245 g. Mean weights in the four groups were similar.
- Housing: The animals were housed individually in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray, containing autoclaved sawdust (SICSA, Alfortville, France), was placed under each
cage.
- Diet: Pelleted maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 50 +/- 20 %
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/12h - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- VEHICLE
The vehicle was purified water, obtained by reverse osmosis using a Milli-Ro 8 plus apparatus (Millipore SA, Saint-Quentin en Yvelines, France).
DOSAGE FORM PREPARATION
The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle in order to achieve the concentrations of 1, 5 and 25 mg/mL and then homogenized using a magnetic stirrer. The test item dosage forms were made for up to 9 days according to the stability results obtained for the study and were stored at +4 °C and protected from light prior to use. The dosage forms were delivered each day to the animal room.
TREATMENT GROUPS
The dose-levels were determined in agreement with the Sponsor, following the results of a
previously conducted preliminary study. In this study, minor to marked matemotoxicity was recorded at 20, 70 and 250 mg/kg/day dose-levels. Consequently, it was decided, in agreement with the Sponsor, to lower the low and intermediate dose-levels in order to obtain a NOAEL at the low dose.
Group // Number of animals // Dose-level (mg/kg/day)
1 // 24 mated females* // 0
2 // 24 mated females* // 10
3 // 24 mated females* // 50
4 // 24 mated females* // 250
*: only the first 20 pregnant females were taken into consideration for fetal examinations.
ADMINISTRATION
The oral route was selected since it was one of the routes of exposure in Man. The test item or control dosage forms were administered by gavage using a plastic syringe fitted with a metal gavage tube. The quantity of the dosage form administered to each animal was adjusted according to the most recently recorded body weight. A constant dosage-volume of 10 mL/kg/day was used. The dosage forms were stirred continuously throughout the dosing procedure. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Chemical analysis of the dosage forms:
Before the start of treatment, the suitability of the proposed formulation procedure was
determined by the analysis of concentration and stability of dosage forms which were prepared
using the procedure.
During the treatment period, a check of the concentration was performed on dosage forms
prepared for use in the study.
Stability
Two dosage forms were prepared at the lowest and highest concentrations used in the study
(1 and 25 mg/mL). Each dosage form was protected from light and sampled after O Gust after
preparation), 4 and 9 days storage at +4 °C.
The aliquots sampled just after preparation ( day 0) were analyzed immediately and those
sampled on day 4 were stored frozen at -20°C pending analysis on last sampling occasion (day 9)
when all samples were assayed. -> The results of the chemical analyses demonstrated a satisfactory stability of both dosage forms prepared over a nine-day period at +4°C (away from light).
Concentration
The concentration of samples taken from each dosage form (including the control) prepared for
use on the first day of treatment and on the last day of treatment was determined. -> Throughout the study, a satisfactory agreement was observed between the nominal and actual concentrations of the test material in the administered dosage forms since the deviations from nominal concentration were in an acceptable range of± 6 %. - Details on mating procedure:
- Females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum.
- Duration of treatment / exposure:
- day 6 to day 19 post-coitum inclusive
- Frequency of treatment:
- daily
- Duration of test:
- until day 20 post-coitum
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- control group
- Dose / conc.:
- 10 mg/kg bw/day (nominal)
- Remarks:
- low dose group
- Dose / conc.:
- 50 mg/kg bw/day (nominal)
- Remarks:
- intermediate dose group
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Remarks:
- high dose group
- No. of animals per sex per dose:
- 24 (females only); only the first 20 pregnant females were taken into consideration for fetal examinations
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: dose levels were determined based on the results of a preliminary study
- Maternal examinations:
- CLINICAL EXAMINATIONS
MORBIDITY AND MORTALITY
Each animal was checked for mortality or signs of morbidity:
- at least twice a day during treatment period,
- at least once a day on other days.
Female found dead on day 16 was subjected to a macroscopic post-mortem examination. The number of corpora lutea and implantation sites was recorded.
CLINICAL SIGNS
Each animal was observed for clinical signs (including evidence of abortion/resorption) at approximately the same time.
FOOD CONSUMPTION
The quantity of food consumed by each female was recorded for the following intervals: 2-6, 6-9, 9-12, 12-15, 15-18 and 18-20 post-coitum.
Food intake per animal and per day was calculated.
BODY WEIGHT
The body weight of each female was recorded on days 2, 6, 9, 12, 15, 18 and 20 post-coitum. The net body weight was calculated (gross body weight minus gravid uterine weight), as well as the gross and net body weight change.
SACRIFICE AND TERMINAL EXAMINATIONS
These examinations were carried out in all females.
