Registration Dossier

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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

The aim of the reproduction screening study according to OECD guideline 421 was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development (LPT, 2020). Furthermore, this study is used as dose range finding study for the ongoing OECD 443 study (LPT, 2021). The test item was administered orally to rats at dose levels of 100, 300 or 750 mg/kg b.w./day. For general toxicity the NOAEL was considered to be 100 mg/ kg b.w./ day, based in clinical signs observed in animals treated with 300 and 750 mg test item/kg b.w./day. The NOAEL for reproductive toxicity is considered to be 300 mg/ kg bw/ day based on the reduced litter and pup weight observed in animals treated with 750 mg test item/kg b.w./day p.o. due to parental toxicity. The NOAEL for adverse effects on the reproductive parameters of the parental females is considered to be above 750 mg/ kg bw/day.

In a final decision on a compliance check dated 21 December 2018 (Decision number CCH-D-2114453561-52-01/F) ECHA requested to perform an extended one-generation reproductive toxicity study (Annex IX, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance specified as follows:

- At least two 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) with extension to mate the Cohort 1B animals to produce the F2 generation;

-  Cohorts 2A and 2B (Developmental neurotoxicity).

 

The OECD 443 study was initially scheduled to be completed in December 2020 before the REACH submission deadline. The dose levels of the OECD 443 study should have been determined based on the results of the DRF study according to OECD 421 (LPT, 2020). In this DRF study, the high dose group (group 4, 1000 mg/kg bw/day) was prematurely terminated due to toxicity and an additional test group was established (group 5, 750 mg/kg bw/day). As the evaluation of group 5 delayed the completion of the study report of the DRF study and therefore, the determination of the dose levels of the OECD 443 study, the start of the OECD 443 study had to be postponed. The start of the OECD 443 study was re-scheduled to 12 February 2020. The end of the In-life phase was 2 September 2020 and the first (unaudited) draft report is expected mid of march 2021. The dossier will be updated in a timely manner and submitted to ECHA.

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
June 2019 to October 2019
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 (LPT, 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:
other: CD/ Crl:CD(SD)
Details on species / strain selection:
For this study CD 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 comminly 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
Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Strain: Rat / CD / Crl:CD(SD)
- Sex: male and female
- Age: Group 1 to 4: 76 days, Group 5: 71 days
- Body weight (at 1st administration): Males: 426.9 g - 459.2 g, Females: 257.0 g - 277.4 g
- Housing: With the exception of the mating period (see Section 3.1.10 'Mating procedure'),
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
The animals were placed in the animal room as follows:
Male animals: On one side of the room with an empty row between groups.
Female animals: On the other side of the room with an empty row between groups.
- Diet (ad libitum): ssniff® R/Z V1324, ssniff Spezialdiäten GmbH, 59494 Soest, Germany
- Water (ad libitum): tap water
- Acclimation period: 7 days

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:
corn oil
Details on exposure:
The test item formulations were prepared for up to seven (7) administration days in advance and were administered orally at a constant administration volume/kg b.w. once daily. The test item was diluted in the vehicle to the appropriate concentrations. Between administrations, the test item formulations were stored at 2°C to 8°C. Prior to each administration, the test item formulations were allowed to warm up to room temperature for at least 30 minutes and stirred on a magnetic stirrer for approximately 3 minutes to ensure homogeneity. The amount of test item was adjusted the animals current body weight 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 were paired monogamously: 1 male and 1 female animal were placed in one cage during the dark period. The female was placed with the same male until pregnancy had occurred or two 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 GD 0) was considered to be the day on which sperm was found.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item-vehicle formulations, two (2) aliquots of approximately 3 mL each were taken at the following times and stored at -20°C ± 10% until analysis at LPT:
- At start of the treatment period (first dosing day): Analysis of concentration and homogeneity,
- At start of administration, during (middle) administration and before administration to the
last animal of each dose level group (3 samples / dose level group; groups 2, 3, 4). Number of samples: 9 (18 aliquots)
- At start of the treatment period of group 5 (first dosing day): Analysis of concentration and homogeneity At start of administration, during (middle) administration and before administration to the last animal of each dose level group (3 samples / dose level group; group 5). Number of samples: 3 (6 aliquots)
Towards the end of the 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, 3, 5). Number of samples: 3 (6 aliquots)
Sum of all samples: 15 (30 aliquots)
The samples were labelled with the study number, species, type of sample, concentration, aliquot number, test day, sampling time and date.
The samples were analysed at LPT using a validated HPLC-UV method. The following parameters were determined: Linearity, Accuracy, Precision, Sensitivity, Specificity, Stability. The test item formulation analysis for the concentration and homogeneity revealed concentrations that ranged between 102.4% and 109.7% of the nominal concentration indicating correctly prepared and homogeneous test item formulations.
Duration of treatment / exposure:
The study animals were treated during the following periods:
Males: 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 43 (one day before sacrifice on test day 44).
Females: 2 weeks prior to mating (from test day 15 until test day 29), during the mating period (from test day 30 until test day 41 at maximum) and during the lactation period until test day 64 to 68 (corresponding to lactation day 13). The last dosing was always one day before
sacrifice.
Frequency of treatment:
Once daily
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 at least once daily, all animals were observed for signs of functional/behavioural toxicity. Oestrus cyclicity is evaluated followed by daily monitoring of vaginal smears from beginning of treatment 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. After counting on PND 4, the litters were adjusted to 10 pups per litter (if possible, 5 pups per sex and litter) by eliminating (culling) surplus pups using a randomization scheme
-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 44
Pups: On lactation day 13
Dams (surviving dams): Between lactation day 14 and 16
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
low dose group
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
intermediate dose group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
high dose group (this dose was
terminated after 3 treatments (males) or 1
treatment (females))
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
high dose group
No. of animals per sex per dose:
10 males and 10 females
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 of this study were selected by the Sponsor based on available toxicological data. In the 90-day and the 16-day gavage studies with the test item, the NOAEL was considered to be 500 mg/kg b.w. (both NTP, 1986). In the 16-day study, at the high dose of 1000 mg/kg b.w., only decreased body weights (13.7% for males, 6.7% for females) were observed. One female rat that received 1000 mg/kg b.w. died in the 90-day gavage study. Final mean body weights for rats were not clearly related to dose in this study. Therefore, 1000 mg/kg b.w. was selected as
the highest dose level in this OECD 421 study.

At 1000 mg test item/kg b.w./day, the female animal no. 73 was found dead after the first administration. Hence, the high dose group (1000 mg test item/kg b.w./day) was terminated immediately for the females and after the third administration for the males and 750 mg test item/kg b.w./day was selected as the new high dose level.

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 recorded twice daily. Animals that died prematurely were necropsied as soon as possible after exitus. In the case of prematurely sacrificed animals, blood sampling for Thyroid hormone determination was performed.
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.

MORTALITY
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 were weighed on each day of dosing for dose adjustment and at sacrifice; the individual body weights were recorded. The report includes weekly values for the male animals (starting on test day 15) and the body weight on the day of sacrifice.
For the female animals the body weights on the following days are given in the report:
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.
Food residue (or total food left) was weighed and recorded as follows:
Study period Males Females
Pre-mating period TD 22 and TD 29 TD 22 and TD 29
Mating period None None
Gestation period Not applicable GD 7, GD 14 and GD 20
Lactation period Not applicable LD 1, LD 8 and LD 13

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
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group

T4 DETERMINATION
Blood samples were taken under isoflurane anaesthesia and processed for serum always at the same time of the day, if possible, as scheduled below. Blood sampling was performed randomised from the parental animals.
Oestrous cyclicity (parental animals):
During the 14-day pre-exposure period (TD 1 to TD 14), the oestrus cycle of the female animals was monitored to yield study groups of at least 10 animals each with a regular oestrus cycle. Animals that failed to exhibit typical 4 to 5 day cycles were excluded from the randomisation process on test day 14 that was performed to allocate the female animals to the test groups of the main study. The female animals that were used for the main study revealed 2 or 3 typical 4 to
5 day cycles during the 14-day pre-exposure period. After the allocation of the animals to the study groups and the start of treatment, the oestrus cycle was further monitored during the pre-mating period and the mating period until a positive mating sign was noted. Finally, a vaginal smear was taken in the morning of the day of scheduled necropsy.
Sperm parameters (parental animals):
Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testicle and one epididymis of all males of groups 1 and 5.
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. Any abnormal behaviour of the offspring would have been recorded. However, no abnormal behaviour was noted for the pups. Live pups were counted, sexed and weighed on post-natal days 1, 4 and 13.
Number of pups absolute
- at birth (alive and dead)
- after 4 and 13 days of life
Number of pups per dam
- at birth (alive and dead)
- after 4 and 13 days of life
Number of male and female pups
- at birth
- after 4 and 13 days of life
Number of stillbirths
- absolute
- per dam
Number of pups with malformations
- absolute
- per dam

- 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 pup size (i.e. 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 (if possible, 5 pups per sex and 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.
Blood samples were taken under isoflurane anaesthesia and processed for serum always at the same time of the day, if possible, as scheduled below. For the blood withdrawal of the pups, the litter sequence was determined by randomization of the dams; pups were selected from the dams in ascending order: On LD 4, the first male and female pup per dam selected for culling were used, if possible. On LD 13, the first male and female pup per dam were used.

- 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 44
Dams (surviving dams): Between lactation day 14 and 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 sulphide for about 10 minutes to stain possible implantation sites in the
endometrium according to SALEWSKI.
The organ weights below of all animals were determined before fixation. Paired organs were weighed individually and identified as left or right.
Organs to be weighed - adult animals: Epididymis (2), Thyroid (1) (including parathyroids), Ovary (2), Prostate and seminal vesicles with coagulating glands, as a whole, Testicle (2), Uterus including cervix

The organs or parts thereof of all adult animals were preserved in the appropriate fixative:
Fixative: modified Davison's solution
-Epididymis (2)
-Testicle (2)

Fixative: 7% neutral buffered formalin
- Gross lesions observed
- Ovary and oviduct (2)
- Prostate
- Seminal vesicles with coagulating glands
- Thyroid including parathyroids
- Uterus with cervix

HISTOPATHOLOGY
Histopathological examination was performed on the organs listed below of all adult animals of the control group and the high dose group (groups 1 and 5).
Sections to be prepared and evaluated - groups 1 and 5 adults:
Organ HE-stained sections PAS-stained sections
Epididymis 2 (left and right) 1
Ovary 2 (left and right) none
Testicle 2 (left and right) 1
The histotechnique was performed at LPT.
Histopathological evaluation was performed by AnaPath GmbH, AnaPath Services according to all relevant AnaPath SOPs.
Detailed histopathological examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure was performed on one testicle and one epididymis of all males of groups 1 and 5.
Postmortem examinations (offspring):
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.
The thyroid of 1 male and 1 female pup from each litter was preserved in 7% formalin. Thyroid weight was determined after fixation.
Statistics:
The statistical evaluation of the parametrical values was done by Provantis(Provantis® integrated preclinical software, version 10.2.1.0, 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 nonnormality 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).

The following statistical methods were used for data not captured by Provantis software (reproductive data): Homogeneity of variances was test using the BARTLETT's test. In case of homogeneity, intergroup comparison was performed by the DUNNETT's test (p ≤ 0.05 and p ≤ 0.01). In case of heterogeneity of variances, a stepwise comparison of the test groups with the control group was performed using a STUDENT's t-test (p ≤ 0.05 and p ≤ 0.01).
Statistically significantly different data are indicated in the summary tables and
in the tables of the result sections.
Reproductive indices:
The following indices were calculated for each group:
Female Fertility Index [%] = (Number of pregnant rats/ Number of rats used) 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 [%] = (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:
effects observed, treatment-related
Description (incidence and severity):
- Males:
In the control group, the male animal no. 2 was noted with a haemorrhagic canthus on TD 27 and TD 28. This observation was considered to be spontaneous.
In the low dose group (100 mg test item/kg b.w./day), slight salivation was noted for 5 male animals on 5 (no. 27), 2 (no. 25) or 1 (nos. 22, 23 and 24) test days between TD 27 and TD 43. Salivation started immediately to 5 minutes after administration and lasted until 20 to 60 minutes after administration. Due to the low severity, the low number of days observed and the short duration salivation was considered to be not test item-related.
At 300 mg test item/kg b.w./day, slight to moderate salivation, slightly reduced motility, a haemorrhagic nose/snout were noted as well as yellow discoloured faeces and urine.
As salivation, reduced motility and the discolouration of the urine was noted inalmost all animals of the intermediate dose group and were noted with increased incidence, severity and number of days in the high dose group, these clinical signs were considered to be test item-related.
In the high dose group (750 mg test item/kg b.w./day), slight to severe salivation, moderately reduced motility, yellow discoloured urine and pale faeces were noted for the male animals,
Salivation, reduced motility and yellow discoloured urine were noted in almost all male high dose animals with an increased severity for salivation and reduced motility in comparison to the intermediate dose group. As also for reduced motility and the discolouration of the urine the number of observed days per animal was increased compared to the intermediate dose group, salivation, reduced motility and the yellow discoloured urine were considered to be test item-related.
The male animals of group 4 (1000 mg test item/kg b.w./day) displayed moderate salivation, moderately reduced motility on TD 16 and severe salivation and severely reduced motility on TD 17 and also yellow discoloured urine was noted on TD 16 and 17 for all male animals. Additionally, the male animal no. 65 was noted with a haemorrhagic nose/snout.
Group 4 was terminated for the male animals on TD 18 due to pronounced signs of toxicity in form of moderate to sevee salivation and moderately to severel reduced motility and on TD 16 for the female animals due to one premature death of one female animal for animal welfare reasons.
- Start and duration of clinical signs (Males):
At 300 mg test item/kg b.w./day, salivation started immediately to 5 minutes after administration and ended between 20 and 60 minutes after administration. Reduced motility started immediately to 5 minutes after administration and ended 20 to 60 minutes after administration or 2 to 6 hours after administration. For the animals of the high dose group (750 mg test item/kg b.w./day), salivation was observed immediately to 5 minutes after administration and ended 20 to 60 minutes after administration. Reduced motility started 5 to 20 minutes after test item administration and lasted for the first administration days consistently during the day. Thereafter, reduced motility ended 6 to 24 hours after administration and in the further course of the study, reduced motility ended 2 to 6 hours after administration.
In group 4 (1000 mg test item/kg b.w./day), salivation and reduced motility started immediately to 5 minutes after test item administration and ended 2 to 6 hours after test item administration.
- Females (Pre-mating period, gestation period, lactation period):
No alterations of the behaviour, external appearance or the faeces were noted in the female animals of the control group. In the low dose group (100 mg test item/kg b.w./day), 6 of 10 females were noted with slight to moderate salivation.
At 300 mg test item/kg b.w./day, 8 of 10 females displayed slight to severesalivation and 3 of 10 animals were noted with slightly reduced motility.
Due to the predominantly low severity, the low number of animals that were affected and as reduced motility was observed for only one day per animal, reduced motility was considered to be not test item-related. However, as salivation was noted for almost all test item-treated animals and was observed for a high number of days for some animals, salivation was considered to be test item-related. In the high dose group (750 mg test item/kg b.w./day), slight to severe salivation was noted for 7 of 9 females in the pre-mating/mating period and in all female animals in the gestation and lactation period. Moderately reduced motility and yellow discoloured urine were noted in all females in all three periods of the study. Due to the high incidence during all three study periods and the high number of days the alterations were observed, salivation, reduced motility and the yellow discoloured urine were considered to be test item-related.
- Start and duration of clinical signs (Females): In the low and intermediate dose group (100 or 300 mg test item/kg b.w./day), salivation started immediately to 5 minutes after administration and ended 20 to 60 minutes after administration for the female animals. For the female animals dosed with 750 mg test item/kg b.w./day, during the premating/ mating, gestation and lactation period, salivation started immediately to 5 minutes after administration and lasted until 20 to 60 minutes after administration.
Reduced motility started on TD 15 of the pre-mating/mating period immediately to 5 minutes after administration and lasted consistently during the day until TD 19. From TD 20 to TD 22 reduced motility started immediately to 5 minutes after administration and ended 6 to 24 hours after administration. From TD 23 until the end the pre-mating/mating period, reduced motility was noted immediately to 5 minutes after administration until 2 to 6 hours after administration. The duration of reduced motility in the gestation and lactation period for the high dose group was also observed immediately to 5 minutes after administration until 2 to 6 hours after administration.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
No prematurely deceased male or female animals were noted in the control group and in the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day).
In the high dose group (750 mg test item/kg b.w./day), two female animals (nos. 96 and 100) were found dead during their gestation day 21 or 20. Necropsy revealed for dam no. 96 a slightly enlarged spleen and for no. 100 thickened thyroids (left and right) and spongeous and dark-red discoloured tissue in the upper thorax region. Additionally, both animals were noted with a discharge of yellowish liquid from the vagina. On the day before and on the day of its death, dam no. 96 was noted with piloerection, slightly laboured breathing and lethargy. Moderate salivation, slightly or moderately reduced motility and yellow discoloured urine were noted on nearly all gestation days. Dam no. 100 as well displayed slightly to moderately reduced motility and yellow discoloured urine on all or several gestation days.
While the death of dam no. 96 was considered to be test item-related, the death of dam no. 100 was deemed not to be directly related to the test item.
The female animal no. 73 of the prematurely terminated group 4 (1000 mg test item/kg b.w./day) was found dead in the morning of TD 16, i.e. after the first administration. Hence, the female animals of group 4 were immediately terminated.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
In the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day),
no test item-related changes in body weight were noted for the male animals in
comparison to the control group.
At 750 mg test item/kg b.w./day, a reduction was noted for the body weight
during the whole treatment period from test day 15 until test day 43 (at maximum
13.7% below the value of the control group on test day 43, statistically significant
for all evaluated test days at p ≤ 0.01). This distinctly reduced body weight noted
for the male animals of the high dose group was considered to be test itemrelated.
No test item-related changes in the body weight gain were noted between the
control group and the low and intermediate dose groups (100 or 300 mg test
item/kg b.w./day).
According to the reduced body weight of the males dosed with 750 mg test
item/kg b.w./day, also the body weight gain was decreased throughout the
treatment period.
Female animals:
For the females, no test item-related influence was noted on the body weight of
the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day) in
comparison to the control group.
At 750 mg test item/kg b.w./day, in the gestation period, a decreased body
weight was noted for the GD 14 and GD 20 (6.4% and 13.0% below the value of
the control group, statistically significant at p ≤ 0.01 on GD 20). In the lactation
period, the decreased body weight was still present at LD 1 (9.3% below the
value of the control group, statistically significant at p ≤ 0.01). Thereafter, the
body weight recovered and no statistically significant decrease was noted on LD 4
and LD 13 (7.9% and 2.5% below the value of the control group). The distinctly
decreased body weight during the gestation period was considered to be test item related.
Also, the body weight of the pups and the litter weights on LD 1 were decreased. Therefore, the
reduced female body weights at the end of the gestation period and at the
beginning of the lactation period were considered to be not only due to a reduction
in the body weight of the parental female animals but also partly due to the
reduced litter weights. Accordingly, no influence on the body weight gain of the female animals was
noted after dosing of 100 or 300 mg test item/kg b.w./day during the pre-mating,
gestation or lactation period in comparison to the control group.
In the high dose group (750 mg test item/kg b.w./day), the decreased body weight
noted for the gestation period led to a decrease in body weight gain during the
gestation period (32.2% body weight in group 5 compared to 53.2% body weight
gain in the control group). Therefore, also the decreased body
weight gain during the gestation period was considered to be test item-related.

Males and females:
No test item-related influence on the body weight at autopsy was noted for the male and female animals of the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day) in comparison to the control group.
In the high dose group (750 mg test item/kg b.w./day), a reduction was noted for the male and female animals (13.9% or 10.7% below the value of the control group, for both statistically significant at p ≤ 0.01). As the reduction for the male and female animals is in accordance with the decreased body weight, the body weight at autopsy was considered to be test item-related.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Males:
During the pre-mating period (TD 15 to TD 29) no test item-related difference in
food consumption was noted between the male rats of the control group and
those of the dose groups (100, 300 or 750 mg test item/kg b.w./day).
Statistically significantly decreased values for the food consumption were noted in
the test week 3 (TD 15 to TD 22) for the low and intermediate dose groups (7.7%
or 11.1% below the value of the control group, statistically significant at
p ≤ 0.05 or p ≤ 0.01).
However, as the food consumption recovered in test week 4 for the low and
intermediate dose group and no statistically significant difference was noted for
the high dose group in both test weeks, the reduced food consumption was
considered to be not test item-related.
The relative food consumption during the pre-mating period is given in Figure 2-1
as the mean daily values per group in test week 3 (TD 15 to TD 22) and test week
4 (TD 22 to TD 29).
Females:
No test item-related differences in food consumption were noted between the
female rats of the control group and the female rats of the dose groups (100, 300
or 750 mg test item/kg b.w./day) during the pre-mating, the gestation and the
lactation period.
Statistically significant differences for the food consumption were noted between
the female animals of the control group and the females of the dose groups. However, these differences in food consumption were consideredto be not test item-related.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual appraisal revealed no test item-related changes in the consumption of
drinking water for the male and female rats treated with 100, 300 or 750 mg test
item/kg b.w./day.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
T4 Determination- Males:
No test item-related changes were noted for the T4 levels of the male animals of the dose groups (100, 300 or 750 mg test item/kg b.w./day).
In the low dose group (100 mg test item/kg b.w./day), slightly increased T4 levels were noted (12.1% above the value of the control group, not statistically significant). However, as no statistical significance and no dose response-relationship was present, the slightly increased T4 levels were considered to be spontaneous. All individual values except from 1 control animal (animal no. 5, 38.9 nmol/L) and two animals of group 5 (animals no. 85 and 86, 41.7 and 42.8 nmol/L, respectively) are within the range of the LPT background data.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In the intermediate dose group (300 mg test item/kg b.w./day), changes in the behaviour in form of salivation and reduced motility were noted for the male animals and salivation was noted for the female animals.
At 750 mg test item/kg b.w./day, salivation, and reduced motility was noted for the male and female animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Males and females:
Histopathological examinations were performed on testes, epididymides and ovaries of
animals from Groups 1 and 5 (0 and 750 mg/kg bw/day, respectively). No treatment-related
microscopic changes that could be attributed to treatment with the test item were observed in
these organs of any animals (including survivors and decedents) examined. All recorded findings were deemed to be incidental or were within the range of normal background alterations that may be recorded in animals of this strain and age.
All findings were rated grade 1 (Minimal / very few / very small) and included mononuclear cell focus/foci, interstitium, unilateral or bilateral, grade 1 in the epididymides of animals from the control group and the high dose group and sertoli cell vacuolation, focal/sporadic (single cell),
unilateral, grade 1 in the testes of animals from the control group and the high dose group.
In conclusion, the histopathology revealed that the test item produced no morphological
evidence of toxicological properties in testes, epididymides and ovaries of adult animals
receiving 750 mg/kg bw/day of the test item.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related 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 dose groups (100, 300 or 750 mg test item/kg b.w./day). None of the females showed a complete oestrus cycle during the mating period.
Deviations from the typically expected sequence of oestrus cycles were noted in the form of longer sequences (more than five consecutive dioestrus stages). Such elongated dioestrus sequences were noted for one female each in the control and the high dose group and for two females of the low dose group. However, these elongated dioestrus sequences were not considered to be test item-related, as the incidences were only small, one control animal was also affected and no influence on the reproductive outcome was observed.
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence on the fertility index of the female rats was noted for any of the dose groups (100, 300 or 750 mg test item/kg b.w./day).
All female rats except animal no. 98 were successfully mated (confirmed by sperm detection; for animal no. 98 the sperm detection gave a false-positive result). However, one female animal of the high dose group did not become pregnant, leading to a fertility index of 89% for the female animals of the high dose group.
The occurrence of one non-pregnant female at the high dose level was considered to be spontaneous and not test item-related.
Males and females:
No premature death was noted for the male and female animals of the low and intermediate dose groups (100 or 300 mg test item/kg b.w./day).
At 750 mg test item/kg b.w./day, two female animals (nos. 96 and 100) were found dead on GD 20 or GD 21.
At 1000 mg test item/kg b.w./day, the female animal no. 73 was found dead after the first administration. Hence, the high dose group (1000 mg test item/kg b.w./day) was terminated immediately for the females and after the third administration for the males.

General toxicity-
Parental male and female animals:
Two female animals dosed with 750 mg test item/kg b.w./day were found dead on GD 20 or GD 21, respectively.
In the intermediate dose group (300 mg test item/kg b.w./day), changes in the behaviour in form of salivation, reduced motility and yellow discoloured urine were noted for the male animals and salivation was noted for the female animals.
At 750 mg test item/kg b.w./day, salivation, reduced motility and yellow discoloured urine was noted for the male and female animals.
In the high dose group (750 mg test item/kg b.w./day), reductions were noted for the body weight and the body weight gain.
No test item-related influence was noted on the food consumption.
No test item-related pathologic findings were observed during macroscopic post mortem examination the male and female animals.
Histopathological examination of the epididymides and the testes of the male and of the ovaries of the female high dose animals revealed no test item-related changes.
No influence on the T4 levels were noted for the male animals.

Reproductive toxicity-
Parental females:
No influence was noted on the fertility of the male and female animals in any of the dose groups.
Dose descriptor:
NOAEL
Effect level:
> 750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
Clinical signs:
no effects observed
Description (incidence and severity):
No abnormal behaviour was noted for the pups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
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 dose groups (100, 300 or 750 mg test item/kg b.w./day).
Furthermore, 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 dose groups.
Pre-cull period:
No difference between the control group and the dose groups (100, 300 or 750 mg test item/kg b.w./day) was noted for the viability index (group level) between lactation days 1 and 4. The viability index from LD 0/1 to LD 4 ranged between 93.1% (low dose group) and 99.3% (control group).