HYSTERECTOMIES
On day 20 post-coitum, all the females were killed by asphyxiation using carbon dioxide. The weight of the gravid uterus was recorded for each pregnant female at hysterectomy (with at least one live fetus). The ovaries and uterus of females were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of implantation sites (or uterine scars).
Early resorptions refer to evidence of implantation without recognizable embryo or fetus; late resorptions refer to dead embryo or fetus with external degenerative changes; scars refer to evidence of implantation without recognizable structure (embryo, fetus or placenta). In apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the Salewski staining technique.
MACROSCOPIC POST-MORTEM EXAMINATION
After hysterectomy, the females were subjected to a macroscopic post-mortem examination of the principal thoracic and abdominal organs. A gross evaluation of placentas was also performed. The number of corpora lutea and implantation sites was recorded in any female that died. - Ovaries and uterine content:
- - Examination of uterine content: Weight of gravid uterus, number or corpora lutea, number and distrubution of implantation sites (or uterine scars), number and distrubution of early and late resorptions, number and distribution of dead and live fetuses
- Fetal examinations:
- These examinations were carried out only in the first 20 litters (females with at least one live fetus). The other litters were discarded without further investigation. All the fetal examinations were conducted without knowledge of treatment group in order to minimize bias. The fetal findings were described according to the glossary of the International Federation of Teratology Societies (IFTS) and classified as malformation or variation: malformation refers to a permanent structural change that is likely to adversely affect the survival or health, variation refers to a change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health (this might include a delay in growth or morphogenesis that has otherwise followed a normal pattern of development).
BODY WEIGHT
The body weight of each live fetus was recorded.
EXTERNAL EXAMINATION
Each fetus was submitted to a detailed external examination, which included the observation of all visible structures, surfaces and orifices. The fetuses were then killed by a subcutaneous injection of thiopental sodium.
SOFT TISSUE EXAMINATION
Approximately half of the live fetuses per litter were fixed with Bouin's fluid. A detailed soft tissue examination was performed according to a free-hand sectioning technique (Wilson technique) which included the observation of all the organs and structures of the head, neck, thorax and abdomen.
SKELETAL EXAMINATION
The remaining live fetuses per litter were fixed in ethyl alcohol. A detailed examination of the skeleton was performed after staining with alizarin red S and alcian blue. This examination included the observation of all the bone structures and cartilage of the head, spine, rib cage, pelvis and limbs.
SEX OF FETUSES
The sex of each live fetus was determined at the time of the evisceration ( after fixation in alcohol) or at the time of the Wilson sectioning. - Statistics:
- Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.
- Indices:
- Data are expressed as group mean values ± standard deviation (maternal body weight and food consumption, fetal body weight and number of corpora lutea, implantations, fetuses and
resorptions) or as proportions (pre-implantation loss, post-implantation loss and fetal findings).
Whenever necessary, the experimental unit of comparison was the litter.
The pre-implantation loss was calculated as follows:
((Number of corpora lutea - Number of implantation sites) / Number of corpora lutea) x 100
The post-implantation loss was calculated as follows:
((Number of implantation sites - Number of live fetuses) / Number of implantation sites) x 100
Fetal findings were analyzed as follows:
(Number of fetuses with a particular finding / Total number of fetuses examined) x 100
In addition, the mean percentage of affected fetuses/litter was calculated. - Historical control data:
- historical control data avaiable for fetal soft tissue variation, fetal skeletal malformations and fetal skeletal variation
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- There were no clinical signs in the control and the 10 and 50 mg/kg/day groups.
At the 250 mg/kg/day dose-level, ptyalism was recorded in most females of this group from
day 11, 12, 13 or 14 post-coitum until hysterectomy. Loud breathing and regurgitation were
recorded in 4 females of this group. All these findings might be the consequence of the corrosive
properties of the test item and the high pH of the dosage forms (pH between 1 0 and 12). - Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- There were no deaths in the control, 10 and 50 mg/kg/day treated groups.
In the 250 mg/kg/day treated group, one female was found dead on day 16 of pregnancy. Loud
breathing caused by regurgitation of the test item was recorded the day before death. No changes
of body weight were noted before death. Whitish foci on the lung were recorded at necropsy.
Consequently, this death was attributed to regurgitation following gavage and was not
considered to be related to the toxicity of the test item. - Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- The body weight gain was similar in the control and the 10 and 50 mg/kg/day groups throughout
the treatment period.
In the 250 mg/kg/day group, a significantly lower body weight gain (-35%, p<0.001) was
recorded after the first three days of treatment. Thereafter, the body weight was similar to that of
the controls. Over the whole treatment period, the difference remained slight (-10%, not
statistically significant).
The net body weight gain was also significantly lower at this dose-level (-25%, p<0.0 1) when
compared to the control group.
Because the reduction in food consumption correlated with a reduction in body weight gain at
the 250 mg/kg/day dose-level, these significant changes were considered to be related to the
treatment with the test item. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- The food consumption was similar in the control and the 10 and 50 mg/kg/day groups.
In the 250 mg/kg/day group, a slightly significant decrease in food consumption was recorded
during the treatment period (-7%, p<0.001 ), with a more marked effect during the first three days
of treatment (-21 %, p<0.001). - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment related findings were observed at any dose level. An exception are whitish foci on the lung in the decedent female.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not examined
- Number of abortions:
- no effects observed
- Description (incidence and severity):
- There were no abortions in any group.
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- Pre-implantation loss: The number of corpora lutea and implantation sites, and therefore the pre-implantation loss were
similar in the control and the treated groups. The fluctuations between the groups including a
slightly lower pre-implantation loss in the high-dose group were among those commonly
recorded for rats of this strain and age. Consequently, because the treatment occurred after the
implantation of the ovum, this was considered to be fortuitous.
Post-implantation loss: The number and rate of resorptions (late and early) were low and similar in the control and the treated groups. - Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- No total resorption in any group
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- no effects observed
- Description (incidence and severity):
- Number pregnant per dose level: Control 24; low- and high-dose 23; mid-dose 22
- Other effects:
- no effects observed
- Details on maternal toxic effects:
- Maternal toxic effects:yes
Details on maternal toxic effects:
- Mortality and day of death: One female of the high-dose group was found dead on day 16. This death was attributed to an effect of hold-up in the
esophagus following gavage. No other mortalities were observed.
- Clinical signs: The only treatment-related clinical sign was ptyalism in most females of the 250 mg/kg bw/day group from day 11, 12, 13, or 14 until hysterectomy. Loud breathing and presence of material in the mouth, probably due to hold-up in the esophagus, were recorded in 4 females. These observations might be the consequence of the corrosive properties of the test item (pH between 10 and 12).
- Number pregnant per dose level:
Control 24; low- and high-dose 23; mid-dose 22
- Number aborting: No abortion in any group
- Number of resorptions: No total resorption in any group
- Pre and post implantation loss: No treatment related findings were observed at any dose level.
- Body weight gain: Not affected in low and mid dose groups. A significant decrease (-35 %) was observed in the high-dose group after the first three days of treatment. Thereafter, the body weight was similar to that of the controls. The net body weight gain was also significantly lower (-25 %) at
this dose level.
- Food/water consumption: Not affected in low and mid dose groups; a slightly significant decrease was observed in the high dose group during the
treatment period.
- Gross pathology incidence and severity: No treatment related findings were observed at any dose level. An exception are whitish foci on the lung in
the decedent female. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 50 mg/kg bw/day
- Basis for effect level:
- other: maternal toxicity
- Fetal body weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The fetal weight was similar in the control and the treated groups. There was only a statistically
significantly higher fetal weight in the 50 mg/kg/day group: 4.10 g vs. 3.81 g (p<0.05). However,
since this difference was not dose-related, a relationship to treatment with the test item was ruled
out. - Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- The number of live fetuses per female was similar in the control and the treated groups.
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- No treatment related findings were observed at any dose level.
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- No treatment related findings were observed at any dose level.
- Changes in postnatal survival:
- no effects observed
- External malformations:
- no effects observed
- Description (incidence and severity):
- No treatment related external malformations or variations were observed at any dose level.
- Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Fetal skeletal malformation:
There was no malformation in the 10 and 250 mg/kg/day treated groups.
One malformation was recorded in the control group (1/130 fetuses with supernumerary lumbar
vertebra) and in the 50 mg/kg/day treated groups (1/132 fetuses with splitted sternebra).
Because of the low incidence of these findings, which was within the range of historical control
data and the absence of dose-relationship, the malformations were not considered to be related to
the treatment with the test substance.
Fetal skeletal variation:
All the findings were recorded without clear dose-relationship and/or statistical significance
and/or occurred at incidences consistent with historical control background data.
The two exceptions were the increase in fetal incidence of incomplete ossification of
5th stemebra in the 250 mg/kg/day group: 106/134 fetuses (79.l %, p<0.0 1) were affected, versus
88/130 (67.7%) in the control group. However, when expressed on a fetus/litter basis, it was not
statistically significant.
There was also a non statistically significant increase in fetal incidence of incomplete
ossification of rib(s) in the same group: 9/134 fetuses (6.7%) were affected, versus 2/130 (1.5%).
The litter incidence was also slightly increased in this group when compared to the control but
there were no statistical differences. - Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no malformations in the control and the 10 and 50 mg/kg/day groups.
In the 250 mg/kg/day treated group, misshapen heart was recorded in 1/126 fetuses.