Post-cull period:
No test item-related influence on the viability index was noted for the dose groups (100, 300 or 750 mg test item/kg b.w./day) after culling on LD 4. One dead pup each was noted in the low and intermediate dose group between LD 5 and LD 13.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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 low and intermediate dose groups (100 or 300 mg test item/kg b.w./day) on LD 1, LD 4 and LD 13.
In the high dose group (750 mg test item/kg b.w./day), a reduced body weight was noted for the male and female pups on LD 1, LD 4 and LD 13 (between 18.2% and 21.9% below the value of the control group, statistically significant for all values at p ≤ 0.01). The distinctly decreased body weight was noted on LD 1, LD 4 and on LD 13 and therefore, was considered to be test item-related.
One runt each was noted in the control group and in the low dose group. The single occurrence of one runt in the control group and in the low dose group was considered to be spontaneous.
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):
T4 determination:
No test item-related changes were noted for the T4 levels of the male and female pups of the dose groups (100, 300 or 750 mg test item/kg b.w./day) on LD 13.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related influence was noted on the ano-genital distance of the male and the female pups of the dose groups (100, 300 or 750 mg test item/kg b.w./day).
A reduction of the absolute ano-genital distance was noted for male and female pups of the high dose group (14.0% and 11.9% below the value of the control group, statistically significant for the male animals at p ≤ 0.01). However, as no difference was noted for the relative ano-genital distance, the reduced absolute ano-genital distance was due to the reduced body weight of the pups. Therefore, the test item was considered to have no influence on the ano-genital distance.
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 (100, 300 or 750 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):
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 100, 300 or 750 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:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
REPRODUCTIVE PARAMETERS No test item-related influence was noted on the number of implantations, the number of resorptions, the number of live born pups and the ratio of male to female pups for any of the dose groups.
PUP BODY WEIGHT In the high dose group (750 mg test item/kg b.w./day), a reduced body weight was noted for the male and female pups on LD 1, LD 4 and LD 13 (between 18.2% and 21.9% below the value of the control group, statistically significant for all values at p ≤ 0.01)

LITTER WEIGHT In the high dose group (750 mg test item/kg b.w./day), reduced litter weights were noted for the male and the female litters on the lactation days 1, 4 and 13 (between 12.3% and 22.6% below the value of the control group, statistically significant for the female litters and for the male and female litters combined on LD 13 at p ≤ 0.01).
EXTERNAL EXAMINATION The external macroscopic examination of the pups after sacrifice revealed no gross abnormalities.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: reduced litter and pup weight observed in animals treated with 750 mg test item/kg b.w./day p.o. due to parental toxicity

Text table 3‑1:      Group size and dose levels.

Group

test item dose #

[mg/kg b.w./day, p.o.]

Number and

sex of animals

Animal number[1]

1

0

(vehicle control)

10

10

m

f

1 - 10

11 - 20

2

100

(low dose group)

10

10

m

f

21 - 30

31 - 40

3

300

(intermediate dose group)

10

10

m

f

41 - 50

51 - 60

4[2]

1000

(high dose group)

10

10

m

f

61 - 70

71 - 80

5

750

(high dose group)

10

10

m

f

81 - 90

91 - 100

#:       no correction factor was used.

m:     male

f:       female

 

[1]             Including spare animals; 8 female animals per group were evaluated.

[2]             All group 4 females were terminated on 20 June 2019 on TD 16, i.e. after the first administration. All group 4 males were terminated on 22 June 2019 on TD 18, i.e. after the third administration.

 

Text table 4‑1:      Reproductive outcome of the female animals per group.

test item

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 5

750 mg/kg

Group 4 #1

1000 mg/kg

Females started dosing

10

10

10

10

10

Females used for pairing

10

10

10

10

n.a.

Females excluded

0

0

0

1 #2

n.a.

Females mated #3

10

10

10

9

n.a.

Females not pregnant

0

0

0

1

n.a.

Females pregnant #4

10

10

10

8

n.a.

Prematurely deceased females

0

0

0

2 #5

1

Females with litters #6

10

10

10

6

n.a.

Females that delivered only stillbirth

0

0

0

0

n.a.

Females with live born pups

10

10

10

6

n.a.

Females surviving the lactation period until scheduled sacrifice

10

10

10

6

n.a.

n.a.:   not applicable

#1:     Group 4 was terminated on TD 16 (females), i.e. after the first administration, or TD 18 (males), i.e. after the third administration due to signs of pronounced toxicity and a premature death (for details see Section 4.2 'Mortality' and 4.3 'Clinical signs'). No data of group 4 are available except for mortality and clinical signs.

#2:     In group 5, for animal no. 98 sperm detection was false-positive and animal no. 98 was erroneously considered to be mated. Therefore, it was excluded from the study (see Section 2.12 'Study Plan deviations').

#3:     Number of animals at start of the pre-mating and mating period.

#4:     Number of evaluated animals at start of the gestation period.

#5:     In group 5, the female animal no. 96 was found dead on GD 21 and the female animal no. 100 was found dead on GD 20.

#6:     Number of evaluated animals at start of the lactation period.

 

Text table 4‑2:      Changes in behaviour, external appearance or faeces noted for male animals of group 3.

Observations in group 3

(300 mg test item/kg b.w./day)

Observation

Affected males

First to last seen

Number of days

observed

(min - max)

Salivation

(slight to moderate)

9 of 10

TD 27 - TD 43

4 - 15

Motility reduced

(slight)

10 of 10

TD 16 - TD 41

10 - 22

Discolouration, urine

(yellow)

8 of 10

TD 20 - TD 24

5

Discolouration, faeces

(yellow) #

10 of 10

TD 16 - TD 17

2

Haemorrhagic nose/snout #

1 of 10

(no. 42)

TD 27

1

#:       Considered to be spontaneous due to low number of affected animals and/or low number of days observed.

 

Text table 4‑3:      Changes in behaviour, external appearance or faeces noted for male animals of group 5.

Observations in group 5

(750 mg test item/kg b.w./day)

Observation

Affected males

First to last seen

Number of days

observed

(min - max)

Salivation

(slight to severe)

9 of 10

TD 21 - TD 44

2 - 21

Motility reduced

(slight to moderate)

10 of 10

TD 15 - TD 43

29

Discolouration, urine

(yellow)

10 of 10

TD 28 - TD 38

11

Discolouration, faeces

(pale) #

1 of 10

(no. 81)

TD 21 - TD 22

2

#:       Considered to be spontaneous due to low number of affected animals and low number of days observed.

 

Text table 4‑4:      Changes in behaviour, external appearance or faeces noted for female animals of group 2.

Test item-related observations in group 2

(100 mg test item/kg b.w./day)

 

Affected females per period #1

 

Incidence

Observation

Pre-mating/

Mating

Gestation

Lactation

First to last seen

Number of days

observed

(min - max)

Salivation

(slight to moderate)

6 of 10

6 of 10

1 of 10

TD 27 - LD 4

1 - 11

 

Text table 4‑5:      Changes in behaviour, external appearance or faeces noted for female animals of group 3.

Observations in group 3

(300 mg test item/kg b.w./day)

 

Affected females per period #1

 

Incidence

Observation

Pre-mating/

Mating

Gestation

Lactation

First to last seen

Number of days

observed

(min - max)

Salivation

(slight to severe)

8 of 10

10 of 10

7 of 10

TD 27 - LD 13

4 - 27

Reduced motility (slight) #²

3 of 10

0 of 10

0 of 10

TD 27

1

#1:         The number of females examined per period is given in Section 4.1.

#²:     Considered to be not test item-related due to low number of affected animals and low number of days observed.

 

Text table 4‑6:      Changes in behaviour, external appearance or faeces noted for female animals of group 5.

Test item-related observations in group 5

(750 mg test item/kg b.w./day)

 

Affected females per period #1

 

Incidence

Observation

Pre-mating/

Mating

Gestation

Lactation

First to last seen

Number of days

observed

(min - max)

Salivation

(slight to severe)

7 of 9

8 of 8

6 of 6

TD 21 - LD 13

5 - 29

Reduced motility (slight to moderate)

9 of 9

8 of 8

6 of 6

TD 15 - LD 13

35 - 53

Discolouration of urine (yellow)

9 of 9

8 of 8

6 of 6

TD 28 - LD 13

17 - 35

Lethargy #²

none

1 of 8

none

GD 20 - GD 21

2

Piloerection #²

none

1 of 8

none

GD 20 - GD 21

2

Laboured breathing

(slight) #2

none

1 of 8

none

GD 20 - GD 21

2

#1:         The number of females examined per period is given in Section 4.1.

#²:     Lethargy, piloerection and laboured breathing were noted only for one animal on the two days before its premature death. Hence, these observations were considered to be pre-mortal symptoms.

 

Text table 4‑7:      Body weight gain during the treatment period (TD 15 - TD 43) - male animals.

Body weight gain - Males #

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 5

750 mg/kg

(TD 15 to TD 43)

+15.8 %

+14.0 %

+12.2 %

+7.2 %

 

Text table 4‑8:      Body weight gain of the female animals during the pre-mating/mating, gestation and lactation period.

Body weight gain - Females

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 5

750 mg/kg

pre-mating period

(TD 15 to TD 29)

+0.5%

+1.7%

+2.6%

+3.6%

gestation period

(GD 0 to GD 20)

+53.2%

+50.8%

+45.2%

+32.2%

lactation period

(LD 1 to LD 13)

+9.4%

+11.2%

+10.8%

+17.6%

 

Text table 4‑9:      Statistically significant differences for the food consumption that were considered to be not test item-related.

Period

Ref. table no.

Group

Test week

Increase  ­

Decrease ¯

(% change to control)

Statistical significance

Reason

Pre-mating period

5-2

3

TD 15 - TD 22

¯ (-12.3%)

p £ 0.01

A

5

TD 15 - TD 22

¯ (-16.1%)

p £ 0.01

A

Gestation period

5-3

3

GD 0 - GD 7

¯ (-11.1%)

p £ 0.01

A, B

5

GD 0 - GD 7

­ (+18.8%)

p £ 0.01

A, B

5

GD 14 - GD 20

­ (+34.7%)

p £ 0.01

A, B

A:      No statistically significant difference in the following test weeks.

B:      No dose response-relationship present.

 

Text table 4‑10:    Stage of the oestrus cycle at necropsy (LD 14).

Stage of oestrus at necropsy #

Group 1

Control

Group 2

100 mg/kg/day

Group 3

300 mg/kg/day

Group 4

750 mg/kg/day

Pro-oestrus

-

1 of 10

-

-

Oestrus

-

-

-

-

Metoestrus

1 of 10

-

1 of 10

-

Dioestrus

9 of 10

9 of 10

9 of 10

6 of 6

- :

not detected

#:

The non-pregnant and the excluded animals were not considered in this table.

 

Text table 4‑11:    Comparison of the T4 levels of the male animals per group with the LPT background data.

Parameter

Values from this study #2

Mean values per group ±SD

(range of individual values)

LPT Background Data #1

obtained from the control groups of 11 OECD 422 / 421 studies performed at LPT from 2016 - 2018

Males

(mean value from 110 control males ± SD

(range of the individual values)

T4 levels

- males -

(nmol/L)

Group 1

51.97 ± 7.20

(38.9 - 59.5)

67.62 ± 13.98

(42.9 - 114.8)

Group 2

58.25 ± 6.74

(48.5 - 69.0)

Group 3

51.55 ± 6.23

(43.7 - 62.9)

Group 5

52.59 ± 7.55

(41.7 - 66.9)

#1:     Data not audited by QAU.

 

Text table 4‑12:    Mean length and mean number of oestrus cycles.

Parameter

Group 1

Control

Group 2

100 mg/kg

Group 3

300 mg/kg

Group 5

750 mg/kg

Pre-mating: Test day 15 (start of treatment) until pairing (test day 29)

Mean cycle length [days]

4.08

4.19

4.30

4.46

Number of cycles

2.1

2.4

2.1

2.0

 

Text table 4‑13:    Fertility indices per group.

Group / Dose level

Fertility index [%]

Pregnant rats / rats used

Group 1

(Control)

100

10 / 10

Group 2

(100 mg/kg)

100

10 / 10

Group 3

(300 mg/kg)

100

10 / 10

Group 5

(750 mg/kg)

  89 #

  8 /   9 #

#:       Including the two pregnant females that were found dead on GD 20 or GD 21.

 

Text table 4‑14:    Gestation indices per group.

Group / Dose level

Gestation index %

Dams with live pups / pregnant rats

Group 1

(Control)

100

10 / 10

Group 2

(100 mg/kg)

100

10 / 10

Group 3

(300 mg/kg)

100

10 / 10

Group 5

(750 mg/kg)

100 #

6 / 6 #

#:       Excluding the two pregnant females that were found dead on GD 20 or GD 21.

 

Text table 4‑15:    Litter weight of the male and female pups per dam on LD 1, LD 4 and LD 13.

Litter weight for the male and female pups combined

Group

Parameter

LD 1

LD 4

LD 13

Group 1

control

mean per dam

99.5 g

140.6 g

292.3 g

difference to control

n.a.

n.a.

n.a.

Group 2

100 mg/kg b.w./day

mean per dam

102.6 g

134.2 g

273.8 g

difference to control

+3.1%

-4.6%

-6.3%

Group 3

300 mg/kg b.w./day

mean per dam

92.5 g

127.2 g

269.9 g

difference to control

-7.0%

-9.5%

-7.7%

Group 5

750 mg/kg b.w./day

mean per dam

83.8 g

111.4 g

235.7 g **

difference to control

-15.8%

-20.8%

-19.4%

n.a.:   not applicable

**:     Statistically significant at p ≤ 0.01 (Dunnett test)

 

Text table 4‑16:    % difference of the T4 levels of the pups in comparison to the control group.

Pup T4 level

% difference to the control group

Group 2

Group 3

Group 5

Male pups

LD 13

-3.0

-2.4

-8.1

Female pups

LD 13

+3.5

-1.6

-3.8

Male + Female pups

LD 13

+0.1

-2.0

-6.0

 

Tabular summary report of effects on reproduction development (Annex 3 of OECD method 421)

OBSERVATIONS

VALUES

Dosage (mg/kg)

0 (control)

100

300

750

Pairs started (N)

10

10

10

10

Oestrus cycle (mean length / number of animals with elongated dioestrus periods per group )

4.1 / 1

4.2 / 2

4.3 / 0

4.5 / 1

Females showing evidence of copulation (N)

10

10

10

10

Females achieving pregnancy (N)

10

10

10

8

Conceiving days 1 - 5 (Pre-coital interval) (N)

10

10

10

5

Conceiving days ≥ 5 (N)

0

0

0

1

Pregnancy ≤ 21 days (N)

0

0

0

0

Pregnancy = 22 days (N)

3

6

6

6

Pregnancy ≥ 23 days (N)

7

4

4

0

Dams with live young born (N)

10

10

10

6

Dams with live young at day 4 pp (N)

10

10

10

6

Implants/dam (mean)

15.6

17.0

16.3

17.3

Live pups/dam at birth (=lactation day 1) (mean)

14.6

16.0

14.7

15.2

Live pups/dam at day 4 (mean)

14.5

14.9

14.5

14.5

Sex ratio (m/f) at birth (=lactation day 1) (mean)

1.09

0.95

1.10

1.02

Sex ratio (m/f) at day 4 (mean)

1.10

0.94

1.13

1.02

Litter weight at birth (=lactat. day 1) (mean, g)

99.53

102.61

92.53

83.77

Litter weight at day 4 (mean, g)

140.59

134.19

127.24

111.37

Pup weight at birth (=lactat. day 1) (mean, g)

6.852

6.428

6.377

5.542

Pup weigh t a t the time of AGD

measurement (lactation day 4)
(mean males , mean females,  g)

10.035 /

9.657

9.244 /

8.699

9.141 /

8.668

7.984 /

7.540

Pup AGD on the same post-natal day,

lactation day 4 (mean m ales , mean females , note PND)

35.75 /

17.14

PND 4

34.54 /

16.77

PND 4

36.21 /

17.62

PND 4

30.76 /

15.24

PND 4

Pup weight at day 4 (mean)

9.853

8.973

8.905

7.747

Pup weight at day 13 (mean)

29.539

27.975

27.242

23.962

Male pup nipple retention at day 13 (mean)

0.26

0.02

0.32

0.40

ABNORMAL PUPS

Dams with 0

10

10

10

6

Dams with 1

0

0

0

0

Dams with ≥ 2

0

0

0

0

LOSS OF OFFSPRING

Pre-natal (implantations minus live births or sum of resorptions and stillbirths)

Females with 0

3

 

 

2

2

 

3

1

2

Females with 1

4

5

5

1

Females with 2

3

1

2

2

Females with ≥ 3

0

1

2

1

Post-natal (prematurely deceased pups between PND 0/1 and PND 13))

Females with 0

9

5

8

2

Females with 1

1

4

1

4

Females with 2

0

0

1

0

Females with ≥ 3

0

1

0

0

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
421. The test item was administered orally to rats at dose levels of 100, 300 or 750 mg/kg b.w./day. For general toxicity the NOAEL was considered to be 100 mg/ kg b.w./ day, based in clinical signs observed in animals treated with 300 and 750 mg test item/kg b.w./day. The NOAEL for reproductive toxicity is considered to be 300 mg/ kg bw/ day based on the reduced litter and pup weight observed in animals treated with 750 mg test item/kg b.w./day p.o. due to parental toxicity. The NOAEL for adverse effects on the reproductive parameters of the parental females is considered to be above 750 mg/ kg bw/day.
Executive summary:

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 421. The test item was administered orally to rats at dose levels of 100, 300 or 750 mg/kg b.w./day.

 

General toxicity

Parental male and female animals

Two female animals dosed with 750 mg test item/kg b.w./day were found dead on GD 20 or GD 21, respectively.

In the intermediate dose group (300 mg test item/kg b.w./day), changes in the behaviour in form of salivation, reduced motility and yellow discoloured urine were noted for the male animals and salivation was noted for the female animals.

At 750 mg test item/kg b.w./day, salivation, reduced motility and yellow discoloured urine was noted for the male and female animals.

In the high dose group (750 mg test item/kg b.w./day), reductions were noted for the body weight and the body weight gain.

No test item-related influence was noted on the food consumption.

No test item-related pathologic findings were observed during macroscopic post mortem examination the male and female animals.

Histopathological examination of the epididymides and the testes of the male and of the ovaries of the female high dose animals revealed no test item-related changes.

No influence on the T4 levels were noted for the male animals.

 

Reproductive toxicity

Parental females

No influence was noted on the fertility of the male and female animals in any of the dose groups.

 

 

Pups

No influence was noted of the pre-natal development (number of implantations, number of resorptions or number of live born pups) at all dose groups.

For the post-natal development, changes in form of a reduced body weight and accordingly in form of a reduced litter weight were noted for the pups of the high dose group (750 mg test item/kg b.w./day). This is considered to be a secondary effect of the reduced body weight of the dams.

No influence on the T4 levels was noted for the male and female pups.

No abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.

 

The following no-observed-adverse-effect levels (NOAEL) were established:

General toxicity (parental animals)

 

 

NOAEL.....................................................................

100 mg test item/kg b.w./day, p.o.

Based on clinical signs observed in animals treated with 300 and 750 mg test item/kg b.w./day p.o.

 

Reproductive toxicity

 

 

 

a) adverse effects on the reproductive parameters of the parental females

NOAEL......................................................................

above 750 mg test item/kg b.w./day, p.o.

 

 

b) adverse effects on pre- and postnatal development

NOAEL.....................................................................

300 mg test item/kg b.w./day, p.o.

Based on the reduced litter and pup weight observed in animals treated with 750 mg test item/kg b.w./day p.o. due to parental toxicity.

Endpoint:
extended one-generation reproductive toxicity - with F2 generation and developmental neurotoxicity (Cohorts 1A, 1B with extension, 2A and 2B)
Type of information:
experimental study
Remarks:
Study started in February 2020. All available information is submitted in this updated dossier by 4 January 2021 as requested in a decision on a cc from ECHA (Decision number CCH-D-2114453561-52-01/F). Draft report will be expected in March 2021.
Adequacy of study:
key study
Study period:
Study started in February 2020. Draft report is expected in March 2021
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 2018-12-21 (Decision number CCH-D-2114453561-52-01/F) the study design of this 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:

- At least two 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) with extension to mate the Cohort 1B animals to produce the F2 generation;
- Cohorts 2A and 2B (Developmental neurotoxicity).
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
adopted June 25, 2018
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 21 Decemberl 2018 (Decision number CCH-D-2114453561-52-01/F). An Extended one-generation reproductive toxicity study (Annex IX, 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:
- At least two 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) with extension to mate the Cohort 1B animals to produce the F2 generation;
- Cohorts 2A and 2B (Developmental neurotoxicity).

The aim of the study is to evaluate the pre- and postnatal effects of the test item on development as well as systemic toxicity in pregnant and lactating females and young and adult offspring.
According to the final decision, a possible mode of action related to endocrine disruption should be clarified: Therefore, possible endocrine disruptor effects
of the test item will be examined by using appropriate laboratory methods (e.g. T4 and TSH hormone ELISA). Furthermore, based on findings reported by Marty et al. (2011), further examinations will be conducted to investigate pubertal development of males, i.e. determination of serum testosterone levels in all male F1 animals on PND 53 and weight of the seminal vesicles including coagulating glands in male animals of F1 generation cohort 2A. In addition, the study will provide and/or confirm information about the effects of a test item on the integrity and performance of the adult male and female reproductive systems.

Additionally, effects on developmental neurotoxicity will be evaluated.

Furthermore, liver weight will also be determined for all F1 cohort 2A animals to establish a timeline for the possible decrease in liver weight
Species:
rat
Strain:
other: CD® / Crl:CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for such studies and required by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: F0: 12-13 weeks
- Number of parental animals:
Pre-exposure period:
At least 120 female animals will be evaluated pre-exposure for oestrus cyclicity to yield 96 females with a regular oestrus cycle for the study.
Main study:
192 animals (96 males and 96 females)
A sufficient number in order to grant at least 20 pregnant females per group for evaluation of the F0 generation.
- Weight at study initiation: Details will be stated in the raw data and the report
- Fasting period before study:
- Housing: With exception of the mating period, the animals are 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 at a room temperature of 22°C ± 3 °C (maximum range) and a relative humidity of 55% ± 10% (maximum range).
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/ Arkeburg, Germany) is used as bedding material in these cages. The cages are cleaned and changed once a week.
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.
- Diet: ssniff® R/M-Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest), ad libitum with the exception of the night before the day of blood withdrawal for laboratory examinations
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature of 22°C ± 3 °C
- Humidity (%): 55% ± 10%
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light cycle
IN-LIFE DATES: From: To: *
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- Route of administration: Oral, via gavage
- Frequency of administration: Once daily
- Vehicle: corn oil
Administration volume: 5 mL/kg b.w.
Dosages: 0, 65, 200, 600 mg/kg bw/d
Selection of route of administration: According to international guidelines.
Details on mating procedure:
Sexually mature male and female rats of the same dose group are paired randomly 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. If mating has not occurred after 2 weeks, the animal will be separated without further opportunity for mating.
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. If there are insufficient males, for example due to male death before pairing, then male(s) which have already mated may be paired with a second female such that all females are paired.

Establishment of F2 generation using Cohort 1B (potential reproductive toxicity):
Cohort 1B animals are maintained on treatment beyond PND 90 and bred to obtain the F2 generation. Mating procedures will be similar to those for the parental animals (F0 generation); sibling mating will be avoided. Reproductive performance of F1 animals will be assessed according to section 4.10 and animals of the F2 generation will be assessed in the same way as F1 animals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item formulations will be prepared for up to seven (7) administration days in advance and will be administered orally at a constant application volume/kg b.w. once daily. The test item will be diluted in the vehicle to the appropriate concentrations. Following the last dosing on each administration day, the test item formulations will be stored at 2 – 8°C. Prior to the first administration of each administration day, the test item formulations will be allowed to warm up to room temperature for at least 30 minutes and stirred on a magnetic stirrer for approximately 3 minutes to ensure homogeneity. The amount of the test item will be adjusted to the animal's current body weight daily.
The control animals receive the vehicle at the same administration volume daily in the same way.
The homogeneity and concentration of the test item formulations will be monitored.

For the analysis of the test item formulations, two (2) aliquots of approximately 3 mL each are taken as scheduled and stored at -20°C ± 10% until analysis at LPT.
At start of the treatment period of the F0 animals (1st dosing day): Analysis of concentration and homogeneity, at the start of administration, during (middle) administration and before administration to the last animal of each dose group. (3 samples/dose group). At a time when most F0 females have delivered: Analysis of concentration, During treatment always before administration to the last animal/dose group (1 sample/dose group). At termination of the F0 dosing period at a time when the majority of animals is dosed: Analysis of concentration, During treatment always before administration to the last animal/dose group (1 sample/dose group). At start of the treatment period of the F1 animals (1st dosing day): Analysis of concentration and homogeneity. At the start of administration, during (middle) administration and before administration to the last animal of each dose group (3 samples/dose group). At termination of Cohort 1A at a time when the majority of animals is dosed: Analysis of concentration, During treatment always before administration to the last animal/dose group (1 sample/dose group). At termination of Cohort 1B at a time when the majority of animals is dosed: Analysis of concentration. During treatment always before administration to the last animal/dose group (1 sample/dose group).
Duration of treatment / exposure:
The study animals will be treated during the following periods:
F0 ANIMALS: Males: 2 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: 2 weeks prior to mating, during the mating, gestation and lactation period and until termination after weaning of their litters (up to and including the day before sacrifice).
F1 ANIMALS: Until weaning, F1 animals are indirectly exposed to the test item through the breast milk. After weaning, dosing will continue in the same way as for the parental generation.
COHORT 1A: Until a dosing period of 10 weeks has been completed (up to and including the day before sacrifice, i.e. around PND 91).
COHORT 1B: Until sacrifice of the F2 generation (up to and including the day before sacrifice, i.e. F2 PND 21).
COHORT 2A: Up to and including the day before sacrifice, i.e. around PND 75 (after behavioural testing).
COHORT 2B: Until sacrifice at PND 21/22, Cohort 2B animals are indirectly exposed to the test item through the breast milk.
F2 ANIMALS: Until sacrifice at PND 21, F2 animals are indirectly exposed to the test item through the breast milk.
Frequency of treatment:
once daily
Details on study schedule:
PARENTAL ANIMALS, PRE-EXPOSURE:
At least 120 female animals will be evaluated pre-exposure for oestrus cyclicity to yield 96 females with a regular oestrus cycle for the study.
MAIN STUDY:
192 animals (96 males and 96 females), a sufficient number in order to grant at least 20 pregnant females per group for evaluation of the F0 generation.

F1 GENERATION:
At weaning, around PND 21, the pups from all available litters per group are randomly selected for further examinations (Cohorts 1A, 1B, 2A and 2B). Obvious runts (animals with a body weight more than two standard deviations below the mean pup weight of the respective litter) will be excluded.