Because this finding was the only soft tissue malformation recorded in the study, it was
considered to be fortuitous in origin. Only two kind of common findings (dilated renal pelvis and dilated ureter) were recorded in the
control or the treated groups. Because the incidence of this finding was not dose-related, not
statistically significant and found among CIT historical control data, it was not considered to be
related to the treatment with the test item. - Other effects:
- no effects observed
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
- Litter size and weights: No treatment related findings were observed at any dose level.
- Sex ratio: No treatment related findings were observed at any dose level.
- External abnormalities: No treatment related external malformations or variations were observed at any dose level.
- Soft tissue abnormalities: No treatment related soft tissue malformations or variations were observed at any dose level.
- Skeletal abnormalities: No statistically significant treatment related skeletal malformations or variations were observed at any dose level.
There was a statistically insignificant increase in fetal incidence of incomplete ossification of the 5th sternebra in the 250 mg/kg bw/day group
(106/134 fetuses = 79.1%, p<0.01 were affected vs. 88/130 = 67.7% in control group).
In the same group there was a statistically nonsignificant increase in fetal incidence of incomplete ossification of the
rib(s) (9/137 fetuses = 6.7 % vs. 2/130 = 1.5% in control group.
When ossification was incomplete, cartilage was generally present, demonstrating that the skeletal variations recorded corresponded to slight
fluctuations in the time of ossification rather than being a persistent alteration. In conclusion, these findings were considered to be incidental and of no toxicological significance. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- > 250 mg/kg bw/day
- Basis for effect level:
- other: embryotoxicity
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- > 250 mg/kg bw/day
- Basis for effect level:
- other: fetotoxicity
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- > 250 mg/kg bw/day
- Basis for effect level:
- other: teratogenicity
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
- Conclusions:
- The test item did not show any teratogenic or embryofetotoxic effects in a gavage study with rats performed in accordance with OECD TG 414 (2001) up to and including the highest tested dose level of 250 mg/kg bw/day. The NOEL for maternal toxicity was 50 mg/kg bw/day, effects at 250 mg/kg bw/day were reduced food consumption and reduced body weight gain. The NOAEL for developmental toxicity is > 250 mg/kg bw/day
- Executive summary:
Based on the results of a dose finding study, three groups of 24 mated female Sprague-Dawley rats received 3-aminomethyl-3,5,5-trimethylcyclohexylamine by daily oral administration (gavage) at 0 (water = control), 10, 50 and 250 mg/kg/day from day 6 to day 19 post-coitum inclusive. On day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The study was designed according to OECD TG 414.There was no treatment-related death in any of the dams. Clinical signs were not observed, except for ptyalism in most females of the 250 mg/kg/day group (from day 11, 12, 13 or 14 post-coitum until hysterectomy; effect not considered as adverse). Loud breathing and hold-up in the esophagus were recorded in 4 females of this group and a significantly lower body weight gain (-35%) was recorded after the first three days of treatment. Thereafter, the body weights were similar to that of the controls. Over the whole treatment period, the difference remained slight (-10 %, not statistically significant). The net body weight gain was also significantly lower at this dose-level (-25 %) when compared to the control group. In the 250 mg/kg/day group, a significant decrease in food consumption was recorded during the treatment period (-7%), with a more marked effect during the first three days of treatment (-21 %).Abortions or total resorptions were not observed in any of the groups, nor were there any macroscopic findings that were ascribed to treatment with the test item. No treatment related effects were observed on pre- or post-implantation loss, fetal weight or sex-ratio. With respect to the fetuses, no test item related external, soft tissue or skeletal malformations or variations were detected. There was an increase in fetal incidence of incomplete ossification of the 5th sternebra in the 250 mg/kg bw/day group (106/134 fetuses = 79.1 %, p < 0.01 were affected vs. 88/130 = 67.7 % in control
group, statistically insignificant on a fetus/litter basis). Because these findings are of low concern and occur only in the presence of maternal toxicity, they are considered to be secondary. In the same group there was a statistically nonsignificant increase in fetal incidence of incomplete ossification of the rib(s) (9/137 fetuses = 6.7 % vs. 2/130 = 1.5 % in control group. When ossification was incomplete, cartilage was generally present, demonstrating that the skeletal variations recorded corresponded to slight fluctuations in the time of ossification rather than being a persistent alteration. In conclusion, these findings were considered to be incidental and of no toxicological significance. The NOEL for maternotoxicity was 50 mg/kg/day and the NOAEL for embryonic development was >250 mg/kg/day.
Referenceopen allclose all
Summary of animals examined
Test item |
Group 1 Control |
Group 2 5 mg/kg |
Group 3 25 mg/kg |
Group 4 50 mg/kg |
Treated females |
4 |
4 |
4 |
4 |
Not examined females (excluded/spare animals) |
none |
none |
none |
1 |
Non-pregnant females |
1 |
1 |
1 |
none |
Deceased animals |
none |
none |
none |
none |
Evaluated pregnant females |
3 |
3 |
3 |
3 |
Dams without viable fetuses (total post implantation loss) |
none |
none |
none |
none |
Dams with abortion |
none |
none |
none |
none |
Evaluated litters with viable fetuses |
3 |
3 |
3 |
3 |
Reproduction data of the dams
Parameter |
Group 1 Control (n=3) |
Group 2 5 mg/kg (n=3) |
Group 3 25 mg/kg (n=3) |
Group 4 50 mg/kg (n=3) |
||||
Corpora lutea |
total mean per dam |
|
31 10.3 |
33 11.0 |
29 9.7 |
39 13.0 |
||
Implantation sites |
total mean per dam |
|
31 10.3 |
30 10.0 |
27 9.0 |
35 11.7 |
||
Total resorptions |
total mean per dam |
|
1 0.3 |
1 0.3 |
2 0.7 |
0 0.0 |
||
Early resorptions |
total mean per dam |
|
1 0.3 |
1 0.3 |
2 0.7 |
0 0.0 |
||
Late resorptions |
total mean per dam |
|
0 0.0 |
0 0.0 |
0 0.0 |
0 0.0 |
||
Fetuses (alive + dead) |
total mean per dam |
|
30 10.0 |
29 9.7 |
25 8.3 |
35 11.7 |
||
Live fetuses |
total mean per dam |
|
30 10.0 |
29 9.7 |
25 8.3 |
35 11.7 |
||
Dead fetuses |
total mean per dam |
|
0 0.0 |
0 0.0 |
0 0.0 |
0 0.0 |
||
Pre-implantation loss |
per group |
|
0.0 |
9.1 |
6.9 |
10.3 |
||
[%] |
mean per dam |
|
0.0 |
7.1 |
6.7 |
8.9 |
||
Post-implantation loss |
per group |
|
3.2 |
3.3 |
7.4 |
0.0 |
||
[%] |
mean per dam |
|
3.3 |
3.0 |
7.2 |
0.0 |
||
* / ** |
Significantly different from the controls at p ≤ 0.05 / p ≤ 0.01, Chi2test, only performed for the group values of the pre- and post-implantation loss |
|||||||
* / ** |
Significantly different from the controls at p ≤ 0.05 / p ≤ 0.01, Dunnett test, performed for the mean values per group |
|||||||
Body weight gain
Body weight gain (mean %)# |
Group 1 Control |
Group 2 5 mg/kg |
Group 3 25 mg/kg |
Group 4 50 mg/kg |
Gestation day 6 to gestation day 29 |
4.5% |
7.8% |
7.4% |
7.4% |
Sex distribution of fetuses
Parameter |
Sex ratio observed in this study (male / female) |
|
Sex ratio of fetuses (male/female) |
Group 1: |
0.76 |
Group 2: |
0.71 |
|
Group 3: |
0.92 |
|
Group 4: |
1.33 |
|
Table: Overview on abortions of the study animals
Group |
Dam no. |
Day of abortion (GD) |
Time of abortion |
Day of sacrifice |
Abortion rate (Aborted animals / evaluated pregnant females; row 5 in text table 4-1) |
1 Control |
1 |
22 |
during the day |
22 |
1 / 21 = 4.8% |
2 10 mg/kg |
- |
- |
- |
- |
0 / 20 = 0.0% |
3 25 mg/kg |
58 |
20 |
during the night |
21 |
2 / 22 = 9.1% |
63 |
26 |
during the night |
27 |
||
4 75 mg/kg |
76 |
26 |
during the night |
27 |
2 / 23 = 8.7% |
90 |
21 |
during the night |
22 |
Table: Comparison of the abortion rate with theLPTbackground data.
Abortions |
Abortion rates in this study (LPT Report 35503)
|
LPT Background Data#1- Values of control groups (n = 18) and test groups (n = 55) not significantly influenced by any test compound; data taken from the years 2013 to September 2018
[Mean value from the mean values of the groups used] [Range of mean values of the individual groups used] |
|||
Abortion rate (%) |
Control: |
4.8 |
4.13 ± 3.81 0.0 - 9.5 |
control groups |
|
Group 2: |
0.0 |
||||
Group 3: |
9.1 |
3.32 ± 3.65 0.0 - 10.0 |
test groups |
||
Group 4: |
8.7 |
||||
#1: |
not audited by QAU |
||||
Table:Summary of the mean body weight gain from GD6 to GD29
Mean Body Weight Gain |
|||
Group |
Time interval Gestation day 6 - 29 (whole study period) |
||
|
Gain in kg |
Gain in % |
Difference to control % |
Control |
0.60 |
14.1 |
n.a. |
Group 2 (10 mg/kg) |
0.64 |
14.7 |
+6.6 |
Group 3 (25 mg/kg) |
0.53 |
12.8 |
-10.8 |
Group 4 (75 mg/kg) |
0.52 |
12.6 |
-12.8 |
Table:Comparison of the resorption rates and the post-implantation loss with the LPT background data.