F2 GENERATION:
Cohort 1B animals are maintained on treatment beyond PND 90 and bred to obtain the F2 generation. Mating procedures will be similar to those for the parental animals (F0 generation); sibling mating will be avoided. Reproductive performance of F1 animals will be assessed according to section 4.10 and animals of the F2 generation will be assessed in the same way as F1 animals

The study animals will be treated during the following periods:
F0 ANIMALS: Males: 2 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: 2 weeks prior to mating, during the mating, gestation and lactation period and until termination after weaning of their litters (up to and including the day before sacrifice).
F1 ANIMALS: Until weaning, F1 animals are indirectly exposed to the test item through the breast milk. After weaning, dosing will continue in the same way as for the parental generation.
COHORT 1A: Until a dosing period of 10 weeks has been completed (up to and including the day before sacrifice, i.e. around PND 91).
COHORT 1B: Until sacrifice of the F2 generation (up to and including the day before sacrifice, i.e. F2 PND 21).
COHORT 2A: Up to and including the day before sacrifice, i.e. around PND 75 (after behavioural testing).
COHORT 2B: Until sacrifice at PND 21/22, Cohort 2B animals are indirectly exposed to the test item through the breast milk.
F2 ANIMALS: Until sacrifice at PND 21, F2 animals are indirectly exposed to the test item through the breast milk.

DURATION OF STUDY:
- at least 5 adaptation days of the F0 generation
- F0 (parental generation):
2 weeks pre-exposure
2 weeks pre-mating
2 weeks mating
3 weeks pregnancy
3 weeks lactation
- F1 Cohort 1A: approx. 10 weeks
- F1 Cohort 1B: approx. 10 weeks
2 weeks mating for establishing of F2 3 weeks pregnancy
3 weeks lactation
- F2 Generation: lactation until PND 211
- F1 Cohort 2A: approx. 10 weeks
- F1 Cohort 2B: lactation until PND 21/22
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control
Dose / conc.:
65 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
intermediate dose group
Dose / conc.:
600 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
F0 generation: 20 m/f per group
F1 generation Cohorts 1A + 1B: 20 m/f per group
F1 generation Cohorts 2A + 2B: 10 m/ f per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels have been selected in agreement with the Sponsor based on available toxicological data and the results of a 16-day repeated dose toxicity study in rats (conducted 1986), a 90-day repeated dose toxicity study in rats (conducted 1986) as well as an OECD 421 study in rats (LPT Study No. 37311, 2020). Additionally, the results and the study design of the Pubertal Development Assay (Marty et al., 2011) were taken into account. In the 16-day and the 90-day study, dose levels of 125, 250, 500 and 1000 mg/kg b.w. per day were employed. In the Pubertal Development Assay, dose levels of 50, 200 and 800 mg/kg b.w. per day were selected. In the current OECD 421 study, dose levels of 100, 300, 750 and 1000 mg/kg b.w. per day were used.

In the 90-day and the 16-day gavage studies with the test item, the NOAEL was considered to be 500 mg/kg b.w. (both NTP, 1986). In the 16-day study, at the high dose of 1000 mg/kg b.w., only decreased body weights were observed. One female rat that received 1000 mg/kg b.w. died in the 90-day gavage study. Final mean body weights for rats were not clearly related to dose in this study. In the Pubertal Development Assay, there are indications of a mode of action related to endocrine disruption based on the effects observed at 800 mg/kg b.w., i.e. reduced seminal vesicle weights, reduced serum testosterone, increased age at preputial separation).
In the current OECD 421 study, female animals treated with 1000 mg/kg b.w. had to be sacrificed after the first dosing and male animals after the third dosing due to animal welfare reasons. Two female animals dosed with 750 mg/kg b.w. were found dead on GD 20 and GD 21. Both male and female animals treated with 750 mg/kg b.w. showed slight to severe salivation, slight to moderately reduced motility and yellow discoloured urine. Furthermore, a reduction in body weight was noted for the male animals during the dosing period, while female animals showed a reduced body weight between GD 14 and GD 20. Accordingly, also the body weight gain was reduced for male and female animals during the respective periods. Reduced body weights were also noted at necropsy for both male and female animals. Therefore, the results of the above-mentioned historical studies could not completely be reproduced under the test conditions of the current DRF OECD 421 study (LPT, 2020). For the pups of animals treated with 750 mg/kg b.w. reduced body weights were noted for male and female animals on lactation days 1, 4 and 13.
Male animals treated with 300 mg/kg b.w. showed slight to moderate salivation, a slightly reduced motility and yellow discoloured urine, while slight salivation was observed in females treated with 300 mg/kg b.w.

Based on the findings in these studies, doses of 1000 and 750 mg/kg b.w. per day were considered too high for the main OECD 443 study due to systemic toxicity resulting in death. Taking all study results into account and considering the extended exposure time of the F0 and F1 generation within the OECD 443 study, 600 mg/kg b.w. per day was considered the maximum tolerated dose for the F0-generation of the OECD 443 study, with no systemic effects to be expected in the F1-generation. Furthermore, 600 mg/kg b.w. per day have been selected as the high dose in order to induce and verify effects on pubertal development described by Marty et al. (2011).

- Fasting period before blood sampling for clinical biochemistry: the night before the day of blood withdrawal for laboratory examinations
Positive control:
no
Parental animals: Observations and examinations:
F0 generation + F1 generation Cohort 1B
CLINICAL SIGNS:
- All animals
Throughout the test period, each animal will be observed for clinical signs at least once daily. The frequency will be increased when signs of toxicitiy are observed. Behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity are recorded.Individual animals will be observed before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment, or illness.Cageside observations will include skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed will be recorded. In addition, animals will be checked regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals will be 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 will be maintained on clinical history sheets for individual animals.
- F0 animals and F1 animals Cohort 1B after weaning
Additionally, a more detailed examination of all F0 and F1 Cohort 1B animals is conducted on a weekly basis. F0 animals will be examined once before the first exposure (to allow for within-subject comparisons) and weekly thereafter until termination. F1 animals will be examined weekly after weaning until termination. Detailed clinical observations will be made in all animals outside the home cage in a standard arena, approximately at the same time of day, each time preferably by observers unaware of the treatment. Signs noted will include changes 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) will also be recorded.

MORTALITY:
Further checks will be made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This will allow post mortem examination to be carried out during the working period of that day. On Saturdays and Sundays a similar procedure will be followed except that the final check will be carried out at approximately 3:30 p.m. Premortal symptoms will be recorded in detail; as soon as possible after exitus, a post mortem examination will be performed. In the case of prematurely sacrificed animals, laboratory examinations will be performed, if possible.

BODY WEIGHT:
P0: The animals will be weighed and the weights recorded as scheduled.
Pre-mating period: Daily, starting on the first day of dosing*
Mating period: Daily*
Gestation period (females): Daily; reported for GD 0, 7, 14, 21
Lactation period (females): Daily, report for PND 4, 7, 14, 21
Post-mating period: Males: Daily*, Females: See gestation and lactation period
*: Weekly values will be reported

Schedule for body weight recordings of offspring animals:
F1/ F2: Lactation period: PND 1, 4, 7, 14, 21
After weaning: Daily, starting on PND 22*
F1 Cohort 1B animals only
Mating period: Daily*
Gestation period (females): Daily; reported for GD 0, 7, 14, 21
Lactation period (females): Daily, report for PND 4, 7, 14, 21
Post-mating period: Males: Daily*, Females: See gestation and lactation period
*: Weekly values will be reported
In addition, all animals will be weighed at sacrifice.

FOOD AND WATER CONSUMPTION:
P0: Food residue is weighed and recorded as follows:
Pre-mating period: weekly
Mating period: none
Gestation period (females): GD 7, 14, 21
Lactation period (females): PND 1, 7, 14, 21
Post-mating period: Males: Weekly*, Females: See gestation and lactation period
*: Starting on a suitable day after the mating period to consolidate all male animals

F1: Food consumption of offspring animals
Cohort 1B: Mating period: none
Gestation period (females): GD 7, 14, 21
Lactation period (females): PND 1, 7, 14, 21
Post-mating period: Males: Weekly* Females: See gestation and lactation period
*: Starting on a suitable day after the mating period to consolidate all male animals
Water consumption is monitored by visual appraisal daily throughout the study.

SEXUAL MATURATION:
F1 Cohort 1B: daily until achieved

TESTOSTERONE DETERMINATION:
F1 Cohort 1B: In order to investigate possible effects of the test item on progression of male sexual maturation, a sufficient amount of blood will be taken from the scheduled animals under isoflurane anaesthesia always at the same time of day, if possible, as scheduled below to obtain 2 x 100 μL serum. Blood will be collected on PND 53 +/- 1.

URINALYSIS:
Urine samples are collected from animals fasted overnight at the following times and the parameters listed below are determined.
At the end of the F0 dosing period: 10 males and 10 females randomly selected from each F0 group (animals also selected for laboratory examinations).
The urine is collected for 16 hours in a URIMAX funnel cage. The collection of urine will be terminated immediately prior to starting the blood withdrawals for the haematological and clinical chemical examinations at study termination.
Parameters: volume, pH, specific gravity
Analytes: protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin (Hb), nitrite
microscopic examination: Epithelial cells, Leucocytes, Erythrocytes, Organisms, Further constituents (i.e. sperm, casts), Crystalluria

REPRODUCTIVE PERFORMANCE - F0 animals
Evaluation/parameters:
- Number of pregnant females
- Pre-coital time
- Gestation length calculated from day 0 of pregnancy
Implantation sites:
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group
Number of pups absolute:
- at birth (alive and dead)
- after 4 days of life, after 21 days
Number of pups per dam:
- at birth
- after 4 days of life, after 21 days
Number of male and female pups:
- at birth
- after 4 days of life, after 21 days
Number of stillbirths:
- absolute
- per dam
Number of pups with malformations:
- absolute
- per dam

REPRODUCTIVE PERFORMANCE - F1 Cohort 1B animals
Evaluation/parameters:
- Number of pregnant females
- Pre-coital time
- Gestation length calculated from day 0 of pregnancy
Implantation sites:
- number per dam
- distribution in the uterine horns
- absolute number per group
- mean per group
Number of pups absolute:
- at birth (alive and dead)
- after 4 days of life
Number of pups per dam:
- at birth
- after 4 days of life
Number of male and female pups:
- at birth
- after 4 days of life
Number of stillbirths:
- absolute
- per dam
Number of pups with malformations:
- absolute
- per dam

s. also reproductive indices
Oestrous cyclicity (parental animals):
Vaginal smears will be taken and the oestrus cycle stages will be determined at the following time points:
F0 ANIMALS: 14-day pre-exposure period to select 96 animals with regular oestrus cycles (4-5 days).
During 2 weeks of premating until evidence of mating and at sacrifice.
F1 ANIMALS, COHORT 1A: Start after onset of vaginal patency until first appearance of cornified cells. Two weeks starting around PND 75 and at sacrifice.
F1 ANIMALS, COHORT 1B: Starting from the time of pairing until mating evidence is detected and at sacrifice.
F1 ANIMALS, COHORT 2A: at sacrifice
Sperm parameters (parental animals):
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:
LITTERING:
Each litter will be examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts5 and the presence of gross abnormalities. Based on these parameters, the reproductive performance of the dams will be evaluated. The pups will be examined as described below. Any abnormal behavior will be recorded.

COUNTING, SEXING AND WEIGHING:
Live pups will be counted and sexed. Live pups are weighed on PND 1 and regularly thereafter.
Schedule for body weight recordings of offspring animals:
F1/ F2: Lactation period: PND 1, 4, 7, 14, 21
After weaning: Daily, starting on PND 22*

ANO-GENITAL DISTANCE:
On post-natal day 4 before litter adjustment the ano-genital distance (AGD) of all pups will be determined using a scale.

NIPPLES/ AREOLAE COUNTING:
Nipples/areolae will be counted in all male pups on PND 13.

SEXUAL MATURATION:
Only F1 animals (Cohort 1A and 2A) are evaluated daily for balano-preputial separation or vaginal patency before expected achievement of these endpoints to detect if sexual maturation occurs early. Any abnormalities of the genitals are recorded. Sexual maturity of the F1 animals is compared to physical development (i.e. body weight and age at balano-preputial separation or vaginal opening).

THYROID HORMONES (T4 AND TSH) DETERMINATION:
Pups, 2 surplus pups per litter, all litters, if possible, PND 4, non-fasted, T4 only
Pups, 2 surplus pups per litter, all litters, non-fasted, T4 and TSH

FOOD CONSUMTION OF OFFSPRING ANIMALS:
Cohort 1A/ Cohort 2A: starting after weaning: weekly

TESTOSTERONE DETERMINATION:
F1 Cohort 1A/ 2A: In order to investigate possible effects of the test item on progression of male sexual maturation, a sufficient amount of blood will be taken from the scheduled animals under isoflurane anaesthesia always at the same time of day, if possible, as scheduled below to obtain 2 x 100 μL serum. Blood will be collected on PND 53 +/- 1.

URINALYSIS:
Urine samples are collected from animals fasted overnight at the following times and the parameters listed below are determined.
At the end of the F1 cohort 1A dosing period: 10 males and 10 females randomly selected from each F1 cohort 1A group (animals also selected for laboratory examinations).
The urine is collected for 16 hours in a URIMAX funnel cage. The collection of urine will be terminated immediately prior to starting the blood withdrawals for the haematological and clinical chemical examinations at study termination.
Parameters: volume, pH, specific gravity
Analytes: protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin (Hb), nitrite
microscopic examination: Epithelial cells, Leucocytes, Erythrocytes, Organisms, Further constituents (i.e. sperm, casts), Crystalluria

NEUROLOGICAL SCREENING OF F1 COHORT 2A ANIMALS:
- PND 24 (±1): Auditory startle response
- PND 63-75: Functional observational battery and motor activity
functional tests: Grip strength, Locomotor activity
Postmortem examinations (parental animals):
LABOBRATORY EXAMINATIONS:
Blood samples will be taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight as scheduled. The blood samples collected will be divided into tubes as follows:
EDTA anticoagulant (whole blood): for haematological investigations
Citrate anticoagulant (plasma): for coagulation tests
LiHeparin anticoagulant (plasma): for clinical chemistry tests

Sampling time: at sacrifice
Animals: 10 males and 10 females randomly selected from each F0 group.

HAEMATOLOGY:
The parameters listed below will be determined:
- Differential blood count (Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes. Large unstained cells will be simultaneously quantified during measurement of the differential blood count.), relative
- Differential blood count (absolute)
- Erythrocytes (RBC)
- Leucocytes (WBC)
- Haematocrit value (HCT)
- Haemoglobin content (HGB)
- Platelets (PLT)
- Reticulocytes (RET)
- Mean corpuscular volume (MCV)
- Mean corpuscular haemoglobin (MCH)
- Mean corpuscular haemoglobin concentration (MCHC)
Following the haematological examinations using the ADVIA system, blood smears will be prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.

COAGULATION:
Prothrombin time (PT)/ Activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY:
- Sodium
- Potassium
- Calcium
- Chloride
- Albumin
- Total bilirubin
- Total cholesterol
- Glucose
- Total protein
- Blood urea (BUN)
- Creatinine
- Alanine amino-transferase (ALAT/GPT)
- Alkaline phosphatase (aP)
- Aspartate aminotransferase (ASAT/GOT)
- Bile acids
- Lactate dehydrogenase (LDH)
- Sodium/Potassium ratio
- Globulin
- Albumin/globulin ratio
- BUN/creatinine ratio

THYROID HORMONES (T4 AND TSH) DETERMINATION:
Blood samples will be taken at sacrifice. The blood is processed for serum.
10 males and 10 females randomly selected from each F0 group

GROSS NECROPSY:
For adult F0 and F1 females a vaginal smear taken on the day of sacrifice shortly before necropsy will be examined to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.
The animals will be euthanised by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, and weighed as scheduled:
F0: Males, all, after weaning of the F1 animals, full necropsy
F0: Females,shortly after weaning of the F1 animals, full necropsy
F1 Cohort 1B: shortyl after weaning of the F2 animals, full necropsy

DISSECTION OF ALL ADULT ANIMALS:
At the time of sacrifice or death during the study, the adult animals will be dissected and examined macroscopically for any abnormalities or pathological changes. Special attention will be paid to the organs of the reproductive system.
All superficial tissues will be 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 will be examined. The condition of the thoracic viscera will be noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera will be examined before and after removal; the urinary bladder will be examined externally and by palpation. The gastro-intestinal tract will be examined as a whole and the stomach and caecum will be incised and examined. The lungs will be removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys will be examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs will be recorded.
Organ weights of selected organs will be determined for each generation and cohort. Organ weights of the animals which die or are sacrificed prematurely will be recorded but not included in the mean value comparison.


ORGAN WEIGHTS:
F0 generation:
The number of implantation sites will be recorded and used to evaluate reproductive performance (s. reproductive indicies).

The weights of the following organs of all adult F0 animals will be determined before fixation, where applicable. Thyroid weight will be determined after fixation. Paired organs will be weighed individually and identified as left or right:
- Adrenal gland (2)
- Testicle (2)
- Brain
- Thyroid (1) (including parathyroid, post-fixation)
- Epididymis (2)
- Thymus
- Heart
- Prostate (dorsolateral and ventral parts combined)
- Kidney (2)
- Seminal vesicles with coagulating glands
- Liver
- Pituitary
- Ovary (2)
- Uterus including cervix
- Oviducts (2)
- Identified target organs
- Spleen
The following organs or parts thereof of all adult male and female animals of the F0 generation will be preserved in an appropriate fixative:
Davidson’s solution:
- Eye with optic nerve (2)
modified Davidson’s solution:
- Epididymis (1)
- Testicle (1)
The second epididymis and testicle will not be preserved but used for the spermiogram
7% buffered formalin:
- Adrenal gland (2)
- Oviducts
- Bone
- Pituitary
- Bone marrow (os femoris)
- Prostate
- Brain (cerebrum, cerebellum, pons)
- Seminal vesicles with coagulating glands
- Gross lesions observed
- Spinal cord (3 sections)
- Heart (3 levels: right and left ventricle, septum)
- Spleen
- Intestine, small (duodenum, jejunum, ileum, including Peyer’s patches, Swiss roll method)
- Stomach
- Intestine, large (colon, rectum)
- Thyroid (2) (including parathyroids)
- Kidney and ureter (2)
- Thymus
- Liver
- Trachea (including larynx)
- Lungs (with mainstem bronchi and bronchioles)
- Urinary bladder
- Mammary gland
- Uterus (including cervix)
- Muscle (skeletal)
- Vagina
- Nerve (sciatic)
- Vas deferens
- Oesophagus
- Identified target organs
- Ovary (2)

F1 generation Cohort 1B:
Determination of organ weight and preservation of the F1 Cohort 1B animals is restricted to the organs listed below:
- Adrenal gland (2)
- Pituitary
- Thyroid (2) (including parathyroids)
- Epididymis (2)
- Ovary (2)
- Prostate
- Seminal vesicles with coagulating glands
- Testicle (2)
- Uterus (including oviducts and cervix)
- Vagina
- Vas deferens
- Identified target organs

BONE MARROW:
During dissection fresh bone marrow will be obtained from the os femoris (3 airdried smears/animal) from 10 male and 10 female randomly selected F0 animals per group and stained according to PAPPENHEIM. The myeloid: erythroid ratio will be determined by cell differentiation (counting of 200 nuclei-containing cells) for animals of groups 1 and 4.

HISTOPATHOLOGY:
The blood smears prepared from all animals during the haematological examination will be available for possible examination of pathological changes but examined and evaluated only depending on necropsy findings and upon agreement with the Study Monitor.
Full histopathology will be performed on the preserved organs of F0 animals: 20 randomly selected animals/sex/group of groups 1 and 4 and of all deceased or prematuraly sacrificed animals
The organs listed in above are examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They are examined microscopically if in the plane of section and in all cases where they are noted as grossly enlarged. In addition, frozen sections of the heart, liver and one kidney are prepared, stained with Oil Red O, and examined.
Detailed histopathologic examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure will be performed on one testicle and one epididymis of the selected F0 males of groups 1 and 4 following H-E and PAS staining.

F1 Cohort 1B animals:
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.

Details of the Histology processing and the Histopatholigical evaluation could be found in the Study Plan attached to this study endpoint record.
Postmortem examinations (offspring):
LABOBRATORY EXAMINATIONS:
Blood samples will be taken from the retrobulbar venous plexus under isoflurane anaesthesia from animals fasted overnight as scheduled. The blood samples collected will be divided into tubes as follows:
EDTA anticoagulant (whole blood): for haematological investigations
Citrate anticoagulant (plasma): for coagulation tests
LiHeparin anticoagulant (plasma): for clinical chemistry tests

Sampling time: at sacrifice
Animals: 10 males and 10 females randomly selected from each F1 cohort 1A group.

HAEMATOLOGY:
The parameters listed below will be determined:
- Differential blood count (Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes. Large unstained cells will be simultaneously quantified during measurement of the differential blood count.), relative
- Differential blood count (absolute)
- Erythrocytes (RBC)
- Leucocytes (WBC)
- Haematocrit value (HCT)
- Haemoglobin content (HGB)
- Platelets (PLT)
- Reticulocytes (RET)
- Mean corpuscular volume (MCV)
- Mean corpuscular haemoglobin (MCH)
- Mean corpuscular haemoglobin concentration (MCHC)
Following the haematological examinations using the ADVIA system, blood smears will be prepared from all samples, dried and stained for possible histopathological examinations in case of pathological findings.

COAGULATION:
Prothrombin time (PT)/ Activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY:
- Sodium
- Potassium
- Calcium
- Chloride
- Albumin
- Total bilirubin
- Total cholesterol
- Glucose
- Total protein
- Blood urea (BUN)
- Creatinine
- Alanine amino-transferase (ALAT/GPT)
- Alkaline phosphatase (aP)
- Aspartate aminotransferase (ASAT/GOT)
- Bile acids
- Lactate dehydrogenase (LDH)
- Sodium/Potassium ratio
- Globulin
- Albumin/globulin ratio
- BUN/creatinine ratio

THYROID HORMONES (T4 AND TSH) DETERMINATION:
Blood samples will be taken at sacrifice. The blood is processed for serum.
- 10 males and 10 females randomly selected from each F1 cohort 1A group
- F1 pups (2 surplus pups per litter, all litters if possible) on PND 4 (T4 only)
- F1 pups (2 surplus pups per litter, all litters) on PND 22
- F2 pups 10 males an 10 females randomly selected at sacrifice PND 21

GROSS NECROPSY:
The animals will be euthanised by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, and weighed as scheduled:
F1: "surplus" pups, on PND 4, gross necropsy
F1: "surplus" pups, on PND 22, gross necropsy including assessment of reproductive organs (All F1 “surplus” pups from PND 22 will be subject to a gross necropsy, however only a limited number of pups will be selected for tissue preservation)
F1 Cohort 1A: at the end of the dosing period (PND 91), full necropsy
F1 Cohort 2A: after behavioural testing, around PND 75
F1 Cohort 2B: on PND 21/ 22, full necropsy
F2 all pups: on PND 21 (Depending on data collected during the study, dissection of F2 pups may be scheduled for PND 4 upon agreement with the Sponsor), Gross necropsy including assessment of reproductive organs

DISSECATION OF ALL ADULT ANIMALS:
At the time of sacrifice or death during the study, the adult animals will be dissected and examined macroscopically for any abnormalities or pathological changes. Special attention will be paid to the organs of the reproductive system.
All superficial tissues will be 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 will be examined. The condition of the thoracic viscera will be noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera will be examined before and after removal; the urinary bladder will be examined externally and by palpation. The gastro-intestinal tract will be examined as a whole and the stomach and caecum will be incised and examined. The lungs will be removed and all pleural surfaces examined under suitable illumination.
The liver and the kidneys will be examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs will be recorded.
Organ weights of selected organs will be determined for each generation and cohort. Organ weights of the animals which die or are sacrificed prematurely will be recorded but not included in the mean value comparison.

ORGAN WEIGHTS:
The following organs or parts thereof of all adult male and female animals of the F1 generation Cohort 1A will be preserved in an appropriate fixative:
Fixative: Davidson’s solution
- Eye with optic nerve (2)
Fixative: modified Davidson’s solution
- Epididymis (1)
- Testicle (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, including Peyer’s patches, Swiss roll method)
- Spleen
- Intestine, large (colon, rectum)
- Stomach
- Kidney and ureter (2)
- Thyroid (2) (including parathyroids)
- Liver
- Thymus
- Lungs (with mainstem bronchi and bronchioles)
- Trachea (including larynx)
- Lymph node (1, cervical)
- Urinary bladder
- Lymph node (1, mesenteric)
- Uterus (including cervix)
- Mammary gland
- Vagina
- Muscle (skeletal)
- Vas deferens
- Nerve (sciatic)
- Identified target organs
- Oesophagus

Determination of organ weight and preservation of the F1 Cohort 2A and 2B animals is restricted to the organs listed below:
Cohort 2A:
- Brain
- Eye with optic nerve and retina (2)
- Liver
- Muscle (skeletal)
- Nerve (sciatic)
- Seminal vesicles with coagulating glands
- Spinal cord (3 sections)
Cohort 2B:
- Brain
Liver and seminal vesicles with coagulating glands will be weighed and preserved from all animals of Cohort 2A in order to verify findings of the Pubertal Developmental Assay (Marty et al. (2011); see section 2.10). Animals of Cohort 2A will be used as there age at dissection is closest to the age of the animals examined by Marty et al. (2011).

BONE MARROW:
During dissection fresh bone marrow will be obtained from the os femoris (3 airdried smears/animal) from 10 male and 10 female randomly selected F1 Cohort 1A animals per group and stained according to PAPPENHEIM. The myeloid: erythroid ratio will be determined by cell differentiation (counting of 200 nuclei-containing cells) for animals of groups 1 and 4.

PHENOTYPIC ANALYSIS OF SPLEEN CELLS:
After weighing, spleens of selected Cohort 1A animals will be split in two halves. The portion of the spleen not preserved for histopathology will be minced using a mechanic dissociator to prepare single cell suspensions. Samples will then be used for the following examinations:
- Splenic lymphocyte subpopulation analysis via FACS using the MACSQuant® Analyzer 10/1614
CD4+ T-Lymphocytes
CD8+ T-Lymphocytes
Pan-T-lymphocytes (CD3+)
B-lymphocytes (CD45RA+)
Natural killer cells (CD161+)

HISTOPATHOLOGY:
Full histopathology will be performed on the preserved organs of F1 animals: groups 1 and 4 of Cohort 1A.
The organs listed above are examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They are examined microscopically if in the plane of section and in all cases where they are noted as grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney are prepared, stained with Oil Red O, and examined.
Detailed histopathologic examination with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure will be performed on one testicle and one epididymis of all F1 Cohort 1A males of groups 1 and 4 following H-E and PAS staining.
Detailed histopathological examination with quantitative evaluation of primordial and small growing follicles as well as corpora lutea will be performed on one ovary of the F1 Cohort 1A females of groups 1 and 4. Therefore, the ovary will be processed as follows:
- Ten (10) 2-4 μm step sections with 50 μm steps in between;
- Each slide will be labelled with the slide number to follow the sequence.
In case of test item-related changes in group 4, the Sponsor will be given sufficient notice before the corresponding organs of the F1 Cohort 1A of the intermediate/low dose level groups (group 2 and 3) are sectioned and examined histopathologically.

Neuro-histopathology of the F1 Cohort 2A and 2B group 1 and 4 animals is restricted to the following organs:
Cohort 2A:
- Brain (olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum)
- Eye with optic nerve and retina (2)
- Muscle (skeletal)
- Nerve (sciatic)
- Spinal cord (3 sections)
Cohort 2B:
-Brain (olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum)
Organs and tissues demonstrating treatment-related changes will be also examined for the animals in the lower dose groups.
In addition, the liver as well as the seminal vesicles with coagulating glands of all F1 Cohort 2A group 1 and 4 animals will be examined histopathologically after preparation of paraffin sections and haematoxylin-eosin staining in order to verify the findings of the Pubertal Development Assay (Marty et al. (2011)).
Organs and tissues demonstrating treatment-related changes will be also examined for the animals in the lower dose groups.

Details of the Histology processing and the Histopatholigical evaluation could be found in the Study Plan attached to this study endpoint record.
Statistics:
A) The following settings will be used for the statistical evaluation of the parametrical values captured by (Provantis®15 Integrated preclinical software, Instem LSS Ltd.): Homogeneity of variances and normality of distribution will be tested using the BARTLETT’s and SHAPIRO-WILK test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values will be performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group will be made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

B) The following statistical methods will be used for data not captured by Provantis software (reproductive data). For the parametric values (number of pups etc.), homogeneity of variances will be tested using the BARTLETT test. In case of homogeneity, intergroup comparison will be performed by the DUNNETT test (p ≤ 0.05 and p ≤ 0.01). In case of heterogeneity of variances, a stepwise comparison of the test groups with the control group will be performed using a STUDENT's t-test (p ≤ 0.05 and p ≤ 0.01). For the non parametric values (for example the indices of reproduction or the survival rates), the statistical analysis will be performed using the FISHER's exact test (n < 100) or the chi2-test with YATES' correction for continuity (n ≥ 100) at significance levels of p ≤ 0.05 and p ≤ 0.01.

All data are evaluated statistically in this manner. Individual results which differ significantly from those of the control group will be indicated in the tables of the report.
The mean values and standard deviations will be 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:
F0 GENERATION:
For each F0 group the gestation index is determined:
Gestation Index = (Number of litters with live pups/ Number pregnant) x 100
Fertility Index female [%] = (Number of pregnant rats/ Number of females used) x 100

F1 GENERATION COHORT 1B:
For each F1 Cohort 1B group the gestation index is determined:
Gestation Index = (Number of litters with live pups/ Number pregnant) x 100
Fertility Index female [%] = (Number of pregnant rats/ Number of females used) x 100
Offspring viability indices:
F0 GENERATION: For each litter and group the following indices are determined:
Birth Index = (Total number of pups born (live +dead)/ Number of implantation scars) x 100
Live Birth Index = (Number of pups born alive on day 0/1/ Total number born (live + dead)) x 100
Viability Index pre-select= (Number of pups alive on day 4 (pre-select)/ Number of pups live on day 0/1) x 100
Viability Indexpost select= (Number of pups alive on day 21 (post-select)/ Number of pups live on day 0/1) x 100
Post-implantation loss [%] = ((Implantations - number of pups born alive)/ Implantations) x 100

F1 GENERATION COHORT 1B:
For each litter and group the following indices are determined:
Birth Index = (Total number of pups born (live +dead)/ Number of implantation scars) x 100
Live Birth Index = (Number of pups born alive on day 0/1/ Total number born (live + dead)) x 100
Viability Indexpre-select= (Number of pups alive on day 4 (pre-select)/ Number of pups live on day 0/1) x 100
Post-implantation loss [%] = ((Implantations - number of pups born alive)/ Implantations) x 100
Dose descriptor:
other: no Dose descriptor derived yet
Remarks on result:
other: in-life phase completed; draft report not yet available, expected in March 2021
Critical effects observed:
not specified
Dose descriptor:
other: no Dose descriptor derived yet
Remarks on result:
other: in-life-phase completed; draft report not yet available, expected in March 2021
Critical effects observed:
not specified
Dose descriptor:
other: no dose descriptor derived yet
Remarks on result:
other: in-life-phase completed; draft report not yet available, expected in March 2021
Critical effects observed:
not specified
Dose descriptor:
other: no Dose descriptor derived yet
Remarks on result:
other: in-life-phase completed; draft report not yet available, expected in March 2021
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
The OECD 443 study started in Februar 2020 and the in-life-phase is completed.
The first draft report of the study is expected to be available in March 2021.
Executive summary:

The OECD 443 study started in Februar 2020 and the in-life-phase is completed. The first draft report of the study is expected to be available in March 2021.

Effect on fertility: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

DRF study according to OECD 421

The aim of the reproduction screening study according to OECD guideline 421 was to obtain information on possible effects of the test item on general toxicity, reproduction and/or development (LPT, 2020). Furthermore, this study is used as dose range finding study for the ongoing OECD 443 study (LPT, 2021). The test item was administered orally to rats at dose levels of 100, 300 or 750 mg/kg b.w./day. For general toxicity the NOAEL was considered to be 100 mg/ kg b.w./ day, based in clinical signs observed in animals treated with 300 and 750 mg test item/kg b.w./day. The NOAEL for reproductive toxicity is considered to be 300 mg/ kg bw/ day based on the reduced litter and pup weight observed in animals treated with 750 mg test item/kg b.w./day p.o. due to parental toxicity. The NOAEL for adverse effects on the reproductive parameters of the parental females is considered to be above 750 mg/ kg bw/day.

Results:

General toxicity

Parental male and female animals

Two female animals dosed with750 mg test item/kg b.w./day were found dead on GD 20 or GD 21, respectively.

In the intermediate dose group (300 mg test item/kg b.w./day), changes in the behaviour in form of salivation, reduced motility and yellow discoloured urine were noted for the male animals and salivation was noted for the female animals.

At750 mg test item/kg b.w./day, salivation, reduced motility and yellow discoloured urine was noted for the male and female animals.

In the high dose group (750 mg test item/kg b.w./day), reductions were noted for the body weight and the body weight gain.

No test item-related influence was noted on the food consumption.

No test item-related pathologic findings were observed during macroscopic post mortem examination the male and female animals.

Histopathological examination of the epididymides and the testes of the male and of the ovaries of the female high dose animals revealed no test item-related changes.

No influence on the T4 levels were noted for the male animals.

Reproductive toxicity

Parental females

No influence was noted on the fertility of the male and female animals in any of the dose groups. 

Pups

No influence was noted of the pre-natal development (number of implantations, number of resorptions or number of live born pups) at all dose groups.

For the post-natal development, changes in form of a reduced body weight and accordingly in form of a reduced litter weight were noted for the pups of the high dose group (750 mg test item/kg b.w./day). This is considered to be a secondary effect of the reduced body weight of the dams.

No influence on the T4 levels was noted for the male and female pups.

No abnormalities (malformations or variations) were noted during the external macroscopic examination of the pups at necropsy.


EOGRTS study according to OECD 443

The requested OECD 443 study in rats and with dose groups of 0, 65, 200, 600 mg/kg bw /day started in Februar 2020, the In-life phase was completed in September 2020 and first draft report is expected in March 2021 (see above). The dossier will then immediately be updated and submitted to ECHA without delay.

Effects on developmental toxicity

Description of key information

OECD 414, Fischer F344 rats and mice (Exxon, 1984)

In two guideline comparable inhalation teratogenicity studies with rats and mice, the NOAECs for maternal toxicity were 289 mg/m3 (based on < 7 % reductions in body weight gains) (Exxon, 1984).

During the conduct of the dose range finding study in rats there was one instance of exencephaly noted in one late resorption at 144 ppm (Exxon, 1984).

During the conduct of the dose range finding study in mice there were three instances of exencephaly noted in mouse fetuses: in a late resorption in one litter and two alive fetuses in a second litter (Exxon, 1984).

Based on the observations made in these studies the authors did not believe that this anomaly is related to the test item.

Within the framework of the dose levels and test methods used, it is concluded, that isophorne was not teratogenic or fetotoxic in rats and mice.

OECD 414, Fischer F344 rats (Vivotecnia, 2020)

In order to clarify the relevance of exencephalies observed in two different species, including in Fischer rats (Exxon, 1984), a full prenatal developmental toxicity study according to OECD 414 in Fischer F344 rats by the inhalation route was performed (Vivotecnia, 2020).

There was no effect of the test item exposure at any level on pregnancy outcome, in terms of pre- or post-implantation loss, litter weight or the incidence of fetal abnormalities and variants. Therefore, a NOAEL for developmental toxicity was determined to be >= 151 ppm (0.86 mg/L). Foetal body weight was reduced in dosed groups, and even though these differences become statistically significant, they were below 5% for groups B and C and considered to be within a normal biological variation. However, the decreased on foetal body weight when exposed at 151 ppm (0.86 mg/L) was 13.6% both statistically and biologically significant. Therefore, a NOAEL for embryo-fetal effects was established at 55 ppm (0.31 mg/L).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2015-10-22 to end of December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study without restriction
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS870.6300
GLP compliance:
no
Remarks:
All techniques and procedures were performed according to the standard operating procedures.Instruments and facilities are routinely supervised and validated according to GLP guidelines.
Limit test:
no
Species:
rat
Strain:
other: Fischer 344 (F344/HanZtn Rj) and Wistar (Crl:Wi(Han))
Details on test animals or test system and environmental conditions:
TEST ORGANISMS: 
- Species: Rat
- Sex: Female
- Strain: Fischer 344 (F344/HanZtn Rj) and Wistar (Crl:Wi(Han))
- Source Fischer 344: Janvier, Route des ChénesSecs, Le Genest Saint-Isle, F-53940 France
- Source Wister: Charles River C/Argenters 7, Local AB 08290 Cerdanyola del Vallés Barcelona – Spain
- Age: 10 - 11 weeks on arrival
- Body weight on day 1 of gestation: Fischer 344: 165.0 - 196.4 g, Wistar: 225.7 - 285.1 g
- Animals per cage (before / after distribution): 5 (1 after distribution and before mating)
- Bedding material: Souralit C16 / 11 (irradiated), Souralit, Spain
- Diet: ad libitum, Global diet, Harlan Teklad Station Road Blackthorn, Bicester Oxon, OX25 1TP United Kingdom
- Water: ad libitum, Tap water
- Acclimatisation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C +/- 3° C
- Humidity (%): 30 - 70 %
- Light cycle: 12:12 – 07.00 to 19.00 CET
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Remarks:
Sham filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a flow-past, nose-only exposure system. Exposure chambers type EC-FPC-232
(anodized aluminium, volume inside compartment: approximately 3 L, equipped with glass exposure tubes were used.
- Method of holding animals in test chamber: The rats were individually exposed in glass tubes matching their size.
- Source and rate of air: Compressed air.
The flow of air at each tube was between 0.5 to 1 L/min, which was sufficient to minimize re-breathing of the test item vapour as it is more than twice the respiratory minute volume of rats.
- Method of conditioning air:
- System of generating particulates/aerosols: Vapors of test item were generated using a VOC (Volatile organic compound) vapor generator
(TSE-systems).
- Temperature, humidity, pressure in air chamber:
Mean chamber temperature was maintained within the recommended range of 22±3°C over the entire exposure period.
Mean chamber relative humidity was maintained within the a range of 14-20% over the entire exposure period, excluding chamber corresponding
to sham air exposed groups (A and D) on day 1 of exposure which showed a relative humidity of 9%.
Throughout the exposure period the oxygen concentration was maintained above 19% (values ranged between 20.8-20.9%) while the CO2
concentration remained below 1% (0.04-0.09%).
- Air flow rate: The airflow rate was maintained at 25 L/min for sham air and low dose exposure chambers over the 18 days of exposure. The
airflow rate for high dose exposure chambers (days 1 and 2 of exposure) was maintained at 50 L/min.
- No diet or drinking water was available during exposure.


TEST ATMOSPHERE
- Brief description of analytical method used:
Vapour samples were captured in a solvent trap containing a defined volume of methanol (cooled with ice) positioned in an empty port of the
inhalation chamber. Based on the results from the technical trials, an additional second solvent trap might be placed immediately after the first
solvent trap to ensure that all test item was captured during sampling. The sampling flow rate matched the air flow rate per port. The defined volume of methanol in the solvent trap was refilled to the start volume after sampling completion. Aliquots of samples of the resulting solution of test item
in methanol were appropriately labelled and stored at 2° - 8°C until analysis or shipment. Test item concentrations were analyzed using an analytical method validated in compliance to GLP at Swiss BioQuant AG

VEHICLE (if applicable)
- Concentration of test material in vehicle: Technical trials were performed without animals and conducted before the animal phase of the study to
establish the conditions for vapour generation and sampling, which included:
- Determination of the target concentration and/or technical limit. Several tests were performed to establish the highest stable vapour
concentration achievable that could be maintained at least for 6 hours. Vapor concentration was aimed to 2.0 mg/L in order to compare the results with a previous non-GLP reproduction toxicity study. A stable respirable vapour at a maximum concentration of 1.25 mg/L could be achieved.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical determination of the vapour concentration was performed thrice during each day of exposure for each test item group. Additional samples
were analysed if considered necessary.
The quantification of test item was performed by column separation with reversed-phase chromatography followed by detection with UV.


Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) to initiate the nightly mating period, until evidence of copulation was
observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The
day of mating was designated day 0 post coitum (GD0).
Male rats of the same source and strain were used only for mating. These male rats are in the possession of Vivotecnia and are not considered part of the test system.
Duration of treatment / exposure:
- Duration of dosing: From day 6 post coitum (implantation) to day 20 post coitum
- Dosing regimen: 6h/day, Groups B and E were exposed together on the same exposure chamber. The same applies to Groups C and F.
Frequency of treatment:
6h/day
Duration of test:
Caesarean section on day 21
No. of animals per sex per dose:
6 females per group, 18 pregnant females per strain were distributed in 3 groups
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for target concentration selection:
The target concentrations were selected based on toxicity information available and the technical limit for the maximal vapour concentration.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
The animals were examined twice daily for mortality and morbidity.
Any clinical signs, discomfort and mortality were recorded in accordance with the humane endpoints guidance document of the OECD.
Monitoring was adjusted accordingly when the condition of the animal gave cause for concern.
Where the condition of an animal required premature sacrifice, it was euthanized by CO2 asphyxiation..

DETAILED CLINICAL OBSERVATIONS: Yes
Clinical observations in response to treatment were performed on all animals once daily, approximately 1h after exposure.
Any visible clinical signs, discomfort and mortality were recorded.
Monitoring was adjusted accordingly when the condition of the animal gave cause for concern.

BODY WEIGHT: Yes
Body weight was recorded daily from day 0 until day 21 post coitum.

FOOD CONSUMPTION: Yes
Food was weighed on days 0, 3, 6, 9, 12, 15, 18 and 21 post coitum.
Data were presented in terms of consumption per animal per day for the periods (days 0 – 3), (3 – 6), (6 – 9), (9 – 12), (12 – 15), (15 – 18) and
(18 – 21).

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
Gross necropsy:
At the scheduled necropsy (day 21 post coitum) females were sacrificed by CO2 asphyxiation and the fetuses removed by Caesarean section.
All females were examined, either at the scheduled necropsy or during the study if death occurs.
Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora
lutea count and position of fetuses in the uterus, was performed and the data recorded.
The uteri (and contents) of all females with live fetuses was weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites

Organ weights and tissue preservation
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight taken.
Any organ with gross lesions was collected and preserved in fixation medium (neutral-buffered 4 % formaldehyde) for histological evaluation if
considered relevant.
Animals dying during the study or sacrificed for animal welfare reasons were also subjected to a gross necropsy. Similarly, nasal cavity, trachea,
larynx and lungs from animals showing relevant signs of irritation during exposure (i.e nasal bleeding) were collected and preserved in fixation
medium (neutral-buffered 4 % formaldehyde) for histological evaluation if considered relevant.
Additionally, lungs from all dams were instilled via trachea with formalin at approximately 30 cm H2O pressure collected and fixed in neutral
phosphate buffered 4% formaldehyde solution for possible further examinations.


Ovaries and uterine content:
Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents,
corpora lutea count and position of fetuses in the uterus, was performed and the data recorded.
The uteri (and contents) of all females with live fetuses was weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible hemorrhagic areas of implantation sites

Fetal examinations:
Fetal Pathology
Fetuses were removed from the uterus by Caesarean section (C-section), sexed, weighed individually, and examined for gross external abnormalities.External examination of foetuses was performed at the test facility. After external examination, foetuses were assigned with a number and sacrificed
by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- Microdissection technique (sectioning/dissection technique). Approximately one half of the foetuses (foetuses with odd numbers) from each litter were fixed in Bouin’s fixative. Then foetuses were sent in the same fixative medium to the test site AnaPath GmbH within 3 weeks. At the test site the fetuses were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed
examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of
glycerin/ethanol (one fetus per container). Descriptions of any abnormalities and variations were recorded.
- The remaining fetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. After fixation in ethanol
(ca.94%), foetuses were sent in the same fixative medium to the test site AnaPath GmbH for further skeletal evaluation. Carcasses were processed
through solutions of glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining
ossified bone) and aqueous glycerin for preservation and storage [see literature (2)]. The skeletons were examined and all abnormal findings and
variations were recorded. The assessment included, but was not limited to all principal skeletal structures including cranium, vertebral column, rib
cage and sternum, pectoral and pelvic girdles. The specimens were preserved individually in small containers.
Peer review of at least 10% of the fetal examinations was performed. Fetuses with abnormalities were photographed, when considered appropriate.
The foetal pathology results were provided to the study director by e-mail and a pathology phase report was sent to the study director upon
completion of the study for inclusion in the final report
Statistics:
Provided that the sample size was considered adequate, the following parameters were subject to statistical analysis:
- Body weight
- Food consumption
Analysis of data from two groups:
Test of normal distribution Yes Un-paired t-test
No Mann-Whitney test
Test of equal variance No Un-paired t-test with Welch’s correction

Analysis of data from more than two groups
Test of normal distribution Yes One-way ANOVA Dunnett’s post test (comparison versus control group)
Tukey post test (comparison of all groups)
No Dunn’s post test
Test of equal variance No Dunn’s post test
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Over the first two days of exposure period (corresponding to gestation days 6 and 7), results obtained were indicative of excessive toxicity in animals exposed to the high concentration (1.25 mg/L) of test item (Group C, F344 strain and Group F, Wistar strain). As a result, 4 females from group C in addition to 4 females from group F were found dead or sacrificed for humane reasons over this period.
On the basis of these results, it was decided for animal welfare reasons to decrease the target concentration of both experimental groups (Groups C
and F). Thereby, from GD 8 to GD20 the 2 surviving females from each group (Group C, animals ID 17 and 18 and Group F, animals ID 35 and 36) were exposed to the low concentration (0.85 mg/L) being administered as well to groups B (Strain F344) and E (Strain Wistar).
Dose descriptor:
NOAEL
Basis for effect level:
other: maternal toxicity
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
NOAEL
Basis for effect level:
other: developmental toxicity
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Dose descriptor:
NOAEL
Effect level:
150 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
150 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
see below
Abnormalities:
not specified
Developmental effects observed:
not specified

Maternal data

Maternal mortality

 

Strain

Group

Treatment

Target concentration (mg/L)

Animal ID

Exposure day 1

Exposure day 2

GD8

GD6

GD7

F344

C

Isophorone

1.25

13

FD 1h post dose

 

 

14

 

S pre-dose

 

15

S 5 hs exposure

 

 

16

FD 6 hs exposure

 

 

Wistar

F

Isophorone

1.25

31

 

 

S pre-dose

32

 

 

S pre-dose

33

S 1h post dose

 

 

34

 

 

S pre-dose

FD: Found dead. S: Humane sacrifice

 

Pregnancy rates

F344 Strain: The pregnancy rate for control animals (Group A) was 100%. In the case of the group B (0.85 mg/L), 1/6 animals showed no implantation sites upon c-section, being pregnancy rate 83.3%. Finally, pregnancy rate achieved by the 2 surviving animals of group C (1.25/0.85 mg/L) was 100%.

Wistar Strain: Pregnancy rate achieved in the three experimental groups was 100%.

Clinical signs and observations

F344 Strain: The main and more frequent clinical signs present in animals belonging to all experimental groups were the presence of chromodacryorrhea (eye red secretion, or Harderian gland secretion) piloerection and wet fur immediately after the end of the daily exposure period. The incidence of these clinical signs was slightly higher in test item exposed groups (B and C, 0.85 mg/L) when compared to control animals from this strain.

Wistar Strain: The main clinical signs evident in females belonging to group D (Wistar rats, control group) from GD6 to GD13, comprised the presence of piloerection, chromodacryorrhea together with dirty fur. From GD14 onwards of exposure period, the only remarkable observation found was the presence of dirty fur. Regarding females belonging to group E (test item, 0.85 mg/L) and F (test item, 1.25/0.85 mg/L), piloerection, chromodacryorrhea and dirty fur were recorded over the entire exposure period. The incidence of these signs observed in animals from group E was slightly higher than the observed in the sham control group.

Comparison of findings in both strains: When comparing clinical signs observed in sham control animals from both strains, F344 rats seemed to be more sensitive to the stress induced by the experimental procedures, since the incidence of the main clinical signs observed over the exposure period (piloerection, chromodacryorrhea and dirty fur) was slightly higher in F344 strain.

Maternal body weight and body weight-gain

F344 Strain: No statistically significant differences were observed among groups in body weights and body weight gains prior to the start of the exposures (GD 0-6). Thereafter, over the early gestation period (GD6 -12) body weight loss was observed in the two experimental groups exposed to test item (Group B, 0.85 mg/L and C 1.25/0.85 mg/L), without statistically significant differences being observed with respect to control group.

Thereafter from GD9 to GD21 in the case of animals from group B (0.85 mg/L) maternal body weight gain was still significantly reduced when compared to control animals. As a result, mean body weight gain for the overall exposure period (GD6-GD21) was statistically lower in animals from group B (reaching a body weight gain of -10.13%) when compared to control group (which achieved a body weight gain of 23.18%). This effect was considered to be attributable to maternal toxicity, leading to effects on embryonic development.

Regarding animals from group C (1.25/0.85 mg/L), lower body weight gain was noted for the remainder exposure period (GD9-GD21) for the two surviving animals compared to control group. Hence, mean body weight gain for the overall exposure period (GD6-GD21) in animals from group C reached nearby the 66% of the achieved by control animals. Given to the limited group size in group C statistical analysis could not be performed.

Wistar Strain: Maternal body weight and weight changes were equivalent across all groups prior to the start of exposures (GD0-GD6) as well as over the exposure period (GD6-GD21), with no statistically significant differences being found among the experimental groups.These results were in accordance with c-section observations, which revealed a 100% of dams with viable foetuses in control and test item exposed groups G (Group B, 0.85 mg/L and C 1.25/0.85 mg/L).

Comparison of findings in both strains: When comparing body weight changes of control animals (Group A, strain F344 and group D, strain Wistar) a similar trend was observed in both strains over the entire observation period. In particular, control animals from both strains exhibited body weight gain prior to the start of the exposures (GD 0-6), reaching on GD6 a 5% and 12% increase with respect GD0 in F344 and Wistar control rats, respectively.

Initiation of exposure led to a decrease of body weight in both strains of similar magnitude. Thus, from GD6 to GD9 a body weight drop nearby a 4.3% was observed in animals belonging to control group A (strain F344). Similarly, over this period (GD6-GD9) animals from control group D (strain Wistar) exhibited a body weight loss up to a 3%.

Thereafter, from GD9 to GD21 body weight increase was observed in both experimental groups, being slightly higher in Wistar rats in terms of percentage of increase across the exposure period (16% F344 control rats vs 25% Wistar control rats), as well as over the entire gestation period (23.2% F344 control rats vs 38.74% Wistar control rats).

Food consumption

F344 Strain: Over the pre-exposure period (GD0-GD6) food consumption was similar across all the experimental groups. Afterwards, in general terms, food consumption of females exposed to test item

(Group B, 0.85 mg/L and group C, 1.25/0.85 mg/L) was slightly lower than the observed from sham control F344 rats (Group A) over the entire exposure period. This effect was more remarkable from GD6 to GD9, stage at which animals from test item exposed groups exhibited a decline in food intake up to a 29.5% when compared to control group. Thereafter, from GD9 to GD 21, the drop of food consumption in females exposed to test item (Groups B and C) ranged from 10% to 17%. This effect did not reach statistical significance.

Wistar Strain: Over the pre-exposure period (GD0-GD6) food consumption was similar in control (Group D) and test item exposed (Group B, 0.85 mg/L and group C, 1.25/0.85 mg/L) Wistar rats. Thereafter, exposure to test item did not seem to have an impact in food consumption of any experimental group. Only animals from group E (test item, 0.85 mg/L) exhibited a transient and statistically significant decline in food consumption from GD15 to GD18 (12% respect to control group).

Comparison of findings in both strains: According to the results disclosed in this section, animals from F344 strains seemed to be more sensitive to the exposure to test item in terms of food consumption. This lower food consumption values in F344 strain correspond with body weight effects in these groups together with c-section findings.