Parameter |
Values observed in this study (LPT Report 35503)
[fetal incidence in % per group] |
LPT Background Data#1- Values of control groups (n = 18) and test groups (n = 55) not significantly influenced by any test compound; data taken from the years 2013 to September 2018
[Mean value from the mean values of the groups used] [Range of mean values of the individual groups used] |
||||
Total resorptions mean % per dam |
Control: |
0.3 |
0.70 ± 0.39 0.1 - 1.3 |
control groups |
||
Group 2: |
0.0 |
|||||
Group 3: |
0.4 |
0.79 ± 0.41 0.0 - 1.6 |
test groups |
|||
Group 4: |
1.5** #2 |
|||||
Early resorptions mean % per dam |
Control: |
0.1 |
0.39 ± 0.30 0.0 - 1.0 |
control groups |
||
Group 2: |
0.0 |
|||||
Group 3: |
0.3 |
0.45 ± 0.32 0.0 - 1.3 |
test groups |
|||
Group 4: |
1.0** #2 |
|||||
Late resorptions mean % per dam |
Control: |
0.3 |
0.33 ± 0.22 (0.0 - 0.7) |
control groups |
||
Group 2: |
0.0 |
|||||
Group 3: |
0.1 |
0.36 ± 0.23 (0.0 - 0.9) |
test groups |
|||
Group 4: |
0.4 |
|||||
Post-implantation loss % per group |
Control: |
2.9 |
7.71 ± 4.66 (0.5 - 16.5) |
control groups |
||
Group 2: |
1.0 #3 |
|||||
Group 3: |
3.8 |
8.83 ± 4.36 (1.0 - 17.4) |
test groups |
|||
Group 4: |
13.8** #3 |
|||||
Post-implantation loss mean % per dam |
Control: |
2.0 |
7.85 ± 4.92 (0.4 - 16.9) |
control groups |
||
Group 2: |
1.0 |
|||||
Group 3: |
3.9 |
8.71 ± 4.30 (1.0 - 15.7) |
test groups |
|||
Group 4: |
15.1** #2 |
|||||
#1: |
not audited by QAU |
|||||
#2*/**: |
(p ≤ 0.05/0.01) Dunnett test |
|||||
#3*/**: |
(p ≤ 0.05/0.01) Chi2test |
|||||
Table: :Summary of the reproduction data
Parameter |
Group 1 Control (n=20) |
Group 2 10 mg/kg (n=20) |
Group 3 25 mg/kg (n=20) |
Group 4 75 mg/kg (n=21) #1 |
|
||||
Corpora lutea |
total mean per dam |
|
225 11.3 |
199 10.0 |
195 9.8 |
231 11.0 |
|
||
Implantation sites |
total mean per dam |
|
205 10.3 |
195 9.8 |
186 9.3 |
225 10.7 |
|
||
Total resorptions |
total mean per dam |
|
6 0.3 |
0 0.0 |
7 0.4 |
31 1.5 ** #1 |
|
||
Early resorptions |
total mean per dam |
|
1 0.1 |
0 0.0 |
5 0.3 |
22 1.0 ** #1 |
|
||
Late resorptions |
total mean per dam |
|
5 0.3 |
0 0.0 |
2 0.1 |
9 0.4 |
|
||
Fetuses (alive + dead) |
total mean per dam |
|
199 10.0 |
195 9.8 |
179 9.0 |
194 9.7 #² |
|
||
Live fetuses |
total mean per dam |
|
199 10.0 |
193 9.7 |
179 9.0 |
194 9.7 #² |
|
||
Dead fetuses |
total mean per dam |
|
0 0.0 |
2 0.1 |
0 0.0 |
0 0.0 #² |
|
||
Post-implantation loss |
per group |
|
2.9 |
1.0 |
3.8 |
13.8 ** |
|
||
[%] |
mean per dam |
|
2.0 |
1.0 |
3.9 |
15.1 ** |
|
||
|
|
|
|||||||
|
* / ** |
Significantly different from the controls at p ≤ 0.05 / p ≤ 0.01, Chi2test, only performed for the group values of the pre- and post-implantation loss (see table7-1). |
|||||||
|
* / ** |
Significantly different from the controls at p ≤ 0.05 / p ≤ 0.01, Dunnett test, performed for the mean values per group (see table7-2). |
|||||||
|
#1 |
A total post-implantation loss (9 early resorptions) was noted in 1 of 21 litters. |
|||||||
|
#2 |
The calculation was based on 20 litters with viable fetuses (see table7-2). |
|||||||
Table :Comparison of the number of dead fetuses with the LPT background data.