Female necropsy data and pregnancy outcome

Macroscopic observations No test item related macroscopic observations were recorded in any animal from any experimental group upon macroscopic necropsy performed on GD21.

Uterine implantation data

F344 Strain: Mean number of corpora lutea was similar across groups. Concerning mean implantation sites and pre-implantation loss, similar results were observed in control animals and in the two animals from group C surviving up to GD21. In particular mean number of implantation sites per animal observed in group A were 10.8, while in group C (1.25/0.85 mg/L) mean number of implantation sites per animal was 10.0. These results involved a mean percentage of pre-implantation loss of 4.7% in control animals (Group A) and equal to 30.5% in group C (1.25/0.85 mg/L).

In the case of group B (0.85 mg/L), mean percentage of pre-implantation loss was 42.4%, being remarkably higher than the 4.70% recorded in control animals. It should be noted that one female belonging to this group (ID11) showed a 100% of pre-implantation loss together with the circumstance that within group variability was large. Accordingly, this effect did not achieve statistical significance.

In terms of post-implantation losses, a clear treatment related effect was observed in groups B (0.85 mg/L) and C (1.25/0.85 mg/L). In particular, mean post-implantation losses per animal in group B was 84.4% while in the case of group C this value reached a 63.7%. These values were remarkably higher than the 17.52% of mean post-implantation losses per animal found in the control group, with no statistically significant differences being observed.

It must be pointed out that, while in control group the major part of the post-implantation losses were classified as early or embryonic resorptions (11/12 losses) in the case of test item exposed animals, most of these losses took place at an earlier stage. In particular, in group B (0.85 mg/L) 24 of the 27 post-implantation losses recorded were defined as empty implantation site, meaning a very early resorption or aborted implantation. Concerning group C, animal ID 17 showed a 100% of post-implantation losses, all of them considered empty implantation sites, whereas animal ID 18 showed 3 post-implantation losses (from a total of 11 implantation sites), being the 3 described as embryonic resorptions.

All together, these circumstances led to a statistically significant decrease in the number of viable foetuses recorded in animals from group B (0.85 mg/L) when compared to control group. In particular, a total of 56 live foetuses were observed upon c-section in animals from group A, while in the case of group B only 1 female presented viable fetuses (a total of 5).

Wistar Strain: Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation losses were similar across all groups. Moreover, post-implantation losses and litter sizes were similar when comparing test item exposed and control animals.

Comparison of findings in both strains: In general terms, comparison of data from control groups showed a similar reproductive performance for both strains concerning to mean pre-implantation losses per animal. Thereafter, F344 females showed a worse reproductive performance compared to Wistar rats, when considering the mean percentage of post-implantation losses per animal (17.52% in F344 strain versus 6.56% in Wistar strain). On the other hand, litter size was higher in Wistar rats (12.8 fetus per litter) than in F344 strain (8.8 fetus per litter).

These results are in agreement with the published literature, reporting relatively poor reproductive performance of the F344 strain (inbred strain) when compared to outbred strains such as Wistar rats, known for their better reproductive capabilities. Therefore, F344 strain would not be the appropriate model of choice for prenatal developmental toxicity studies.

Gravid uterine weight data

F344 Strain: Upon c-section the 6 females belonging to control group (Group A) were determined to be gravid. By contrast, in the case of group B (Test item, 0.85 mg/L) from the 6 animals surviving until GD21 only 1 (ID7) was found to be gravid on GD21 and showed viable fetuses. Finally, regarding from the two females from group C (Test item 1.25/0.85 mg/L) surviving the entire observation period, only one of them (ID18) was determined to be gravid and showed viable festuses.

Therefore, on the basis of the limited group size available, no remarkable differences were found in gravid uterine weight between control and test item exposed groups or neat terminal body weight. A decline in neat body weight gain from GD6 to GD21, was found in all experimental groups, ranging between -7.7% to -9.5%.

Wistar Strain: Upon c-section conducted on GD21, the 6 females from groups D (Control group) and group E (Test item, 0.85 mg/L) were determined to be gravid. In addition, the two surviving females from group F (Test item, 1.25/0.85 mg/L) well found as well to be gravid.

Gravid uterine weight, neat terminal body weight together with neat body weight gain was very similar among the three experimental groups, with no statistically significant differences being observed. A decline in neat body weight gain from GD6 to GD21, was found in all experimental groups, ranging between - 4.2 % to - 4.9%.

Comparison of findings in both strains: Control animals from both strains showed a decrease in neat body weight gain from GD6 to GD21, being this effect more noticeable in F344 rats. In particular, over the exposure period F344 control females exhibited a neat body weight gain up to a -9.4%, while in Wistar rats neat body weight gain was - 4.2%.

Conclusions:
Fischer 344 strain:
Based on the results of this study, a no observed-adverse-effect level (NOAEL) for maternal effects and embryo-fetal development could not be established for this strain.
Wistar strain:
On the basis of these results, it was considered that a no observed-adverse-effect level (NOAEL) for maternal effects and embryo-fetal development was evident at 0.85 mg/L (150 ppm).
Strain comparison:
- The F344 rat is an inbred strain which was used for years as a suitable animal model for reproduction and development
toxicity studies. Nevertheless, over time these animals developed several health issues inherent to the strain, involving among
others decreased fecundity (King-Herbert and Thayer 2006), reasons why their use within this type of studies was
discouraged. By contrast, outbred strains such as Wistar rat are known for their hybrid vigor, large litter size, long
lifespan, resistance to disease, and low neonatal mortality (Hedrich 2006).
- In the present study, F344 rats exposed to test item vapour by inhalation (nose-only) at 0.85 mg/L, 6 hours/day from
gestation day 6 to gestation day 20 showed an exposure related effect on pregnancy outcome, in terms of pre and
post-implantation loss. By contrast, such effects were not observed in Wistar rats following the same exposure regimen.
Thereby, inherent strain reproductive performance characteristics may have enhanced the test item effect on
embryo-fetal development observed in F344 rats.
- Results of the present study together with the information existing in the published literature, provide clear evidence that F344
rats are not a suitable strain for prenatal developmental toxicity studies.
Executive summary:

Objective

In a first instance this study was designed to assess the potential effects of the test item on pregnancy and embryo-fetal development in F344 and Wistar rats, following daily inhalation exposure (nose-only) at concentrations of 0.85 (150) and 1.25 mg/L (220 ppm), during 6 hours per day, from implantation to one day prior to scheduled caesarean section (day 6 to day 20 post-coitum, inclusive). Therefore this study aimed to characterize maternal toxicity at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

Furthermore, these results should serve as the basis for the selection of the exposure concentrations to be used in a further regulatory prenatal and developmental toxicity study to be performed according to guideline OECD 414.

Study Design

For these purposes, 18 pregnant females per strain were distributed in 3 groups (6 animals per group). F344 rats were allocated into groups A-C. Animals from group A were exposed to filtered air (Group A, sham controls), whereas animals allocated in group B were exposed to the low concentration of the test item (0.85 mg/L) and animals from group C were exposed to the high concentration

(1.25 mg/L).

Similarly, Wistar rats were allocated into groups D-F. Group D served as sham control, while animals from groups D

and E were exposed to the low (0.85 mg/L) and high (1.25 mg/L) doses of test item, respectively.

Over the first two days of exposure (Gestation day 6-7), the high concentration of the test item was reduced from 1.25 mg/L to 0.85 mg/L (low concentration) due to unscheduled mortality and adverse clinical signs observed at this exposure concentration in both strains.

All animals were observed twice daily for mortality and clinical observations. Body weights, and food consumption were recorded at appropriate intervals. On GD 21, a cesarean section was performed on each female and selected organs were collected and weighed. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Study Results

F344 strain

Over the first two days of exposure (GD6-GD7), 4 females from the high concentration group (Group C, 1.25 mg/L) were found dead (2/4) or sacrificed for humane reasons (2/4). Thus, only 2/6 animals from this group survived until the end of the exposure period, involving exposure to a concentration of 1.25 mg/L from gestation day (GD) 6 to GD 7 and to a concentration of 0.85 mg/L thereafter, from GD8 to GD20.

Exposure related clinical signs (such as piloerection and wet fur) were found in all experimental groups, which a slight higher incidence in test item treated animals. Over the early pregnancy (GD6-GD12) no statistically significant differences were observed in body weight or body weight gain between sham control and test item exposed animals. Thereafter, from GD9 to GD121 a statistically significant body weight reduction was observed in females from group B (0.85 mg/L). This effect was considered to be attributable to maternal toxicity, leading to effects on embryonic development.

In general terms, food consumption of test item exposed females tended to be slightly lower of the observed for sham control animals over the entire observation period. Nevertheless, these differences did not achieve statistical significance.

No test item related macroscopic findings were noted in animals from group B and C. A test item related effect was observed regarding pre and post-implantation losses in animals from both experimental groups (0.85 and 1.25 / 0.85 mg/L), which showed remarkably higher percentages of losses with respect to control group.

Upon c-section (GD21) 5/6 females from group B and 2/2 females from group C showed implantation sites and/or viable fetuses. On the basis of the limited group size available, no remarkable differences were found in gravid uterine weight or in neat body weight gain (GD6-GD1) when comparing test item treated animals with sham control groups.

Finally, there was no effect of exposures in fetal weight at GD21 and no external, visceral or skeletal abnormalities or variations were noted.

Wistar strain

Over the first two days of exposure (GD6-GD7), 4 females from the high concentration group (Group F, 1.25 mg/L) were sacrificed for humane reasons. Thus, only 2/6 animals from this group survived until the end of the exposure period, involving exposure to a concentration of 1.25 mg/L from gestation day (GD) 6 to GD 7 and to a concentration of 0.85 mg/L thereafter, from GD8 to GD20.

No test item exposure related effects were observed on maternal observations, gestational parameters or fetal evaluations.

Conclusion

Taken together the results disclosed in the present report, it can be concluded that under these experimental conditions:

F344 strain

- Exposure of F344 pregnant rats to test item vapour by inhalation (nose-only) at 1.25 mg/L (220 ppm), 6 hours/day

from gestation day 6 to gestation day 7, resulted in maternal toxicity, which involved clinical signs of toxicity and mortality.

- Exposure of F344 pregnant rats to test item vapour by inhalation (nose-only) at 0.85 mg/L (150 ppm), 6 hours/day

from gestation day 6 to gestation day 20, did induce maternal toxicity (statistically significant body weight loss).

- Accordingly, a no observed-adverse-effect level (NOAEL) for maternal effects could not be established in this study.

- Exposure of F344 pregnant rats to test item vapour by inhalation (nose-only) at 0.85 mg/L, 6 hours/day from gestation day 6

to gestation day 20 led to an exposure related effect on pregnancy outcome, in terms of pre and post-implantation loss.

These effects are considered to be due to overt maternal toxic effects. On the basis of the very limited analysis performed under

the conditions of this study, due to the low number of foetuses available in females exposed to test item vapour by inhalation

at 0.85 mg/L, exposure was not associated with any effect on fetal weight or the incidence of fetal abnormalities and variants.

- On the basis of these results, a NOAEL for embryo-fetal effects could not be established for this strain.

Wistar strain

- Exposure of Wistar pregnant rats to test item vapour by inhalation (nose-only) at 1.25 mg/L (220 ppm), 6 hours/day

from gestation day 6 to gestation day 7, resulted in maternal toxicity, which involved clinical signs of toxicity and mortality.

- Exposure of Wistar pregnant rats to test item vapour by inhalation (nose-only) at 0.85 mg/L (150 ppm), 6 hours/day

from gestation day 6 to gestation day 20, did not induce maternal toxicity and did not have any effect in pregnancy outcome,

in terms of pre- or post-implantation loss, fetal weight or the incidence of fetal abnormalities and variants.

- On the basis of these results, it was considered that a no observed-adverse-effect level (NOAEL) for maternal effects

and embryo-fetal development was evident at 0.85 mg/L (150 ppm).

Strain comparison

- The F344 rat is an inbred strain which was used for years as a suitable animal model for reproduction and development

toxicity studies. Nevertheless, over time these animals developed several health issues inherent to the strain, involving among

others decreased fecundity (King-Herbert and Thayer 2006), reasons why their use within this type of studies was

discouraged. By contrast, outbred strains such as Wistar rat are known for their hybrid vigor, large litter size, long

lifespan, resistance to disease, and low neonatal mortality (Hedrich 2006).

- In the present study, F344 rats exposed to test item vapour by inhalation (nose-only) at 0.85 mg/L, 6 hours/day from

gestation day 6 to gestation day 20 showed an exposure related effect on pregnancy outcome, in terms of pre and

post-implantation loss. By contrast, such effects were not observed in Wistar rats following the same exposure regimen.

Thereby, inherent strain reproductive performance characteristics may have enhanced the test item effect on

embryo-fetal development observed in F344 rats.

- Results of the present study together with the information existing in the published literature, provide clear evidence that F344

rats are not a suitable strain for prenatal developmental toxicity studies.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2018-07-23 to 2018-08-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS870.6300
GLP compliance:
no
Remarks:
All techniques and procedures were performed according to the standard operating procedures.Instruments and facilities are routinely supervised and validated according to GLP guidelines.
Limit test:
no
Specific details on test material used for the study:
Physical state (at 20ºC): Neat Liquid
Purity / composition: ≥98.0 to ≤ 99.6%
Molecular weight: 138.20 g/mol
Colour: Clear
Storage conditions: Room temperature (ca. 23ºC); protected from light
Expiry date: July, 2019
Handling conditions: Personnel will wear gloves and auto-ventilated equipment equipped with filters appropriate vapours
Other: Sensitive to air, highly volatile, odour of peppermint.
Avoid inhalation and contact with eyes and skin. Test item may cause irritation of respiratory tract, eyes and skin
The neat substance without solvent will be tested
Species:
rat
Strain:
other: Fischer 344 (F344/HanZtn Rj)
Remarks:
Although the F 344 strain is normally discouraged in reproduction studies (OPPTS 870.6300), the authorities specificaly asked for their use in order to compare the results with previous non-GLP reproduction toxicity studies, in which this strain was used
Details on test animals or test system and environmental conditions:
TEST ORGANISMS: 
- Species: Rat
- Sex: Female
- Strain: Fischer 344 (F344/HanZtn Rj)
- Source Fischer 344: Janvier, Route des ChénesSecs, Le Genest Saint-Isle, F-53940 France
- Age: 9 - 10 weeks on arrival; 12 - 13 weeks on first item exposure
- Animals per cage (before / after distribution): Up to 5 (1 after distribution and before mating)
- Bedding material: Souralit C16 / 11 (irradiated), Souralit, Spain
- Diet: ad libitum, Global diet, 2914C, Envigo Teklad
- Water: ad libitum, Tap water
- Acclimatisation period: at least 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 °C - 24.6° C
- Humidity (%): 28 - 61 %
- Light cycle: 12 hours light, 12 hours dark
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Remarks:
Sham filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

Preparation of the exposure system
Previous to exposure onset, a complete setup of the system and flow/pressure characterization was performed according to the corresponding internal SOPs. The tasks were the following:
- Making an outline of each line including identification of the corresponding equipment
- Leak test through the vapour conveyor glass tubes
- Checking of working air temperature and relative humidity
- Flow of working air entering the machine and internal pressure thereof
- Pump status: measuring puff volume, pump settings
- Theoretical calculations regarding dilution and Venturi tubes to be used
- Vacuum system checking in sampling ports: capillary installed, vacuum pressure measurement, sampling flow
- Exposure test: Determination of pressure inside the inhalation chamber versus room pressure and difference of pressure between both chamber compartments. Determination of aerosol flow in different positions of the chamber randomly selected: six ports from the high level, six ports from the mid level and six ports from the low level. Calculation of coefficient of variation (%CV) within each level and across the three levels. Within each level, the CV will not be higher than 5%, whereas the CV among the three levels will not exceed 10%.
- All these determinations were also performed in the sham exposed group with the exception of the flow through chamber.

Test item Vapour generation and characterisation
- A vapour was generated from the liquid test item using a Volatile Organic Compound (VOC) generator (TSE-systems).
- The test item was placed inside the generator vessel in liquid state. It was heated at a controlled temperature of approximately 55ºC by means of a recirculating water bath equipped with a temperature probe to generate a vapour phase.
- In addition, the liquid was stirred by means of a magnetic stirrer in order to increase the evaporation.
- At the same time, a source of compressed air at a controlled temperature was delivered into the VOC generator. This compressed air was mixed with the vapour phase of test item extracting the formulation of the product in a controlled amount of air (L/min) and concentration.
- The vapour was conveyed via glass tubing from the generator to the exposure chambers.
- The flow rate through the exposure chamber was adjusted as necessary.
- In order to prevent exposure of the animals to test item droplets from condensation, a filter will be placed immediately before the exposure chamber. For sham exposure, filtered, compressed fresh air will be provided to the exposure chamber.

Exposure conditions
- Inhalation exposure was performed using a flow-past, nose-only exposure system.
- The animals were confined separately in restraint glass tubes matching their size which are positioned radially around the exposure chamber.
- The exposure system ensures a uniform distribution and provides a constant flow of test material to each exposure tube.
- The flow of air at each tube was within 1.0 ± 0.5 L/min.
- The position of the rats in each group within the exposure chamber was changed daily according to an insertion schedule.
- Sham-exposed rats were exposed to filtered, conditioned air, the exposure conditions being the same as those for the test item-exposed rats.
- Exposure chambers type EC-FPC-232 (anodized aluminium, volume inside compartment: approximately 3 L, equipped with glass exposure tubes) were used. The rats were individually exposed in glass tubes matching their size.
- Before starting the inhalation period, the animals were not adapted to the glass tubes to avoid pre-implantation losses due to the stress caused by the procedure itself.
- The temperature of the test atmosphere in the exposure chamber was maintained at 22 ± 5 °C and the relative humidity as required by experimental conditions.
- Air flow per animal was monitored regularly.
- No diet or drinking water was available during exposure.
- The time spent during animal shifting was not included within the exposure time.
- Before the animal exposures, technical trials were performed in order to set-up the conditions.
- A vapour was generated for up to 6 hours for two consecutive days. During this period, the concentrations as well as the temperature and relative humidity of the chamber were monitored.
- The set-up conditions and results were similar to those obtained during the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Characterisation of test atmosphere
In order to characterize the test atmospheres and to check the reproducibility of aerosol generation, several parameters were determined at defined intervals.
Determination of the nominal vapour concentration
The test item usage was determined daily by volume difference in the vapour generator before and after each exposure. The weight used, calculated by means of the specific gravity, was then divided by the total air-flow feeding the vapour generator to give the nominal concentration. These data were used for the purpose of monitoring the performance of the generation system.
Determination of test item concentration
Test item vapour concentration was determined three times a day on each day of exposure for each test item group by high performance liquid chromatography (HPLC) after collecting the vapour in a solvent trap containing 50 mL of methanol (cooled with ice) positioned in an empty port of the inhalation chamber. The analytical method was validated afterwards in Vivotecnia study B-02791. Sampling time was 5 minutes at a flow rate of approximately 1 L/min. Trap volume was determined before and after sampling in order to evaluate discrepancies in the final volume. Aliquots of the samples were analysed freshly immediately after the sampling process.
The solvent trap capacity was evaluated in Vivotecnia study number N-02130 by sampling the vapour in two in-line solvent traps. No test item was detected in the second trap. Thus, the capacity of one single trap was considered adequate at the concentrations performed in this study.
Chromatographic method see "other information on materials and methods".
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) to initiate the nightly mating period, until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day of mating was designated day 0 post coitum (GD 0).
Male rats of the same source and strain were used only for mating. These male rats were not considered as part of the study.
Only animals that meet the inclusion criteria will be recruited for the study and distributed into experimental groups.
Duration of treatment / exposure:
Duration of dosing: Daily from day 6 post coitum (implantation, GD 6) to day 20 post coitum (GD 20)
Dosing regimen: 6h/day
Frequency of treatment:
6h/day
Duration of test:
Caesarean section on day 21
Dose / conc.:
0 mg/L air
Remarks:
Control Group (Group A), Sham filtered air
Dose / conc.:
0.1 mg/L air
Remarks:
Low Group, 17 ppm (Group B)
Dose / conc.:
0.3 mg/L air
Remarks:
Mid Group,, 50 ppm (Group C)
Dose / conc.:
0.85 mg/L air
Remarks:
High Group, 150 ppm (Group D)
No. of animals per sex per dose:
8 females per group, 32 pregnant females were distributed into four experimental groups. One control group and three different dose groups (low, mid and high vapour concentration).
Control animals:
yes, concurrent vehicle
Details on study design:
- Introduction and study outline
The objective of this dose range finding (DRF) study was to assess the potential effects of the test item on pregnancy and embryo-foetal development in F344 rats resulting from repeated administration of the test item through the inhalation route (nose-only) from day 6 post coitum (implantation, GD 6) to day 20 post coitum (the day prior to Caesarean section, GD 20). Therefore, this study aimed to characterize maternal toxicity at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity. Furthermore, these results should serve as the basis for the selection of the exposure concentrations to be used in a further regulatory prenatal and developmental toxicity study to be performed according to guideline OECD 414.
For those purposes, 32 pregnant female rats were distributed into four experimental groups (A to D). Animals were exposed to diluted vapour from the test item (groups B to D) or to filtered compressed fresh air (Group A; control) 6 hours/day from day 6 post coitum (implantation, GD 6) to day 20 post coitum (the day prior to Caesarean section, GD 20).
Animals from group A were exposed to filtered compressed fresh air and served as control and animals from groups B to D were exposed to diluted vapour from the test item at nominal concentrations of 0.1, 0.3 and 0.85 mg/L air, respectively. The vapour was generated from the liquid test item using a Volatile Organic Compound (VOC) generator (TSE-systems). In order to characterize the test atmosphere and to check the reproducibility of the vapour generation and dilution, the following analytical parameters were determined at defined intervals: test item concentrations, temperature, relative humidity as well as oxygen and carbon dioxide concentrations through exposure chamber.
All animals were observed twice daily for mortality and clinical observations before and within 1 hour after exposure. Body weights, and food consumption were recorded at appropriate intervals. On GD 21, a caesarean section was performed on each female and selected organs were collected and weighed. The uteri, placentae, and ovaries were examined, and the numbers of foetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded. The foetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations

- Rationale for selection of species / strain: Although the Fisher 344 strain is normally discouraged in reproductive toxicity studies (OPPTS 870.6300), the authorities specifically asked for their use in both the DRF and main studies in order to compare the results with previous non-GLP reproduction toxicity studies, in which the Fischer 344 strain was used.

- Rationale for target concentration selection: The target concentrations were selected by the sponsor according to previous prenatal development toxicity studies (Exxon, 1984 and VVT, 2016) available for this test item.

- Inclusion criteria
Only animals that meet the inclusion criteria will be recruited for the study and distributed into experimental groups.
Inclusion criteria: All animals were subjected to individual veterinary examinations. Only animals in good health and with pregnancy status confirmed were included in the study
Stratification method: Animals were randomly distributed by means of the body weight stratification method at least 2 days before first administration

- Determination of the temperature
The temperature in the chamber was measured hourly during exposure using a thermohygrometer. The target range is 22±5ºC.
- Determination of the relative humidity
The relative humidity in the chamber was measured hourly during exposure using a thermohygrometer.
- Determination of oxygen and CO2 concentration
The oxygen concentration of the test atmosphere was measured hourly during each day of exposure using an appropriate device. The oxygen and CO2 concentrations were maintained above 19% and below 1% respectively, during the exposure period.
- Airflow rate
The exposure airflow rate was adjusted as appropriate before the start of the exposure using the pressure difference over a Venturi tube. Air flow per animal was monitored regularly (at least 3 times per group and day) during the daily exposure period.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
Experimental data concerning body weight, general clinical signs, organ weights and gross necropsy findings (dams) and ovaries and uteri examination were recorded through Provantis 8.5.2.1TM (Instem LSS Ltd., Staffordshire, United Kingdom) preclinical software.

Mortality and premature sacrifice
All animals were examined daily for mortality and morbidity. Any clinical signs, discomfort and mortality were recorded in accordance with the human endpoints guidance document to the OECD (ENV/JM/MONO (2000)7).


DETAILED CLINICAL OBSERVATIONS: Yes
Clinical observations were recorded before and within 60 minutes after finishing exposure in all animals.
Any visible clinical signs, discomfort and mortality was recorded in accordance with the humane endpoints guidance document of the OECD (ENV/JM/MONO(2000)7). Observations included changes in skin, eyes, and mucous membranes. Alterations in respiratory pattern or behaviour as well as changes in posture and response to handling and the presence of abnormal movements were also recorded.


BODY WEIGHT: Yes
All animals were weighed on day GD0, every 3 days during the inhalation period and on day 21 post coitum.
Additional body weights were obtained as necessary for animal welfare reasons.


FOOD CONSUMPTION: Yes
Food consumption was recorded in all animals on GD 0, 3, 6, 9, 12, 15, 18 and 21 post coitum. The consumption was calculated by cage, estimating the average food intake for each animal.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
Gross necropsy
At the scheduled necropsy (GD 21 post coitum) females were sacrificed by CO2 asphyxiation and the foetuses were removed by caesarean section.
All females were examined at the scheduled necropsy. Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count, implantations sites and position of foetuses in the uterus, was performed and the data recorded. The uteri (and contents) of all females with live foetuses was weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.

Organ weights and tissue preservation
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight was recorded.
Any organ with gross lesions was collected and preserved in fixation medium (neutral-buffered 4 % formaldehyde) for histological evaluation if considered relevant. Similarly, nasal cavity, trachea, larynx and lungs from animals showing relevant signs of irritation during exposure (i.e nasal bleeding) were collected and preserved in fixation medium (neutral-buffered 4 % formaldehyde) for histological evaluation if considered relevant.
Additionally, lungs from all dams were weighted and instilled via trachea with formalin at approximately 30 cm H2O pressure collected and fixed in neutral phosphate buffered 4% formaldehyde solution for possible further examinations.
Ovaries and uterine content:
Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count, implantations sites and position of foetuses in the uterus, was performed and the data recorded. The uteri (and contents) of all females with live foetuses was weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
Fetal examinations:
Fetal Pathology
Foetuses were removed from the uterus by Caesarean section (C-section), sexed, weighed individually and examined for gross external abnormalities. External examination of foetuses was performed at Vivotecnia. After external examination, foetuses were assigned with a number, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- Microdissection technique (sectioning/dissection technique). Approximately one half of the foetuses (foetuses with odd numbers) from each litter were fixed in Bouin’s fixative. Then foetuses were sent in the same fixative medium to the test site AnaPath GmbH within 3 weeks. At the test site the foetuses were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissues were preserved in a solution of glycerin/ethanol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
- The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. After fixation in ethanol (ca.94%), foetuses were sent in the same fixative medium to the test site AnaPath GmbH for further skeletal evaluation. Carcasses were processed through solutions of glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage . The skeletons were examined, and all abnormal findings and variations were recorded. The assessment included,but was not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles. The specimens were preserved individually in small containers.
Foetuses with abnormalities were photographed, when considered appropriate. The foetal pathology phase report is attached to this study endpoint record.
Statistics:
Provided that the sample size was considered adequate, the following parameters will be subject to statistical analysis:
- Body weight
- Food consumption

Analysis of data from two groups:
Test of normal distribution Yes Un-paired t-test
No Mann-Whitney test
Test of equal variance No Un-paired t-test with Welch’s correction

Analysis of data from more than two groups
Test of normal distribution Yes One-way ANOVA Dunnett’s post test (comparison versus control group)
Tukey post test (comparison of all groups)
No Kruska-Wallis Dunn's post test
Test of equal variance No Kruska-Wallis Dunn’s post test
A value of P < 0.05 will be considered statistically significant
Indices:
DRF, Fertility Index, Viability Index, Resorption Index, Pre-Implantation Loss Index, Post-Implantation Loss Index, Runts Index, Variation Index,
Number of litters having abnormalities, Number of abnormalities per litter
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The most frequent clinical sign present in animals belonging to all experimental groups, including sham, was piloerection. This sign is commonly observed in nose-only inhalation studies and, since it is equally present in the control group, it can be considered not to be caused by the test item.
Low dose group showed punctual wet fur at the beginning of the exposure. Additionally, incidental bleeding in vagina was observed on isolated days of animals belonging to groups B – D (< 2 animals per group). Since both signs were single observations (< 2 and not in consecutive days) and there was no dose-dependent relationship, these observations are considered incidental and probably not test item related.
Mortality:
no mortality observed
Description (incidence):
None of the animals died during exposure. All animals survived until the schedule necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No statistically significant differences were found in mean body weight among the four experimental groups neither during the exposure period nor at the end of the study.

No statistically significant differences were found in mean body weight gain among the four experimental groups neither during the exposure period nor at the end of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was similar among the four experimental groups (no significant differences) over the course of the study.
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
Description (incidence and severity):
No statistical differences were recorded neither in Terminal body weight nor in gravid uterus weight. The adjusted body weight to gravid uterus was also similar.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant macroscopic abnormalities related to the exposure to the test item were observed.
Red spots in the uterus were observed for dams 11 and 15 (group B) as well as dams 23 and 24 (Group C). However, this finding could be considered incidental and therefore not related to test item exposure since it was not observed in any of the dams belonging to the high dose groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in number of corpora lutea, implantation sites or litter size.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
An increase trend was however observed in pre-implantation loss and post-implantation loss (not statistically significant in any case) for dams of groups C and D. Although it cannot be ruled out a potential test item effect, since this effect was more relevant for pre-implantation loss than for post-implantation loss, it is thought that it could be also caused by the stress due to test item odour or slight irritability on respiratory tract, eyes and skin associated to the test item .
After the main study (B-02670) which included a higher number of animals (24 dams per group) but exposed at same concentrations, no statistical differences were found neither in pre-implantation nor in post-implantation losses.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in number of corpora lutea, implantation sites or litter size.
Early or late resorptions:
no effects observed
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
There was a slight decrease in the number of foetuses alive in both mid and high-dose groups. In addition, the litter weight was also low in these groups, but it is mainly related with the litter size as the mean of foetal weight was similar in all groups. Similar trend with no statistically significant data was found after the Main study.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Caesarean section (C-section) on day 21.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
All females in the groups were pregnant. No abortions were recorded.
Other effects:
not examined
Details on maternal toxic effects:
There were no statistically significant differences in number of corpora lutea, implantation sites or litter size.
An increase trend was however observed in pre-implantation loss and post-implantation loss (not statistically significant in any case) for dams of groups C and D. Although it cannot be ruled out a potential test item effect, since this effect was more relevant for pre-implantation loss than for post-implantation loss, it is thought that it could be also caused by the stress due to test item odour or slight irritability on respiratory tract, eyes and skin associated to the test item.
After the main study (B-02670) which included a higher number of animals (24 dams per group) but exposed at same concentrations, no statistical differences were found neither in pre-implantation nor in post-implantation losses.
Key result
Dose descriptor:
NOAEL
Remarks:
Taking into account all observations in this DRF study, for maternal effects, NOAEL could not clearly achived based on the small size of the groups.
Fetal body weight changes:
no effects observed
Description (incidence and severity):
There was no effect of the exposures on foetal weight at GD 21.
Reduction in number of live offspring:
effects observed, non-treatment-related
Description (incidence and severity):
There was a slight decrease in the number of foetuses alive in both mid and high-dose groups. In addition, the litter weight was also low in these groups, but it is mainly related with the litter size as the mean of foetal weight was similar in all groups. Similar trend with no statistically significant data was found after the Main study.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
No statistical differences were found in litter weight. There was a slight decrease in the number of foetuses alive in both mid and high-dose groups. In addition, the litter weight was also low in these groups, but it is mainly related with the litter size as the mean of foetal weight was similar in all groups. Similar trend with no statistically significant data was found after the Main study.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related findings were noted during external examination of foetuses at any exposure level.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No malformation was observed during the skeletal examination at any dose level and no test item-related findings were noted.
The alterations observed during the skeletal examination of foetuses were assigned to
- bone ossification stage/supernumerary ribs, which were related to a change from the expected ossification state of rat bones in this stage.
- bone or cartilage variations, which were used to describe structural changes (as changes in shape or size).

Ossification stage/supernumerary ribs
Fusion of zygomatic arch was observed in 2 foetuses in 2 litters in group C and in 1 foetus in 1 litter in group D, this finding was isolated and did not have any dose-related pattern.
In group D, there was a slightly increased incidence of litters with Thoracic ribs/supernumerary one rudimentary (5 litters in comparison with 3 litters in group A). Due to low incidence and the absence of a dose-dependency, this finding was considered to be incidental.
All remaining findings recorded were observed in structures known to be the most likely to show variable stages of ossification at this post coital age in rat. Therefore, there was no test item effect on the stage of development at any dose level.

Bone and cartilage variations:
Sternebra bipartite ossification was observed only in one foetus of group D, therefore it was considered to be incidental and not to be test item-related effect.
Costal cartilage 11 interrupted, long or supernumerary site was observed in group B (5 foetuses in 4 litters), in group C (3 foetuses in 2 litters) and in group D (8 foetuses in 6 litters). This finding was considered not to be test item related but a result of biological variability, since no corresponding or further ribs showed any comparable findings.
All remaining variations were comparable to the concurrent control and they were commonly seen in rats at this post coital age, therefore they were considered to be incidental and not test item-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
During visceral examination of the foetuses, findings were noted in:
42% examined foetuses in 88% litters in Sham filtered air
48% examined foetuses in 100% litters in Isophorone (Group B)
57% examined foetuses in 88% litters in Isophorone (Group C)
50% examined foetuses in 88% litters in Isophorone (Group D)

No test item-related findings were noted.
Malformation such kidney and ureter absent was found in one foetus in group A and in one foetus in group B. It was considered to be incidental and not test item related, as the incidence was similar in both groups.
Moderate urinary bladder distended was observed in 2 foetuses in 2 litters of group C and in 2 foetuses in 2 litters of group D. Due to the low and similar incidence in both treated groups, this finding was considered not to be test item-related.
The remaining variations observed were isolated incidences and did not indicate any test item effect. Thus, they were considered not to be test item-related.
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
- No statistically significant differences on reproductive parameters were observed, although a trend in percentage of pre and post implantation loss was recorded in the high dose group. However, due to the very low number of animals examined in this dose group, a scientifically valid interpretation of these results is not possible. After the main study (B-02670) which included a higher number of animals (24 dams per group) but exposed at same concentrations, no statistical differences were found neither in pre-implantation nor in post-implantation losses.
- Although a test item related effect cannot be ruled out, as this effect was more relevant for pre-implantation loss than for post-implantation loss, it could be also caused by the stress due to test item odour or slight irritability on respiratory tract, eyes and skin associated to the test item.
- No effects on external, visceral or skeletal abnormalities or variations were noted.
Key result
Dose descriptor:
NOAEL
Effect level:
0.84 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Key result
Developmental effects observed:
no

Terminology Used in the Assessment of the Data

Term

Description

Empty Implantation Site:

Very early resorption or aborted implantation

Early Resorption:

Amorphous mass being resorbed

Late Resorption:

Clearly defined foetal body being resorbed

Dead Foetus:

Appearance of live foetus at C-section but without induced respiration or movement

Live Foetus:

Breathing and/or moving foetus

Malformation:

A structural change in a foetus that would probably impair its health or development

Variation:

A foetal change that is unlikely to adversely affect survival or health. This includes a delay in growth or morphogenesis that has otherwise followed a normal pattern of development

 Calculations

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

             

Uterine and implantation data

Sex :Females

Group A

(Control)

Group B

(0.1 mg/L)

Group C

(0.3 mg/L)

Group D

(0.85 mg/L)

Number with Foetuses

8

8

8

7

Number of Corpora Lutea

Sum

112

111

107

97

Mean

14.0

13.9

13.4

13.9

SD

2.1

1.9

1.4

1.7

Number of Implantations

Sum

105

100

95

74

Mean

13.1

12.5

11.9

10.6

SD

2.0

2.1

2.2

3.1

% Pre-implantation Loss

Mean

5.9

9.9

10.6

23.4

SD

8.8

9.8

17.0

21.4

Early resorption

Sum

7

3

10

1b

Mean

0.9

0.4

1.3

0.1

SD

0.8

0.5

2.8

0.4

Late resorption 

Sum

0

0

0

1b

Mean

0

0

0

0.1

SD

0

0

0

0.4

Number of Dead Foetuses

Sum

0

0

0

0

Mean

0

0

0

0

SD

0

0

0

0

Number of Live Foetuses

Sum

98

96

82

67

Mean

12.25

12.00

10.25

9.57

SD

2.25

2.20

2.87

4.31

%Post-Implant Loss

Mean

6.85

4.16

12.61

12.61

SD

6.52

4.63

20.51

22.93

Live Foetus %Implant

Mean

93.2

95.8

87.4

87.4

SD

6.5

4.6

20.5

22.9

Litter size / foetal data

Sex :Females

Group A (Control)

Group B

(0.1 mg/L)

Group C

(0.3 mg/L)

Group D

(0.85 mg/L)

 

n

8

8

8

7

Total live Foetuses

Sum

98

96

82

67

Mean

12.25

12.00

10.25

9.57

SD

2.25

12.00

10.25

9.57

Live male foetuses

Sum

43

54

42

29

Mean

5.38

6.75

5.25

4.14

SD

1.92

2.55

2.05

2.41

Live female foetuses

Sum

55

42

40

38

Mean

6.88

5.25

5.00

5.43

SD

2.53

2.12

1.85

2.57

% Male foetuses

Mean

44.44

55.73

50.95

43.34

Litter weight

Mean

57.92

56.74

48.94

47.14

SD

10.33

9.11

11.82

20.67

Mean foetal weight (M+F)

Mean

4.74

4.75

4.84

4.95

SD

0.30

0.33

0.31

0.24

Mean foetal weight (M)

Mean

4.89

4.87

4.91

4.93

SD

0.28

0.34

0.31

0.33

Mean foetal weight

(F)

Mean

4.62

4.58

4.75

4.95

SD

0.28

0.31

0.33

0.27

Placenta weight

Mean

0.52

0.49

0.49

0.53

SD

0.07

0.06

0.06

0.10

Test atmosphere related parameters

Test item concentration in vapour

Throughout the study period, the variation of the mean test item concentrations with respect to the target values was within the ± 11% and the Relative Standard Deviation (RSD) on study means was 25%, 12% and 11% in groups B, C and D, respectively.

The deviations from target concentration on the daily individual atmosphere determinations in Groups C and D were < ±20% accepted criteria according the OECD guidelines except for 3 and 2 not consecutive days, respectively, when deviation from target was always < 24%.

Therefore, even though the deviations from target concentration on the daily individual atmosphere determinations were above the ±20% accepted criteria (according the OECD guidelines), the results obtained did not impact the objective of the study.

 

Summary of the mean test item concentrations (mg/L air) in test atmosphere:

 

Group

Exposure

Target test item conc.

(mg/L air)

Test item vapour concentration (mg/L air)

Mean

SD

RSD (%)[DS1] 

% Deviation from target

A

Air

-

-

-

-

-

B

Low dose

0.1

0.11

0.03

25

11

C

Mid dose

0.3

0.31

0.04

12

2

D

High dose

0.85

0.84

0.09

11

0.7

 

Nominal concentration

The nominal concentration was calculated from the daily consumed test item and the air flow feeding the vapour generator and thus it was determined for the overall inhalation system. Under consideration of the respective dilution air flow at every chamber, an efficiency of approximately 50 % or higher could be estimated for all groups. Overall, this value is within the expected range for this type of atmosphere generation, especially taking into account the actual concentrations determined at the representative sampling ports for every group.

Temperature and relative humidity

Mean values for exposure temperature were between 23.4 and 23.5 ºC for all groups. Mean Relative humidity was in the range 14.5 – 17.6 % for the different groups. These values are below the target range for husbandry. The difference was considered due to technical reasons. However, it was considered not to have affected the outcome of the study. 

Oxygen and carbon dioxide (CO2)

Mean oxygen concentration was 20.9 % for all groups. Carbon dioxide was always < 1% (mean concentration of 0.04 % for all groups).

Flow per animal

The flow per animal was between 0.5 and 1.5 L/min, which is sufficient to avoid re-breathing as it was more than twice the minute volume of rats.

Conclusions:
It was considered that the determination of a no observed-adverse-effect level (NOAEL) embryo-foetal development was suggested at (0.84 mg/L (149 ppm). However, for maternal effects, NOAEL could not clearly be achieved.
Executive summary:

Introduction and study outline

The objective of this dose range finding (DRF) study was to assess the potential effects of the test item on pregnancy and embryo-foetal development in F344 rats resulting from repeated administration of the test item through the inhalation route (nose-only) from day 6 post coitum (implantation, GD 6) to day 20 post coitum (the day prior to Caesarean section, GD 20). Therefore, this study aimed to characterize maternal toxicity at the exposure levels tested and to determine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

Furthermore, these results should serve as the basis for the selection of the exposure concentrations to be used in a further regulatory prenatal and developmental toxicity study to be performed according to guideline OECD 414.

For those purposes, 32 pregnant female rats were distributed into four experimental groups (A to D). Animals were exposed to diluted vapour from the test item (groups B to D) or to filtered compressed fresh air (Group A; control) 6 hours/day from day 6 post coitum (implantation, GD 6) to day 20 post coitum (the day prior to Caesarean section, GD 20).

Animals from group A were exposed to filtered compressed fresh air and served as control and animals from groups B to D were exposed to diluted vapour from the test item at nominal concentrations of 0.1, 0.3 and 0.85 mg/L air, respectively. The vapour was generated from the liquid test item using a Volatile Organic Compound (VOC) generator (TSE-systems).

In order to characterize the test atmosphere and to check the reproducibility of the vapour generation and dilution, the following analytical parameters were determined at defined intervals: test item concentrations, temperature, relative humidity as well as oxygen and carbon dioxide concentrations through exposure chamber.

All animals were observed twice daily for mortality and clinical observations before and within 1 hour after exposure. Body weights, and food consumption were recorded at appropriate intervals. On GD 21, a caesarean section was performed on each female and selected organs were collected and weighed. The uteri, placentae, and ovaries were examined, and the numbers of foetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded. The foetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Results

The composition and characteristics of test atmospheres were stable throughout the study period in Groups C and D. There was a slightly higher variation in Group B. The mean concentrations obtained deviated < 11% from the respective target.

Concentrations of 0.11, 0.31 and 0.84 mg/L air corresponding to 20, 54 and 149 ppm were achieved in Groups B, C, and D, respectively.

 

Maternal toxicity

None of the animals died during exposure.

The most frequent clinical sign present in animals belonging to all experimental groups, including sham, was piloerection. This sign is commonly observed in nose-only inhalation studies and, since it was equally present in the control group, it can be considered not to be caused by the test item.

No statistically significant differences were found in mean body weight/body weight gain and food consumption among the four experimental groups neither during the exposure period nor at the end of the study.

No relevant necropsy findings related to the test item exposure were recorded for the dams. There were no effects of the exposure on the pregnancies and/or gravid uterus weight. There were no statistically significant differences in pre- and post-implantation losses, however, a trend was observed especially for pre-implantation loss (%) where a 4-fold increase was recorded for dams of the high dose group ( 149 ppm, 0.84 mg/L) comparted to control group.

Post-implantation loss (%) was higher for mid and high-dose group compare to control group. In addition, the number of live foetuses was smaller in the mid- and high dose groups if compare to the control.

With regards to litter size, it was similar in all the groups.

Foetal toxicity

There was no effect of the exposures on foetal weigh at GD 21. Finally, no external, visceral or skeletal abnormalities or variations were noted.

Conclusions

No statistically significant differences on reproductive parameters were observed, although a trend in percentage of pre and post implantation loss was recorded in the high dose group. However, due to the very low number of animals examined in this dose group, a scientifically valid interpretation of these results is not possible. After the main study (B-02670) which included a higher number of animals (24 dams per group) but exposed at same concentrations, no statistical differences were found neither in pre-implantation nor in post-implantation losses.

Although a test item related effect cannot be ruled out, as the observed effect was more relevant for pre-implantation loss than for post-implantation loss, it could be also caused by the stress due to test item odour or slight irritability on respiratory tract, eyes and skin associated to the test item.

No effects on external, visceral or skeletal abnormalities or variations were noted. Therefore, under the conditions described for this DRF study, the test item did not reveal any teratogenic potential up to and including the highest dose level of 149 ppm (0.84 mg/L).

On the basis of these results and under the conditions of this study, it was considered that the determination of a no observed-adverse-effect level (NOAEL) embryo-foetal development was suggested at 149 ppm (0.84 mg/L). However, for maternal effects, NOAEL could not clearly be achieved.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-03-12 to 2019-04-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS870.6300
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Physical state (at 20ºC): Neat Liquid
Purity / composition: ≥98.0 to ≤ 99.6%
Molecular weight: 138.20 g/mol
Colour: Clear
Storage conditions: Room temperature (ca. 23ºC); protected from light
Expiry date: July, 2019
Handling conditions: Personnel will wear gloves and auto-ventilated equipment equipped with filters appropriate vapours
Other: Sensitive to air, highly volatile, odour of peppermint.
Avoid inhalation and contact with eyes and skin. Test item may cause irritation of respiratory tract, eyes and skin
The neat substance without solvent will be tested
Species:
rat
Strain:
other: Fischer 344 (F344/HanZtn Rj)
Remarks:
Although the F 344 strain is normally discouraged in reproduction studies (OPPTS 870.6300), the authorities specificaly asked for their use in order to compare the results with previous non-GLP reproduction toxicity studies, in which this strain was used
Details on test animals or test system and environmental conditions:
TEST ORGANISMS: 
- Species: Rat
- Sex: Female
- Strain: Fischer 344 (F344/HanZtn Rj)
- Source Fischer 344: Janvier, Route des ChénesSecs, Le Genest Saint-Isle, F-53940 France
- Age: 8 - 13 weeks on arrival; 11 - 12 weeks on first item exposure
- Animals per cage (before / after distribution): Up to 5 (1 after distribution and before mating)
- Bedding material: Sodispan (SR-CHOPO-T) (autoclavable)
- Diet: ad libitum, Global diet, 2914C, Envigo Teklad
- Water: ad libitum, Tap water
- Acclimatisation period: at least 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.3 - 24.6° C
- Humidity (%): 28 - 61 %
- Light cycle: 12 hours light, 12 hours dark
Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Remarks:
Sham filtered air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

Preparation of the exposure system
Previous to exposure onset, a complete setup of the system and flow/pressure characterization was performed according to the corresponding internal SOPs. The tasks were the following:
- Make an outline of each set up including identification of the corresponding equipment.
- Leak test through the atmosphere conveyor glass tubes.
- Identification of each group (name, dose, total number of animals, total flow of vapour/air).
- Check air temperature and relative humidity.
- Theoretical calculations regarding dilution.
- Check vacuum system in sampling ports: capillary installed, vacuum pressure measurement, sampling flow.
- Exposure test: determination of pressure inside the inhalation chamber versus room pressure and difference of pressure between both chamber compartments. Determination of atmosphere flow and vapour concentration at different positions of the chambers was obtained to confirm a homogenous distribution of the vapour.
- Six ports from the high level, six ports from the mid level and six ports from the low level. Calculation of coefficient of variation (%CV) within each level and across the three levels. Within each level, the CV was not higher than 5%, whereas the CV among the three levels will not exceed 10%.


Test item generation
- A vapour was generated from the liquid test item using a Volatile Organic Compound (VOC) generator (TSE-systems).
- The test item was inside the generator vessel in liquid state.
- It was heated at a controlled temperature by means of a water bath equipped with a temperature probe to generate a vapour phase.
- At the same time, a source of compressed air at a controlled temperature was delivered into the VOC generator. This compressed air was mixed with the vapour phase of test item extracting the formulation of the product in a controlled amount of air (L/min) and concentration.
- The vapour was conveyed via glass tubing from the generator to the exposure chambers.
- The flow rate through the exposure chamber was adjusted as necessary.
- In order to prevent exposure of the animals to test item droplets from condensation, a filter was placed immediately before the exposure chamber.
- For sham exposure, filtered, compressed fresh air was provided to the exposure chamber.


Exposure conditions
- Inhalation exposure was performed using a flow-past, nose-only exposure system.
- The animals were confined separately in restraint glass tubes matching their size which are positioned radially around the exposure chamber.
- The exposure system ensures a uniform distribution and provides a constant flow of test material to each exposure tube.
- The flow of air at each tube was between 0.5 to 1.5 L/min, which was sufficient to minimize re-breathing of the test item vapour as it is more than twice the respiratory minute volume of rats.
- The position of the rats in each group within the exposure chamber was changed daily according to an insertion schedule.
- Sham-exposed rats were exposed to filtered, conditioned air, the exposure conditions being the same as those for the test item-exposed rats.
- Exposure chambers type EC-FPC-232 (anodized aluminium, volume inside compartment: approximately 3 L, equipped with glass exposure tubes) were used. The rats were individually exposed in glass tubes matching their size.
- Before starting the inhalation period, the animals were not adapted to the glass tubes to avoid pre-implantation losses due to the stress caused by the procedure itself.
- The temperature of the test atmosphere in the exposure chamber was maintained at 22 ± 5 °C and the relative humidity as required by experimental conditions.
- Air flow per animal was monitored regularly.
- No diet or drinking water was available during exposure.
- The time spent during animal shifting was not included within the exposure time.

Technical trials
- Technical trails were performed without animals and conducted before the animal phase of the study to establish the conditions for vapour generation and sampling.
- A vapour was generated for up to 6 hours for four days.
- During this period, the concentrations as well as the temperature and relative humidity of the chamber were monitored.
- The set-up conditions and results were similar to those obtained during the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Characterisation of test atmosphere
In order to characterize the test atmosphere and to check the reproducibility of aerosol generation, several parameters were determined at defined intervals.
- Determination of the nominal vapour concentration
The test item usage was determined daily by weighing the amount in the syringe pump and adjacent lines before and after each exposure to determine the quantity of test item used. The weight used was then be divided by the total air-flow volume to give the nominal concentration. The nominal concentration for different groups was calculated from the nominal concentration of high concentration chamber and the dilution factor for each group, respectively. These data were used for the purpose of monitoring the performance of the generation system.
- Analytical determination of the vapour concentration
Analytical determination of the vapour concentration was performed three times on each day of exposure for each test item group (groups B - D). Additional samples were analysed if considered necessary.
Vapour samples were captured in a solvent trap containing a defined volume of methanol (cooled with ice) positioned in an empty port of the inhalation chamber. Based on the results from the N-02130 study previously conducted at Vivotecnia, no additional solvent trap was necessary to place immediately after the first solvent trap, since it was demonstrated that all test item was expected to be captured during sampling. The sampling flow rate matched the air flow rate per port. The defined volume of methanol in the solvent trap was refilled to the start volume after sampling completion. Aliquots of samples of the resulting solution of test item in methanol were appropriately labelled and stored ca. 5°C until analysis.
Test item concentrations were analysed at Vivotecnia using an HPLC analytical method transferred by Swiss BioQuant AG during a previous study conducted at Vivotecnia. Samples of test item trapped in methanol were analysed within 3 days after sampling. The analytical method was validated at Vivotecnia in the study B-02791 .
Test item vapour concentration was determined three times a day on each day of exposure for each test item group by high performance liquid chromatography (HPLC) after collecting the vapour in a solvent trap containing 50 mL of methanol (cooled with ice) positioned in an empty port of the inhalation chamber. The analytical method was validated afterwards in Vivotecnia study B-02791. Sampling time was 5 minutes at a flow rate of approximately 1 L/min. Trap volume was determined before and after sampling in order to evaluate discrepancies in the final volume. Aliquots of the samples were analysed freshly immediately after the sampling process.
Details on mating procedure:
After acclimatization, females were housed with sexually mature males (1:1) to initiate the nightly mating period, until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed. The day on which mating was detected was designated gestation day 0 (GD0).
Male rats of the same source and strain were used only for mating. These male rats were in the possession of Vivotecnia and were not considered part of the test system.
Duration of treatment / exposure:
Duration of dosing: Daily from day 5 post coitum (implantation, GD 05) to day 19 post coitum (GD 19)
Dosing regimen: 6h/day
Frequency of treatment:
6h/day
Duration of test:
Caesarean section on day 20
Dose / conc.:
0 ppm (nominal)
Remarks:
Control group (Group A), Sham filtered air
Dose / conc.:
17 ppm (nominal)
Remarks:
Low dose group (Group B)
Dose / conc.:
53 ppm (nominal)
Remarks:
Mid dose group (Group C)
Dose / conc.:
150 ppm (nominal)
Remarks:
High dose group (Group D)
Dose / conc.:
0 ppm (nominal)
Remarks:
Control group (Group E), Pregnancy reference group
No. of animals per sex per dose:
24 pregnant female rats per dose
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Details on study design:
The present study was designed to assess the potential effects of the test item 3,5,5-trimethylcyclohex-2-enone (CAS 78-59-1) on pregnancy and embryo-foetal development in F344 rats resulting from repeated administration through the inhalation route (nose-only) from day GD 05 to GD 19 (the day prior to Caesarean section). This study has helped to demonstrate any dose-related response and to establish the no-observed adverse effect level (NOAEL).
For this purpose, 120 pregnant female rats were distributed into 5 different experimental groups (A to E). Group E was a pregnancy reference group not exposed. Animals were exposed to diluted vapour from the test item (groups B to D) or to filtered compressed fresh air (Group A) 6 hours/day from GD05 to GD19 (the day prior to Caesarean section).
All animals were observed once daily for mortality and clinical observations. Body weight and food consumption were recorded at appropriate intervals. On gestational day 20, a caesarean section was performed on each female and selected organ were collected and weighed. The uteri, placentae, and ovaries were examined, and the numbers of foetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The foetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

Rationale for target concentration selection: T
he target concentrations were selected by the sponsor according to two previous dose range finding prenatal development toxicity studies (N-02669, VVT 2019 and N-02130, VVT 2016) available for this test item.
Within these dose range finding studies, mortality was observed at 1.25 mg/L (220 ppm), while exposure at 0.85 mg/L (150 ppm) did induce maternal toxicity (statistically significant body weight losses) in one of the dose range finding studies. Based on these results and due to the fact that the observed effects at 220 ppm shows a very steep dose-response curve of the test substance, 150 ppm was chosen as highest dose concentration for the OECD 414 main study.
This concentration level is slightly above 144 ppm, which was requested by ECHA in order to compare the results with the previous inhalation teratogenicity studies (Exxon, 1984 and Exxon, 1983). In these prior studies, isolated instances of exencephaly were noted in rat and mice foetuses at 144 ppm.