Parameter |
Values observed in this study (LPT Report 35503)
|
LPT Background Data#1- Values of control groups (n = 18) and test groups (n = 55) not significantly influenced by any test compound; data taken from the years 2013 to September 2018 |
|||
Dead fetuses (total number per group) |
Control: |
0 |
0 - 1 #2 |
control groups |
|
Group 2: |
2 |
||||
Group 3: |
0 |
0 - 5 |
test groups |
||
Group 4: |
0 |
||||
Dead fetuses (mean per dam) |
Control: |
0.0 |
0.01 ± 0.02 #4 (0.0 - 0.1) #3 |
control groups |
|
Group 2: |
0.1 ± 0.4 |
||||
Group 3: |
0.0 |
0.06 ± 0.09 (0.0 - 0.4) |
test groups |
||
Group 4: |
0.0 |
||||
#1: |
not audited by QAU |
||||
#2 |
Range of the total number of dead fetuses in the control groups. |
||||
#3 |
Range of the mean number of dead fetuses per dam in the cpntrol groups. |
||||
#4 |
Mean value of the mean numbers of dead fetuses per dam in the control groups. |
||||
Table: :Overview of the runts per group
IPDA |
|||||||
Group 1: Control |
Group 2: 10 mg/kg |
Group 3: 25 mg/kg |
Group 4: 75 mg/kg |
||||
Dam no. |
Runt Fetus no. |
Dam no. |
Runt Fetus no. |
Dam no. |
Runt Fetus no. |
Dam no. |
Runt Fetus no. |
7 |
6 m |
40 |
5 m |
56 |
10 f |
81 |
7 m |
7 f |
7 f |
- |
- |
120 |
5 f |
||
12 |
5 f |
44 |
5 f |
- |
- |
- |
- |
18 |
2 f |
8 f |
- |
- |
- |
- |
|
7 f |
- |
- |
- |
- |
- |
- |
|
Table :Background data of statistically significant incidences of skeletal retardations
Skeletal retardations |
Values observed in this study (LPT Report 35503)
[fetal incidence in % per group] |
LPT Background Data#1- Values of control groups (n = 18) and test groups (n = 55) not significantly influenced by any test compound; data taken from the years 2013 to September 2018
[fetal incidence in % per group] [Mean value ±SD (range)] |
||||
Sternebra(e) unossified |
Control: |
24.1 |
12.8 ± 7.8 2.2 - 25.7 |
control groups |
||
Group 2: |
38.5 ** #² |
|||||
Group 3: |
19.0 |
13.5 ± 7.8 4.0 - 39.0 |
test groups |
|||
Group 4: |
25.3 |
|||||
Skull incompletely ossified |
Control: |
0.0 |
0.37 ± 0.85 0.0 - 3.4 |
control groups |
||
Group 2: |
1.0 |
|||||
Group 3: |
1.7 * #² |
0.53 ± 0.84 0.0 - 2.9 |
test groups |
|||
Group 4: |
1.5 |
|||||
Total fetal skeletal retardations |
Control: |
68.3 |
56.4 ± 14.9 (20.7 - 77.1) |
control groups |
||
Group 2: |
80.0 ** #2 |
|||||
Group 3: |
66.5 |
53.6 ± 13.9 (25.2 - 80.5) |
test groups |
|||
Group 4: |
73.7 |
|||||
#1: |
not audited by QAU |
|||||
#2: |
Considered to be not test item-related as withinLPTbackground data range. |
|||||
*/**: |
(p ≤ 0.05/0.01) Fisher or Chi2- test |
|||||
No teratogenic or embryofetotoxic effects were recorded at any dose level.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 75 mg/kg bw/day
- Species:
- rabbit
- Quality of whole database:
- Klimisch 1 (guideline studies)
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
Studies in animals
OECD 414, rats
Partly cited from SIAR for SIAM 18 (Paris, April 2004):
"Based on the results of a dose finding study, three groups of 24 mated female Sprague-Dawley rats received 3-aminomethyl-3,5,5-trimethylcyclohexylamine by daily oral administration (gavage) at 0 (water = control), 10, 50 and 250 mg/kg/day from day 6 to day 19 post-coitum inclusive. On day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The study was designed according to OECD TG 414.There was no treatment-related death in any of the dams. Clinical signs were not observed, except for ptyalism in most females of the 250 mg/kg/day group (from day 11, 12, 13 or 14 post-coitum until hysterectomy; effect not considered as adverse). Loud breathing and hold-up in the esophagus were recorded in 4 females of this group and a significantly lower body weight gain (-35%) was recorded after the first three days of treatment. Thereafter, the body weights were similar to that of the controls. Over the whole treatment period, the difference remained slight (-10 %, not statistically significant). The net body weight gain was also significantly lower at this dose-level (-25 %) when compared to the control group. In the 250 mg/kg/day group, a significant decrease in food consumption was recorded during the treatment period (-7%), with a more marked effect during the first three days of treatment (-21 %). Abortions or total resorptions were not observed in any of the groups, nor were there any macroscopic findings that were ascribed to treatment with the test item. No treatment related effects were observed on pre- or post-implantation loss, fetal weight or sex-ratio. With respect to the fetuses, no test item related external, soft