Inclusion criteria: All animals were subjected to individual veterinary examinations. Only animals in good health and with pregnancy status confirmed were included in the study
Stratification method: Animals were randomly distributed by means of the body weight stratification method at least 2 days before first administration

- Determination of the temperature
The temperature in the chamber was measured hourly during exposure using a thermohygrometer. The target range is 22±3ºC.
- Determination of the relative humidity
The relative humidity (%) in the vapour was measured hourly in each group during each day of exposure using a thermohygrometer.
- Determination of oxygen and CO2 concentration
The oxygen concentration of the test atmosphere was measured hourly during each day of exposure using an appropriate device. The oxygen and CO2 concentrations were maintained above 19% and below 1% respectively, during the exposure period.
- Airflow rate
The exposure airflow rate was adjusted as appropriate before the start of the exposure. The actual airflow rate was monitored hourly in each group during each exposure. Additional measurements were performed if considered necessary.
Maternal examinations:
Experimental data concerning, body weight, food consumption, general clinical signs, thyroid weight, gross necropsy findings (dams) and ovaries and uteri examination were recorded through Provantis 8.5.2.1TM (Instem LSS Ltd., Staffordshire, United Kingdom) preclinical software.

CAGE SIDE OBSERVATIONS: Yes
The animals were examined once daily by cage side observation for mortality and morbidity. Monitoring was adjusted accordingly when the condition of the animal gave cause for concern.
Where the condition of an animal required premature sacrifice, it was euthanized by a sodium pentobarbital overdose. Any decision regarding premature sacrifice were made after consultation with the Study Director (or a nominated person) and with the study monitor, if possible.

DETAILED CLINICAL OBSERVATIONS: Yes
On exposure days, clinical observations were recorded before exposure, just after exposure and 1h after exposure. Monitoring was adjusted accordingly when the condition of the animal gave cause for concern.
Any visible clinical signs, discomfort and mortality was recorded in accordance with the humane endpoints guidance document of the OECD (ENV/JM/MONO(2000)7). Observations included changes in skin, eyes, and mucous membranes. Alterations in respiratory pattern or behaviour as well as changes in posture and response to handling and the presence of abnormal movements were also recorded.

BODY WEIGHT: Yes
Body weight were recorded on gestation day 0 (GD0), at least every 3 days and on GD20. In addition, body weight after sacrifice was also recorded.
Additional body weights were obtained as necessary for animal welfare reasons.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
Food was weighed on GD 0, 3, 5, 8, 11, 14, 17 and 20. Food consumption was calculated on these defined intervals by cage, estimated the daily food intake for each animal.
Data was presented in terms of consumption per animal per day for the periods (days 0 – 3), (3 – 5), (5 – 8), (8 – 11), (11 – 14), (14 – 17) and (17 – 20).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Gross necropsy
At the scheduled necropsy (GD20) females were sacrificed by CO2 asphyxiation and the foetuses were removed by caesarean section. For all animals the thoracic and abdominal cavities were opened by a ventral mid-line incision and the major organs examined. Organs or tissues showing any macroscopic abnormalities were removed and fixed in appropriate fixative. The uterus of any apparently non-pregnant female was stained with ammonium sulphide to accentuate possible haemorrhagic areas of implantation sites and position of foetuses in the uterus.
- Organ weights and tissue preservation
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight were recorded. From all dams, the weight of the thyroid gland was recorded and preserved in 4 % neutral-buffered formaldehyde for histopathological assessment. Weight of gravid uteri including the cervix was recorded.
Any organ with gross lesions was collected and preserved in fixation medium (neutral-buffered 4 % formaldehyde) for histological evaluation if considered relevant.
Additionally, lungs from all dams will be instilled via trachea with formalin at approximately 30 cm H2O pressure collected and fixed in neutral phosphate buffered 4% formaldehyde solution for possible further examinations.

- Endocrine disrupter relevant endpoints
Concentration of Thyroid hormones, including T4, T3 and thyroid-stimulating hormone (TSH), were measured by the appropriate analytical method from all animals. Blood samples were collected from the abdominal aorta at the end of the study in non-treated tubes and serum samples were processed following the appropriate SOP.
Serum samples of all animals were examined at the end of treatment for an evaluation of test item-related effects on thyroid hormones at BSL BIOSERVICE Scientific Laboratories Munich GmbH (delegated phase). After arrival of serum tubes (a total of 360 tubes = 3 aliquots per animal and sampling a two-times 30µl and one-time 100µl) shipped on dry ice, tubes was stored at < -70°C.
Parameter Units
T3 pg/mL or ng/mL
T4 pg/mL or ng/mL
TSH pg/mL or ng/mL
Hormone determination was performed on the MagPIX, Luminex or on the DRG:HYBRID-XL Analyzer, DRG.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
For pregnant females the following observations were made:

- Number of corpora lutea
- Number and distribution of implantations in each uterine horn, classified as early intrauterine deaths, late intrauterine deaths, dead foetuses or live foetuses. The implantations were numbered separately for the right and left horns. Numbering was sequential, commencing at the ovarian end through to the cervix.
- Gravid uterus weight: The uteri (and contents) of all females with live foetuses were weighed during necropsy on day GD 20 to enable the calculation of the corrected body weight

- Placental weight
The live foetuses and their placentae were removed.


Organ weights and tissue preservation
At the scheduled sacrifice, placentas were trimmed from any adherent tissue, and their wet weight were recorded. From all dams, the weight of the thyroid gland was recorded and preserved in 4 % neutral-buffered formaldehyde for histopathological assessment. Weight of gravid uteri including the cervix was recorded.
Fetal examinations:
Foetal Pathology
Foetuses were removed from the uterus by Caesarean section (C-section), sexed, weighed individually and examined for gross external abnormalities. External examination of foetuses was performed at Vivotecnia. The anogenital distance (AGD) was measured in all live rodent foetus. After external examination, foetuses were assigned with a number, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- Microdissection technique (sectioning/dissection technique)]. Approximately one half of the foetuses (foetuses with odd numbers) from each litter will be fixed in Bouin’s fixative. Then foetuses were sent in the same fixative medium to the test site AnaPath GmbH within 3 weeks. At the test site the foetuses were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissues were preserved in a solution of glycerin/ethanol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
- The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. After fixation in ethanol (ca. 94%), foetuses were sent in the same fixative medium to the test site AnaPath GmbH for further skeletal evaluation. Carcasses were processed through solutions of glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined, and all abnormal findings and variations were recorded. The assessment included but was not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles. The specimens were preserved individually in small containers.
Foetuses with abnormalities were photographed, when considered appropriate.

The foetal pathology phase report is attached to this endpoint study record.

Terminology Used in the Assessment of the Data
Term Description
Empty Implantation Site: Very early resorption or aborted implantation
Early Resorption: Amorphous mass being resorbed
Late Resorption: Clearly defined foetal body being resorbed
Dead Foetus: Appearance of live foetus at C-section but without induced respiration or movement
Live Foetus: Breathing and/or moving foetus
Malformation: A structural change in a foetus that would probably impair its health or development
Variation: A foetal change that is unlikely to adversely affect survival or health. This includes a delay in growth or morphogenesis that has otherwise followed a normal pattern of development


Calculations
Comparison Group A and E, against Groups B, C and D.
Mean foetal body weights were calculated separately by sex for each litter; group mean body weights were calculated (separately by sex) from the litter means.
The percentage of foetuses in each litter exhibiting each classification of abnormality was calculated; group mean percentages were calculated from the litter percentages. The percentage of male foetuses, out of the total number of foetuses, was calculated for each litter.

Pre-implantation loss (%) = (# of corpora lutea –# of implantation sites) x 100/ no. corpora lutea

Post-implantation loss (%) = (# of implantation sites – # of live foetuses) x 100/ #. of implantation sites

Statistics:
Provided that the sample size was considered adequate, the following parameters will be subject to statistical analysis:
- Body weight
- Body weight change
- Gravid uterine weight
- Body weight change corrected for gravid uterine weight
- Food consumption
- Absolute and relative organ weights
- T3, T4 and TSH levels
- Anogenital distance statistically evaluated by gender

Analysis of data from two groups:
Test of normal distribution Yes Un-paired t-test
No Mann-Whitney test
Test of equal variance No Un-paired t-test with Welch’s correction

Analysis of data from more than two groups
Test of normal distribution Yes One-way ANOVA Dunnett’s post test (comparison versus control group)
Tukey post test (comparison of all groups)
No Kruska-Wallis Dunn's post test
Test of equal variance No Kruska-Wallis Dunn’s post test
A value of P < 0.05 will be considered statistically significant
Indices:
Fertility Index, Viability Index, Resorption Index, Pre-Implantation Loss Index, Post-Implantation Loss Index, Runts Index, Variation Index,
Number of litters having abnormalities, Number of abnormalities per litter
Historical control data:
Although the Fisher 344 strain is normally discouraged in reproduction studies (OPPTS 870.6300), the authorities specifically asked for their use in both the DRF and main studies in order to compare the results with previous non-GLP reproduction toxicity studies, in which the Fischer 344 strain was used.
Due to the very limited number of historical control data on Fischer 344 rats this OECD 414 study was performed using two control groups and three dose-level groups in order to receive scientifically meaningful results.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The most frequent clinical signs present in animals belonging to all experimental groups, including sham, were chromorrhinorrea, chromodacryorrhea and wet fur. These signs are commonly observed in nose-only inhalation studies and, since it was equally present in the control group, it can be considered not to be caused by the test item. In addition, it was observed loss of stability at the beginning of the studio in animals exposed to the high dose, however this sign disappeared after the first days of exposure.
Additionally, incidental bleeding through the vagina was observed from days 12 to 16 of gestation in animals belonging to groups A, B, C and D. Even though the number of females with occasional bleeding was higher in the high dose group, these signs were single observations (< 2 and not in consecutive days) and there was not a dose-dependent relationship. Therefore, these observations were considered incidental and probably not test item related.
Mortality:
no mortality observed
Description (incidence):
None of the animals died during exposure. All animals survived until the schedule necropsy.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The exposure regimen resulted in only transient reductions in maternal weight in group D,151 ppm (approximately 3.5% reduced from Control Group A and 12.3% reduced from Pregnancy Reference Group E) on day 17 of Gestation.
No statistically significant differences were found in mean body weight gain among the four experimental groups neither during the exposure period nor at the end of the study. Body weight gain was reduced at GD 14-17, but this difference was not statistically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was decreased between the days 5 and 8 of the exposure period in Group D (151 ppm) when compared to Group Control A (up to approximately 15%) and Pregnancy Reference Group E (30.9%), p<0.01. Even though it cannot be ruled out a test item effect on food consumption at the beginning of the study, after these transient reductions there were a similar consumption among the five experimental groups (no significant differences) at the end of the study. Therefore, the variation on food consumption at the beginning of the exposure was considered incidental and devoid of any toxicology effect.
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
Description (incidence and severity):
No statistical differences were recorded neither in terminal body weight nor in gravid uterus weight when compare the Group Control A with the exposed groups. The adjusted body weight to gravid uterus was also similar among exposed groups at concentrations of 18, 55 and 151 ppm, respectively. However, when compared Group E pregnancy reference group with both Control A and exposed Groups, there were some statistically significant increased. Nevertheless, these differences were considered to be related with the procedure itself rather than with the exposure.
Uterine weight in Group C (55 ppm) and D (151 ppm) was reduced (96% of the control group value) but this reduction was not considered adverse because the differences were slight, within normal animal variation and there were no statistically significant differences between the treated groups and control group.

Absolute and relative organ weights of thyroid gland were similar among all experimental groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No relevant macroscopic abnormalities related to the exposure to the test item were observed.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Under the conditions of this study all animals survived their scheduled period and there were no necropsy findings or histology changes in the thyroid glands that could be related to the treatment with the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Endocrine disrupter relevant endpoints
The serum levels of total T3 were elevated in dosed groups C and D (exposed to 55 and 151 ppm 0.86 mg/L of test Item) when compared to Control Group A. This elevation was not due to enhanced T4 to T3 conversion. In addition, when compared with Reference Group E, only difference with Group D became significant. Individual data showed a great variability between animals from the same group. Taking into consideration that not differences were observed in TSH and T4 levels, the values were considered incidental findings and devoid of toxicological significance or not treatment-related. When compared with historic control data from BSL Bioservice, thyroid hormone levels were within the normal range.
Number of abortions:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in number of corpora lutea, implantation sites or litter size.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A slight increase on post-implantation loss (not statistically significant in any case) was recorded for dams of groups C (55 ppm) and D (151 ppm). In addition, 2 dams from group D (151 ppm) presented total resorption. These differences were not significant and were considered not test item related. In addition, the post-implantation losses (%) where within the observed range (6.86%-18.46%) from vivotecnia historical control data in Fischer 344 sham filtered exposed.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
A total of two pregnant dams with total resorption were recorded in the high dose group (151 ppm ).
Early or late resorptions:
effects observed, non-treatment-related
Description (incidence and severity):
A total of two pregnant dams with total resorption were recorded in the high dose group (151 ppm ).
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no significant decrease in the number of foetuses alive in any of the dose groups.
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Caesarean section (C-section) on day 20.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
From the 120 dams enrolled in the study, 117 were pregnant.
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 151 ppm (analytical)
Based on:
test mat.
Basis for effect level:
other: no adverse and statistically significant maternal toxicity was observed, in addition no differences on reproductive parameters were observed
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
There were some marginal differences of the exposures on foetal weight at GD 20 post coitum between control group A and exposed groups B (18 ppm) and C (55 ppm). The very marginal (3-5%) difference while achieving statistical significance in total foetal, male and female weights, was considered to be within the normal biological variation and therefore, devoid of toxicological significance or not treatment-related. On the other hand, the statistical difference in foetal body weight when compared to controls at the high dose-level showed a decrease of 13.6%, consequently a test item effect on foetal weight in the high dose group D cannot be excluded.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
The number of foetuses alive were similar among dose groups.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The percentage of male foetuses were also similar between all dose groups.
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
No statistical differences were found in litter weight, although a decreased of 9% was observed in group D (151 ppm) compared with Control group A.
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Description (incidence and severity):
No treatment-related findings were noted during external examination of foetuses at any exposure level.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No malformation was observed during the skeletal examination at any dose level and no test item-related findings were noted.
The alterations observed during the skeletal examination were assigned to
• bone ossification stage/supernumerary ribs, which were related to a change from the expected ossification state of rat bones in this stage.
• bone or cartilage variations, which were used to describe structural changes (as changes in shape or size).
The type and frequencies of recorded variations were similar at all dose levels, in the control groups, group A (Sham filtered air) and group E (pregnancy reference).

- Ossification stage/supernumerary ribs
Ossification stage of foetal skeleton and number of supernumerary ribs showed no indication of any test item-related effect at any dose level.
At dose levels of 55 ppm (group C) and 151 ppm (group D), a higher number of incomplete ossified supraoccipital bone was observed when calculated either on a foetus or a litter basis. At dose levels of 18 ppm and 151 ppm a higher number of non-ossified caudal vertebrae was observed when calculated either on a foetus or a litter basis. For both alteration no dose dependency was noted, therefore these findings were considered not to be test item related but a result of biological variability.

- Bone and cartilage variations:
Bone and cartilage variations showed no indication of any test item-related effect at any dose level.
Among bone and cartilage variations, an increase in the incidence of sternebrae offset ossification sites was noted at 18 ppm and 55 ppm when compared to the incidence in pregnancy reference (group E) (20% and 22% respectively, versus 4% in the pregnancy reference group). Whereas, no significant difference was observed when compared to the concurrent control (Sham filtered air). Furthermore, this finding was noted only in 2% of foetuses of group D (151 ppm) when compared to the concurrent control (Sham filtered air), as no dose-dependency was noted, this finding was considered to be incidental and not test item-related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
During visceral examination of the foetuses, findings were noted in:

33% examined foetuses in 78% litters in Sham filtered air (Group A)
41% examined foetuses in 88% litters in 3,5,5-trimethylcyclohex-2-enone (Group B,18 ppm)
35% examined foetuses in 96% litters in 3,5,5-trimethylcyclohex-2-enone ( Group C, 55 ppm)
35% examined foetuses in 75% litters in 3,5,5-trimethylcyclohex-2-enone ( Group D 151 ppm)
53% examined foetuses in 96% litters in Pregnancy Reference (Group E)

No test item related findings were noted in any treated group.

Malformation such as renal pelvis dilated severe was observed in group B (litter 31, foetus 356) in one of 143 foetuses examined. Since this alteration occurred only in one foetus, it was considered to be incidental and not test item related.
The overall incidences of foetuses or litters with any remaining finding were isolated incidences and did not indicate any test item effect. Thus, they were considered not to be test item-related. The incidence of one single variations in form of thymus long and testis malpositioned were increased on foetal and litter basis when compared to group A (Sham filtered air) and they were comparable to those observed in group E (pregnancy reference). These specific findings were known to be normally associated with a slight immaturity of development; therefore, they were considered to be not related to treatment.
Other effects:
no effects observed
Description (incidence and severity):
No differences were observed in anogenital distance for both females and males.
Details on embryotoxic / teratogenic effects:
Taking into account the test item effect on foetal body weight, a NOAEL for embryo-foetal effects was established at 55 ppm (0.31 mg/L). However, under the conditions of the study the test item did not reveal any teratogenic potential up to and including the highest dose level (NOAEL, teratogenic > 151 ppm).
Key result
Dose descriptor:
NOAEL
Effect level:
>= 151 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations
skeletal malformations
visceral malformations
Dose descriptor:
NOAEL
Effect level:
55 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Key result
Abnormalities:
no effects observed
Description (incidence and severity):
Under the condition of this inhalation OECD 414 study in Fischer F344 rats the test item did not reveal any teratogenic potential up to and including the highest dose level of 151 ppm (0.86 mg/L).
Key result
Developmental effects observed:
no

Summary Foetal External examinations

No treatment-related findings were noted during external examination of foetuses at any exposure level.

The table that summarizes the macroscopic findings observed at necropsy (only foetuses with findings are included in the table) is attached to this endpoint study record.

Inhalation Technical Data: Tables see attached document to this endpoint study record

In-Life Data: Tables: see attached document to this endpoint study record

Test atmosphere related parameters

 Test item concentration in vapour

Throughout the study period, the variation of the mean test item concentrations with respect to the target values was within the ± 3% and the Coefficient of Variation (CV) on study means was 8%, 10% and 7% in groups B, C and D, respectively.

The deviations from target concentration on the daily individual atmosphere determinations in Groups B, C and D were <± 20% accepted criteria according the OECD guidelines except for 2 not consecutive days, in group C, when deviation from target was always < 22%.

Therefore, even though the deviations from target concentration on the daily individual atmosphere determinations were punctually above the ± 20% accepted criteria (according the OECD guidelines), the results obtained did not impact the objective of the study.

Summary of the mean test item concentrations (mg/L air) in test atmosphere:

Group

Exposure

Target test item conc.

Test item vapour concentration (mg/L air)

(mg/L air)

Mean

SD

RDS (%)

% Deviation from target

A

Air

-

-

-

-

-

B

Low dose

0.1

0.10

0.01

8

2

C

Mid dose

0.3

0.31

0.03

10

3

D

High dose

0.85

0.86

0.06

7

1

 

Nominal concentration

The nominal concentration was calculated from the daily consumed test item and the air flow feeding the vapour generator and thus it was determined for the overall inhalation system and for each exposure concentration. Overall, this value is within the expected range for this type of atmosphere generation, especially taking into account the actual concentrations determined at the representative sampling ports for every group.

 

Temperature and relative humidity

Mean values for exposure temperature were between 21.78 and 21.55 °C for all groups. Mean relative humidity was in range 6.25 - 7.23 % for the different groups. These values are below the target range for husbrandy. The difference was considered due to technical reasons. However, it was considered not to have affected the outcome of the study.

   

Oxygen and carbon dioxide (CO2)

Mean oxygen concentration was 20.9 % for all groups. Carbon dioxide was always < 1 % (mean concentration of 0.04 % for all groups).

Flow per animal

The flow rate per animal was between 0.5 and 1.5 L/min, which is sufficient to avoid re-breathing as it was more than twice the minute volume of rats.

Conclusions:
Under the conditions of the inhalation OECD 414 study with Fischer rats a no-observed-adverse-effect level (NOAEL) for maternal effects may be established at 151 ppm (0.86 mg/L).
The test item did not reveal any teratogenic potential up to and including the dose level of 151 ppm (0.86 mg/L) and therefore, the NOAEL for developmental effects was determined to be >= 151 ppm (0.86 mg/L). As an effect on foetal body weight at 151 ppm (0.86 mg/L), a NOAEL for embryo-fetal effects was established at 55 ppm (0.31 mg/L).
Executive summary:

Introduction and study outline

The objective of this developmental toxicity study was to assess the potential effects of the test item on pregnancy and embryo-foetal development in F344 rats resulting from repeated administration of the test item through the inhalation route (nose-only) during the period of organogenesis, from gestation day GD05 to day GD 19 (the day prior to Caesarean section).

Therefore, this study aimed to characterize maternal and foetal toxicity at the exposure levels tested and todetermine a no-observed-adverse-effect level (NOAEL) for maternal and developmental toxicity.

 

For this purpose, 120 pregnant female rats were distributed into 5 different experimental groups (A to E). Group E was a pregnancy reference group not exposed. Group Ewas included to be able to identify the possible findings related with the inhalation procedure itself, as the stress of the procedure could affect the pregnancy outcome and related variables. Therefore, differences between group E and groups A, B, C and D are just related with the procedure itself and not with the test item exposure. Furthermore, Group E served as second control group. Animals were exposed to diluted vapour from the test item (groups B to D) at nominal concentrations of 17, 53 and 150 ppm (0.1, 0.3 and 0.85 mg/L air, respectively), respectively, or to filtered compressed fresh air (Group A) 6 hours/day from GD05 to GD19 (the day prior to Caesarean section).

The vapourwas generated from the liquid test item using a Volatile Organic Compound (VOC) generator (TSE-systems).

In order to characterize the test atmosphere and to check the reproducibility of the vapour generation and dilution, the following analytical parameters were determined at defined intervals: test item concentrations, temperature, relative humidity as well as oxygen and carbon dioxide concentrations through exposure chamber.

 

All animals were observed three times daily for mortality and clinical observations before, just after and within 1 hour after exposure. Body weight and food consumption were recorded at appropriate intervals.

On gestational day 20, a caesarean section was performed on each female. The animals were subjected to a macroscopic postmortem evaluation and corpora lutea/implantation data were recorded and the gravid uterus and thyroid gland were weighed. The foetuses were examined for externally visible abnormalities, sexed and weighed. Approximately one-half of the fetuses (alternating fetuses) in each litter were examined for soft-tissue abnormalities using a microdissection procedure. The other half of the fetuses were then stained with Alizarin Red S and examined for skeletal abnormalities and ossification variations.

 

Results

The composition and characteristics of test atmospheres were stable throughout the study period in Groups A, B, C and D. The mean concentrations obtained deviated 3% from the respective target. The overall mean (± standard deviation) analytical exposure concentrations for the respective exposure groups were as follows: 0.10 ± 0.1, 0.31 ± 0.031, and 0.86 ± 0.05 mg/L corresponding to 18, 55 and 151 ppm.

Maternal toxicity

None of the animals died during exposure.

The most frequent clinical signs present in animals belonging to all experimental groups, including sham, were chromorrhinorrea, chromodacryorrhea and wet fur. These signs are commonly observed in nose-only inhalation studies and, since it was equally present in the control group, it can be considered not to be caused by the test item. In addition, it was observed loss of stability at the beginning of the study in animals exposed to the high dose, however this sign disappeared after the first days of exposure.

The exposure regimen resulted in only transient reductions in maternal weight in high dose group D (approximately 3.5% reduced from Control Group A and 12.3% reduced from Pregnancy Reference Group E).Body weight gain was also reducedat GD 14-17, but this difference was not statistically significant. Food consumption was decreased at the beginning of the exposure in Group D (151 ppm) when compared to Group Control A (up to approximately 15%) and Pregnancy Reference Group (30.9%) between the day 5 and 8 of the exposure period. However, the Pregnancy Reference Group E, had a significantly increased in food consumption when compare with all the exposed groups including sham filtered control group A. Consequently, the difference with group E were considered to be related with the exposure procedure and not with the test item.

The Reference Pregnancy Group when compared with the test item exposure groups, showed differences in Body weight, Bodyweight gain and food consumption, however there were also differences when compare the Control Group A with the Pregnancy Reference Group E. Therefore, it was considered that the differences in Body weight, Bodyweight gain and food consumption with group E were related with the stress of the procedure itself and not with the exposure to the test item.

The serum levels of total T3 were elevated in dams from groups C and D (exposed to 55 and 151 ppm of test Item), this elevation was not due to enhanced T4 to T3 conversion. In addition, no differences were observed in thyroid weight and after the histopathology evaluation and when compare with historic control data from BSL Bioservice the T3 levels were within the normal range.

No relevant necropsy findings related to the test item exposure were recorded for the dams. There were no effects of the exposure on the pregnancies and/or gravid uterus weight. There were no statistically significant differences in pre- and post-implantation losses, even though, post-implantation loss (%) was higher for mid and high-dose group compared to control group, where a 2-fold increase was recorded for dams of group D (high dose, 151 ppm) compared to control group. These differences were not significant and were considered not test item related. In addition, two dams from group D had complete resorption which increased the total percentage of post-implantation losses for the group. In addition, the post-implantation losses (%) where within the observed range (6.86%-18.46%) in Fischer 344 sham filtered exposed from vivotecnia historical control data.

Foetal toxicity

There was no foetal death recorded during the study and the sex ratio for live fetuses were unaffected by treatment.

The number of total and live foetuses was similar among all groups. With regards to litter size, it was similar in all the groups. Even though litter weight was smaller in the group D (151 ppm), approximately 9% smaller than the control group A, this difference was not statistically significant. On the other hand, Fetal body weight per litter (male, female, or total) was significantly reduced in all the exposed groups, these differences were below 5% for groups B (18 ppm) and C (55 ppm) and considered devoid of any toxicological significance, but a reduction of 13.6% on foetal body weight, when exposed at 151 ppm (Group D), was both statistically and biologically significant.

Finally, no external, visceral or skeletal abnormalities or variations were noted, and it was concluded that no test item related findings were noted in any treated group, neither during skeletal examination, nor during visceral examination. Therefore, the test item did not reveal any teratogenic potential up to and including the dose level of 151 ppm (0.86 mg/L). As an effect on foetal body weight at 151 ppm (0.86 mg/L), a NOAEL for embryo-foetal effects was established at 55 ppm (0.31 mg/L).

Conclusion

    

-   No adverse and statistically significant maternal toxicity was observed during the study, in addition no differences on reproductive parameters were observed. Only transient reductions in maternal weight gain on GD17 and food consumption between day 5 and 8 of gestation and an incidental increased on T3 serum levels. It was considered that a no-observed-adverse-effect level (NOAEL) for maternal effects may be established at 151 ppm (0.86 mg/L).

 

-        There was no effect of the exposures at any level on pregnancy outcome, in terms of pre- or post-implantation loss or the incidence of fetal abnormalities and variants. Therefore, under the conditions of this study, the test item did not reveal teratogenic potential up to and including the dose level and the NOAEL for developmental effects was determined to be >= 151 ppm (0.86 mg/L).

Nevertheless, taking into account the test item effect on foetal body weight, a NOAEL for embryo-foetal effects was established at 55 ppm (0.31 mg/L).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983-12-21 to 1984-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Principles of method if other than guideline:
Teratogenicity Test
GLP compliance:
not specified
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals or test system and environmental conditions:
TEST ORGANISMS
- Source: Charles River Labs (Kingston Facility)
- Age at study initiation: 9 weeks
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 15 days
- Housing: animals were contained in a 12.9 m3 stainless steel and glass chamber and exposed either to conditioned air or to an atmosphere of
isophorone suspended in air
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
ADMINISTRATION / EXPOSURE
- Vehicle: no vehicle
- Concentrations: 0 / 25 / 50 / 115 ppm (corresponds to 0, 144, 289 and 664 mg/m3)
- Type or preparation of particles: vapour
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All chambers were routinelly sampled on approximately an hourly basis by gas chromatograpgy.
Study mean concentrations as determined by GC were: group I = 0 ppm, group II = 27 ppm, group III = 52 ppm, group IV = 111 ppm
Details on mating procedure:
MATING PROCEDURES
Virgin female CD-1 mice (approximately 9 weeks of age)  were paired with similar males for mating. Females were confirmed to have mated by
observation of a copulatory plug in the vagina or by observation  of sperm in a vaginal rinse. Confirmed mated females were assigned to groups
randomly.
Duration of treatment / exposure:
6th to 15th day of gestation
Frequency of treatment:
6 h/d
Duration of test:
Mice were sacrified by methoxyflurane inhalation and examined by gross necropsy on the 18th day of gestation.
No. of animals per sex per dose:
22 female mice
Control animals:
other: yes, concurrent conditioned air
Details on study design:
Sex: female
Duration of test: section on the 18th d of gestation
Maternal examinations:
PARAMETERS ASSESSED DURING STUDY: 
- Body weight gain: each 3rd day
- Food consumption: not determined
- Clinical observations: each 3rd day

Ovaries and uterine content:
- Examination of uterine content: identified as live fetuses, dead  fetuses, late resorptions, and early resorptions at end of study (day 18  of gestation).
  The uterus of each animal was stained in 10 % aqueous ammonium sulfide and further examined for confirmation of implantation  sites. 
Corporalutea were counted.
Fetal examinations:
- Examination of fetuses: Live and dead fetuses were weighed, examined  externally for gross abnormalities, and crown-rump distances were  
determined.  One half of the fetuses from each litter were decapitated, the heads were preserved and subsequently sectioned and examined, viscera of these fetuses were examined by the Staples technique. All fetuses were eviscerated and processed for skeletal staining . Only those fetuses which
had not been decapitated were examined for skeletal malformations and ossification  variations.

Statistics:
STATISTICAL METHODS: 
- Bartlett's test of homogeneity of variance: body weight, body weight  change, number of implantation sites, ratio of live fetuses to  implantation 
sites, ratios of resorptions to implant sites, malformations  per litter.
- Kruskal-Wallis test if variances were not equivalent.
- Standard nested analysis of variance for fetal weights.
Indices:
no data
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MATERNAL TOXIC EFFECTS BY DOSE LEVEL: 
- Mortality and day of death: no mortalities
- Number pregnant per dose level: 22
- Number of resorptions: no statistically significant differences between  treated and control
- Number of implantations: no statistically significant differences  between treated and control
- Number of corpora lutea: no statistically significant differences  between treated and control
- Duration of Pregnancy: not statistically significant differences  between treated and control
- Body weight: reduced in day 18 mice in 664 mg/m3 dose group (-5.6 %,  corrected for uterine weight)
- Clinical signs: Unremarkable
Dose descriptor:
NOAEL
Effect level:
289 mg/m³ air
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL DATA: 
- Litter size and weights: no statistically significant differences  between treated and control
- Number viable: no statistically significant differences between treated  and control
- Sex ratio: no statistically significant differences between treated and  control
- Grossly visible abnormalities: no statistically significant differences  between treated and control
- External abnormalities: no statistically significant differences  between treated and control
- Soft tissue abnormalities: no statistically significant differences  between treated and control
- Skeletal abnormalities (control, 144, 289, 664 mg/m3): 24/106, 26/120,  31/111, 36/110   
no statistically significant differences between treated and control. 
- Other examinations: Three instances of exencephaly were noted in mouse  fetuses (no data on doses). These effects are regarded as not to be  
compound-related by the authors.
Dose descriptor:
NOAEL
Effect level:
>= 664 mg/m³ air
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

NOAEL (maternal): based on reduced body weight




Conclusions:
In this inhalation teratogenicity study with mice, isophorone elicited a clinical effect in the pregnant dams in the form of lower body weights (664 mg/m3, day 18 of gestation). During the conduct of the probe study there were three instances of exencephaly noted in mouse fetuses. Based on the
observations made in this study the authors do not believe that these anomalies were related to the test material.
Within the framework of the dose levels and test methods used, it is concluded, that isophorne was not teratogenic or fetotoxic in mice.
Executive summary:

Pregnant CD-1 mice were treated daily for 6 hours by inhalation (whole body) with isophorone from day 6 to day 15 of gestation to examine potential developmental toxicity effects of the test substance. Dosages used were 0 (conditioned air control), 144, 289 and 664 mg/m3, respectively (22 animals per dose level).

Body weight was reduced in mice of the highest dose group (gestation day 18, corrected for uterine weight: -5.6 %). Additionally, in one animal a dose related increase in alopecia was observed, as well as a discoloration of the cervical and aogenital region. Adverse effects on the fetuses were not observed.

Therefore, under the conditions of this inhalation teratogenicity study with mice, the NOAEL for maternal toxicity was determined to be 289 mg/m3 (based on < 6 % reduction in body weight gain). Isophorone was neither embryotoxic nor teratogenic up to the highest test concentration of 664 mg/m3 isophorone.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983-12-21 to 1984-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Principles of method if other than guideline:
Method: guideline comparable inhalation teratogenicity test
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on test animals or test system and environmental conditions:
TEST ORGANISMS
- Source: Harlan Sprague Dawley Inc.
- Age at study initiation: 11 weeks
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 15 days
- Housing: animals were contained in a 12.9 m3 stainless steel and glass chamber and exposed either to conditioned air or to an atmosphere of
isophorone suspended in air
Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
ADMINISTRATION / EXPOSURE
- Vehicle: no vehicle
- Concentrations: 0 / 25 / 50 / 115 ppm (corresponds to 0, 144, 289 and 664 mg/m3)
- Type or preparation of particles: vapor
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All chambers were routinelly sampled on approximately an hourly basis by gas chromatograpgy.
Study mean concentrations as determined by GC were: group I = 0 ppm, group II = 27 ppm, group III = 52 ppm, group IV = 111 ppm

Details on mating procedure:
MATING PROCEDURES: 
Virgin female Fischer rats (approximately 11 weeks of  age) were paired with similar males for mating. Females were confirmed to  have mated by 
observation of a copulatory plug in the vagina or by  observation of sperm in a vaginal rinse. Confirmed mated females were assigned to groups
randomly.
Duration of treatment / exposure:
6th to 15th day of gestation
Frequency of treatment:
6 h/d
Duration of test:
Rats were sacrified by methoxyflurane inhalation and examined by gross necropsy on the 20th day of gestation.
No. of animals per sex per dose:
22 female rats
Control animals:
other: yes, concurrent conditioned air
Details on study design:
Sex: female
Duration of test: section on the 20th d of gestation
Maternal examinations:
PARAMETERS ASSESSED DURING STUDY: 
- Body weight gain: each 3rd day
- Food consumption: 3 day intervals
- Clinical observations: each 3rd day

Ovaries and uterine content:
- Examination of uterine content: identified as live fetuses, dead  fetuses, late resorptions, and early resorptions at end of study
(day 20  of gestation). The uterus of each animal was stained in 10 % aqueous  ammonium sulfide and further examined for confirmation of 
implantation  sites. Corpora lutea were counted.
Fetal examinations:
Examination of fetuses: Live and dead fetuses were weighed, examined  externally for gross abnormalities, and crown-rump distances were  
determined. One half of the fetuses from each litter were decapitated, the heads were preserved and subsequently sectioned and examined, viscera
of these fetuses were examined by the Staples technique. All fetuses were eviscerated and processed for skeletal staining . Only those fetuses which
had not been decapitated were examined for skeletal malformations and ossification  variations.
Statistics:
STATISTICAL METHODS: 
- Bartlett's test of homogeneity of variance: body weight, body weight  change, food consumption, number of implantation sites,  ratio of live 
fetuses to implantation sites, ratios of resorptions to implant sites,  malformations per litter.
- Kruskal-Wallis test if variances were not equivalent.
- Standard nested analysis of variance for fetal weights.
Indices:
no data
Historical control data:
no data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MATERNAL TOXIC EFFECTS BY DOSE LEVEL: 
- Mortality and day of death: no mortalities
- Number pregnant per dose level: 22
- Number of resorptions: no statistically significant differences between  treated and control groups
- Number of implantations: no statistically significant differences  between treated and control groups
- Number of corpora lutea: no statistically significant differences  between treated and control groups
- Duration of Pregnancy: no statistically significant differences between  treated and control groups
- Body weight: reduced in days 12 (-6.1 %) and 15 (-6.8 %) rats in 664  mg/m3 dose group
- Food/water consumption: reduced food consumption in 664 mg/m3 dose group
- Clinical signs: alopecia and cervical or anogenital staining (each  dose-related).
Dose descriptor:
NOAEC
Effect level:
289 mg/m³ air
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
FETAL DATA: 
No statistically significant differences between treated and control  groups:
- Litter size and weights
- Number viable
- Sex ratio
- Grossly visible abnormalities
- External abnormalities
- Soft tissue abnormalities
- Skeletal abnormalities
Dose descriptor:
NOAEC
Effect level:
>= 664 mg/m³ air
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

NOAEL (maternal): based on reduced body weight, clinical signs are not considered

Conclusions:
In this inhalation teratogenicity study with rats, isophorone elicited a clinical effect in the pregnant dams in the form of decreased food consumption
(664 mg/m3, days 6-20 and 0-20), lower body weights (664 mg/m3, days 12 and 15 of gestation), and dose related increases in alopecia and
staining of the cervical and anogenital areas.
During the conduct of the probe study there was one instance of exencephaly noted in a rat fetus. Based on the observations made in this study the
authors do not believe that this anomaly was related to the test material.
Within the framework of the dose levels and test methods used, it is concluded, that isophorne was not teratogenic or fetotoxic in rats.
Executive summary:

Pregnant Fischer 344 rats were treated daily for 6 hours by inhalation (whole body) with isophorone from day 6 to day 15 of gestation to examine potential developmental toxicity effects of the test substance. Dosages used were 0 (conditioned air control), 144, 289 and 664 mg/m3, respectively (22 animals per dose level).

There was a significant reduction in food consumption of rats of the highest dose group. Body weight was also reduced in animals of the highest dose group (gestation day 12: -6.1%; gestation day 15: -6.8%). Additionally, a dose related increase in alopecia was observed, as well as a discoloration of the cervical and aogenital region. Adverse effects on the fetuses were not observed.

Therefore, under the conditions of this inhalation teratogenicity study with rats, the NOAEC for maternal toxicity was determined to be 289 mg/m3 (based on < 7% reduction in body weight gain). Isophorone was neither embryotoxic nor teratogenic up to the highest test concentration of 664 mg/m3 isophorone.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Species:
rat
Quality of whole database:
Klimisch 1
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Exxon, 1984 and 1983 (rat and mice)

In the first inhalation teratogenicity study with rats, isophorone elicited a clinical effect in the pregnant dams in the form of decreased food consumption (111 ppm (640 mg/m3), days 6-20 and 0-20), lower body weights (111 ppm 640 mg/m3, days 12 and 15 of gestation), and dose related increases in alopecia and staining of the cervical and anogenital areas. During the conduct of the DRF study (Exxon, 1983) there was one instance of exencephaly noted in a rat fetus at 144 ppm. Based on the observations made in this study the authors do not believe that this anomaly was related to the test material. Within the framework of the dose levels and test methods used, it is concluded, that isophorone was not teratogenic or fetotoxic in rats (Exxon, 1984).

In the second inhalation teratogenicity study with mice, isophorone elicited a clinical effect in the pregnant dams in the form of lower body weights (111 ppm (640 mg/m3), day 18 of gestation). During the conduct of the DRF study (Exxon, 1983) there were three instances of exencephaly noted in mouse fetuses at 144 ppm. Based on the observations made in this study the authors do not believe that these anomalies were related to the test material. Within the framework of the dose levels and test methods used, it is concluded, that isophorone was not teratogenic or fetotoxic in mice (Exxon, 1984).

DRF for OECD 414 (inhalation), F344 and Wistar rats (Vivotecnia, 2016)

According to the decision on substance evaluation dated 17 June 2015 (SEV-D-2114303205-67-01/F; “original decision”) the registrants were requested to perform a pre-natal developmental toxicity study (test method: EU B.31./OECD 414 as limit test) in Fischer 344 rats by the inhalation route at doses above 144 ppm (maximum tolerable and attainable concentration) on order to compare the results with the previous inhalation teratogenicity studies (Exxon, 1984 and Exxon ,1983).

 

The registrants decided to perform a dose-range finding study in advance to estimate the maximum tolerable and attainable concentration as requested in the ECHA decision. Based on occurrence of exencephalies in the two preliminary prenatal developmental toxicity studies at 144 ppm (Exxon, 1983 and 1984), the registrants decided to conduct the DRF study with target concentrations of 150 and 220 ppm. The estimation for the technical limit for the maximal vapour concentration is based on the physico-chemical properties of the test material. The actual technical limit was conducted to be at 220 ppm during technical trails and was used at high dose. Due to the unsuitability of F344 rats as well as due to the absence of historical control data, Wistar rats were also tested in the DRF study in order to be able to generate scientifically established results. (Remark: Due to the fact that no historical controls are available F344 rats and the differences in timing of developmental events compared to strains like Wistar which are commonly tested in developmental/reproductive toxicity studies, the assessment of results is hardly possible. These two points do not allow to differentiate between strain specific and/or incidental findings and substance related effects. For this reason, EPA guideline explicitly points out that Fischer 344 strain should not be used for developmental studies (see EPA Health effects Test Guidelines OPPTS 870.6300, August 1998). Therefore, in this study Wistar rats were used for quality control and comparison of findings in both strains and with available historical control data from Wistar rats.) The legal basis for this test strategy is the ECHA Factsheet "Follow up to dossier evaluation decisions p.2; ECHA-13 -FS-05".

Within this DRF study a high and promptly mortality was observed in F344 rats at the high dose (220 ppm) as well as a statistically significant lower body weight and body weight gain was observed at low dose (150 ppm) when compared to air-treated F344 rats (control group). Therefore, both concentrations were associated with overt maternal toxicity in F344 rats but no major toxicity was observed in Wistar rats. For animal welfare reason the high dose exposure (220 ppm) was stopped for both strains. However, this means that 150 ppm is the maximum tolerated concentration while 220 ppm is the maximum attainable concentration for F344 rats. No test item-related abnormalities were noted during the visceral or skeletal examinations of fetuses of both treated strains (F344 and Wistar rats).

Due to the overt maternal toxicity reproductive effects (post implantation loss) were observed in F344 rats but not in Wistar rats. No test-item related abnormalities were noted during the visceral or skeletal examinations of foetuses of both strains. In so far, the results of the Exxon studies were not reproducible. In particular against the background of all problems and difficulties with Fisher rats and due to animal welfare reasons, at this time, the registrants decided not to carry out a main OECD 414 study with the F344 strain because no scientifically based results could be generated. Additionally, no developmental effects were reported at lower concentration (111 ppm) in the Exxon study (Exxon, 1984).

DRF for OECD 414 (inhalation), F344 rats (Vivotecnia, 2019)

Based on the decision from ECHA of 10 April 2018 as a response to the updated dossier and the decision SEV-D-2114303205-67-01/F (“original decision”), ECHA came to the conclusion that the original decision was not met, and the registrants are still required to provide a full prenatal developmental toxicity study (according to OECD 414) in F344 rats by the inhalation route.

For this, a further DRF study was performed in Fischer 344 rats. 32 pregnant females were distributed into four experimental groups (A to D). Based on the results of the DRF in 2016 (Vivotecnia, 2016) animals were exposed to diluted vapour from the test item (groups B to D) in concentrations of 20, 54 and 149 ppm or to filtered compressed fresh air (Group A; control) 6 hours/day from day 6 post coitum (implantation, GD 6) to day 20 post coitum (the day prior to Caesarean section, GD 20). Within this DRF study, no statistically significant differences on reproductive parameters were observed, although a trend in percentage of pre- and post implantation loss was recorded in the high dose group. However, due to the very low number of animals examined in this dose group, a scientifically valid interpretation of these results was not possible. After the main study (Vivotecnia, 2020) which included a higher number of animals (24 dams per group) but exposed at same concentrations, no statistical differences were found neither in pre-implantation nor in post-implantation losses. Although a test item related effect cannot be ruled out, as this effect was more relevant for pre-implantation loss than for post-implantation loss, it could be also caused by the stress due to test item odour or slight irritability on respiratory tract, eyes and skin associated to the test item. No effects on external, visceral or skeletal abnormalities or variations were noted. Therefore, under the conditions described for this DRF study, the test item did not reveal any teratogenic potential up to and including the highest dose level of 149 ppm (0.84 mg/L). On the basis of these results and under the conditions of this study, it was considered that the determination of a no observed-adverse-effect level (NOAEL) embryo-foetal development was suggested at 149 ppm (0.84 mg/L). However, for maternal effects, NOAEL could not clearly be achieved.

OECD 414 (inhalation), F344 rats (Vivotecnia, 2020)

In the main OECD 414 inhalation study 120 pregnant female F344 rats were distributed into 5 different experimental groups (A to E). Group E was a pregnancy reference group not exposed. Group E was included to be able to identify the possible findings related with the inhalation procedure itself, as the stress of the procedure could affect the pregnancy outcome and related variables. Therefore, differences between group E and groups A, B, C and D are just related with the procedure itself and not with the test item exposure. Furthermore, due to the lack on historical control data, Group E served as second control group. Animals were exposed to diluted vapour from the test item (groups B to D) at concentrations of 18, 55 and 151 ppm (0.1, 0.31 and 0.86 mg/L air, respectively), respectively, or to filtered compressed fresh air (Group A) 6 hours/day from GD05 to GD19 (the day prior to Caesarean section). The target concentrations were selected by the registrants according to the two previous dose range finding prenatal development toxicity studies (Vivotecnia 2016 and 2019): Within these dose range finding studies, mortality was observed at 220 ppm), while exposure at 150 ppm did induce maternal toxicity (statistically significant body weight losses) in one of the dose range finding studies. Based on these results and due to the fact that the observed effects at 220 ppm show a very steep dose-response curve of the test substance, 150 ppm was chosen as highest dose concentration for the OECD 414 main study. This concentration level is slightly above 144 ppm, which was requested by ECHA in order to compare the results with the previous inhalation teratogenicity studies (Exxon, 1984 and Exxon, 1983). In these prior studies, isolated instances of exencephaly were noted in rat and mice foetuses at 144 ppm.

Maternal toxicity

Maternal toxicity was determined by the occurrence of a decrease in net body, weight gain, decrease in food intake and evaluation of intrauterine parameters. Summarizing the results of this study it can be concluded that no relevant clinical signs or necropsy findings related to the test item exposure were recorded for the dams. In addition,only transient reductions were observed for the dosed group D (151 ppm) compared to control group A for body weight/body weight gain and food consumption. Food consumption between gestation day 5 and 8 day decreased in 14.5% (p<0.01) when compared to sham air exposed control at the high dose-level (151 ppm). Nevertheless, no other difference in food consumption was observed over the study period. On the other hand, body weight on day 17 of gestation was reduced in group D (151 ppm) by 3.5% (p<0.05) when compared to control group A. However, on day 20 of gestation no difference was observed.

The serum levels of total T3 were elevated in dosed groups C and D (exposed to 55 and 151 ppm of test Item) when compared to Control Group A. This elevation was not due to enhanced T4 to T3 conversion. In addition, no differences were observed in thyroid weight and after the histopathology evaluation. When compared with Reference Group E, only difference with Group D became significant. Individual data showed a great variability between animals from the same group. Taking into consideration that not differences were observed in TSH and T4 levels,the values were considered incidental findings and devoid of toxicological significance or not treatment-related.

There was no effect of the exposures on the pregnancies. No statistical differences were observed in pre- and post-implantation losses and litter size were similar in all exposed and control groups.

It was considered that a no-observed-adverse-effect level (NOAEL) for maternal effects was evident at 151 ppm (0.86 mg/L).

 

Foetal toxicity

There was no effect of the test item exposure on litter weight or litter size at GD 20. Although foetal body weight per litter (male, female, or total) was significantly reduced in all the exposed groups, this difference was lower than 5% for groups B (18 ppm) and C (55 ppm) and therefore was considered to be within the normal biological variation. Foetal body weight in group D (151 ppm) was 13.6% lower than in group A and statistically significant, therefore a test item effect at 151 ppm (0.86 mg/L) cannot be ruled out.

Finally, no external, visceral or skeletal abnormalities or variations were noted, therefore it was concluded that no test item related findings were noted in any treated group. Therfore, the test item did not reveal teratogenic potential up to and including the dose level of 151 ppm (0.86 mg/L). However, considering the test item effect on foetal body weight, a NOAEL for embryo-fetal effects was established at 55 ppm (0.31 mg/L).

Accordingly, it can be concluded:

- Exposure of pregnant rats to atmospheres of 0 (Air Control), 18 ppm (0.10 mg/L), 55 ppm (0.31 mg/L) or 151 ppm (0.86 mg/L) 6 hours/day on gestation days 5-19, resulted in only transient reductions in maternal weight gain and food consumption during the exposure period, and an incidental increased on T3 serum levels. These changes were almost negligible at 55 ppm and most noticeable at 151 ppm. It was considered that a no-observed-adverse-effect level (NOAEL) for maternal effects may be established at 151 ppm (0.86 mg/L) 

- There was no effect of the test item exposure at any level on pregnancy outcome, in terms of pre- or post-implantation loss, litter weight or the incidence of fetal abnormalities and variants. Therefore, a NOAEL for developmental toxicity was determined to be >= 151 ppm (0.86 mg/L). Foetal body weight was reduced in dosed groups, and even though these differences become statistically significant, they were below 5% for groups B and C and considered to be within a normal biological variation. However, the decreased on foetal body weight when exposed at 151 ppm (0.86 mg/L) was 13.6% both statistically and biologically significant. Therefore, a NOAEL for embryo-fetal effects was established at 55 ppm (0.31 mg/L).

- As no test-item related abnormalities were noted during the visceral or skeletal examinations of foetuses of Fischer 344 rats up to and including the dose level of 151 ppm, the observed exencephalies in the two preliminary prenatal developmental toxicity studies (Exxon, 1983 and 1984) cannot be confirmed in any of the three prenatal toxicity studies (Vivotecnia, 2016, 2019 and 2020).

Justification for classification or non-classification

Based on the result of all available studies the test substance is not classified with reagard to reproduction according to the criteria of CLP Regulation 1272/2008.

Additional information