tissue or skeletal malformations or variations were detected. There was an increase in fetal incidence of incomplete ossification of the 5th sternebra in the 250 mg/kg bw/day group (106/134 fetuses = 79.1 %, p < 0.01 were affected vs. 88/130 = 67.7 % in control group, statistically insignificant on a fetus/litter basis). Because these findings are of low concern and occur only in the presence of maternal toxicity, they are considered to be secondary. In the same group there was a statistically nonsignificant increase in fetal incidence of incomplete ossification of the rib(s) (9/137 fetuses = 6.7 % vs. 2/130 = 1.5 % in control group. When ossification was incomplete, cartilage was generally present, demonstrating that the skeletal variations recorded corresponded to slight fluctuations in the time of ossification rather than being a persistent alteration. In conclusion, these findings were considered to be incidental and of no toxicological significance."
The NOEL for maternal toxicity was 50 mg/kg/day and the NOAEL for embryonic development was >250 mg/kg/day (CIT, 2002).
OECD 414, rabbits
ECHA requested to perform the OECD 414 study in a second species.
In this prenatal developmental toxicity study, the test item test item was administered orally to female rabbits at dose levels of 10, 25 or 75 mg/kg b.w./day from the 6th to 28th day of pregnancy.
Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 25 mg test item /kg b.w./day for the dams:
No test item-related premature death was noted for any of the dose groups. For the high dose group (75 mg test item /kg b.w./day), a reduction was noted for the body weight or the body weight gain. Also, a decrease was noted in food consumption at 75 mg test item /kg b.w./day. No changes were noted for the drinking water consumption. No changes in behaviour, external appearance or faeces were noted for the treatment groups. No test item-related pathological findings were noted during necropsy for the treatment groups. At 75 mg test item /kg b.w./day, a slight decrease was noted for the gravid uterus and the carcass weight.
The no-observed-adverse effect level (NOAEL) was above 75 mg test item /kg b.w./day for the fetuses and 25 mg test item /kg b.w./day for the rate of early resorptions/post-implantation loss: At the maternaltoxic dose level of 75 mg test item /kg b.w./day (reduced body weight, reduced body weight gain, reduced carcass weight and reduced food consumption), an increased number of early resorptions and accordingly an increased post-implantation loss were noted. Due to the increased number of early resorptions, the total resorption rate was slightly above the LPT historical control data (up to 1.3). The early resorption rate and the percentages of post-implantation loss were at the upper range but still within the LPT historical control data. No test item-related influence was noted on the mortality or body weight of the fetuses in the treatment groups. No test item-related deaths of the fetuses and no test item-related malformations, variations or retardations were noted.
Under the conditions of the study, the test item did not show any teratogenic potential.
The treatment with the test substance at 75 mg /kg b.w./day revealed increased incidences of early and total resorptions, and in consequence an increased percentage of post-implantation loss, which were statistically significantly different from the control. A treatment-related effect for the statistically significantly increased rate of early resorptions and the statistically significantly increased percentage of postimplantation loss cannot totally be excluded since both values were at the upper end of the LPT historical control data.
Although treatment-related and adverse effects cannot totally be excluded, the main reason for those increases is animal no. 82 that showed a total post-implantation loss. When assessing the number of live fetuses, the high dose group showed no remarkable difference to the control group (194 vs. 199 fetuses out of 20 dams with viable fetuses each). A feto-toxic effect of the test substance can be excluded, since no test item-related malformations, variations or retardations were evident.
Toxicity to reproduction: other studies
Description of key information
no other studies available
Justification for classification or non-classification
Because of the results of all available studies (OECD 414 in rats and rabbits, OECD 422 and OECD 443 in rats) the test item is not classified according to CLP regulation (1272/2008).
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
