Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Oct 2017 - 22 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
At current, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Version / remarks:
2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: See below under Principles of method if other than guideline
Principles of method if other than guideline:
In addition, the procedures described in this study plan essentially conform to the following
guidelines:
- OECD 421, Reproduction/Developmental Toxicity Screening Test, 2016.
- EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, 2000.
- EC No 440/2008, B.7 Repeated Dose (28 days) Toxicity (oral), 2008.
- OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2008.
- EPA OPPTS 870.3050, Repeated Dose 28-day Oral Toxicity Study in Rodents, 2000.
GLP compliance:
yes
Limit test:
no
Justification for study design:
At current, studies in laboratory animals provide the best available basis for extrapolation to humans and are required to support regulatory submissions. Acceptable models which do not use live animals currently do not exist.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical appearance: yellow liquid
- Storage conditions: at 20 ±5 °C, in the dark

Test animals

Species:
rat
Strain:
other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks (males) and 13 weeks (females)
- Weight at study initiation: 275 - 307 g (males) and 198 - 241 g (females)
- Fasting period before study: No
- Housing: On arrival and following the pretest (females only) and pre-mating period, animals were gr oup housed (up to 5 animals of the same sex and same dosing group together) in Macrolon cages. During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon cages. During the post-mating phase, males were housed in Macrolon cages with a maximum of 5 males/cage. Females were individually housed in Macrolon cages. During the lactation phase, females were housed in Macrolon plastic cages. Pups were housed with the dam, except during locomotor activity monitoring of the dams. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage enrichment, bedding material, food and water.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum. The feed was analyzed by the supplier for nutritional components and environmental contaminants.
- Water: tap water, ad libitum. During motor activity measurements, animals had no access to water for a maximum of 2 hours. Periodic analysis of the water was performed.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-22
- Humidity (%): 42-73
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 04 Oct 2017 To 27 Nov 2017

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING FORMULATIONS
Test item dosing formulations (w/w) were homogenized by swirling to visually acceptable levels at appropriate concentrations to meet dose level requirements. Bulk formulations sufficient for one week of dosing were prepared once a week as clear colorless solutions. After homogenizing the bulk formulations to a visibly acceptable level, each (bulk) formulation was then divided into 7 aliquots for daily dosing and stored in the refrigerator protected from light (maximum storage in the refrigerator for 8 days after preparation). The dosing formulations were removed from the refrigerator for at least 30 minutes before dosing for adjustment to room temperature.
On each day of use the test item dosing formulations were kept at room temperature until dosing. To avoid foam formation and optimal homogeneity, the dosing formulations were swirled shortly before use for dosing. An adjustment was made for specific gravity of the test item.

DOSE VOLUME
5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
After 14 days of treatment, animals were cohabitated on a 1:1 basis within the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. Detection of mating was not confirmed in first instance for one female. Apparently, mating was overlooked in the assessment of the vaginal lavage in first instance. The mating date of this animal was determined a few days later based on detection of sperm cells on the vaginal lavage. Consequently, this couple was separated 4 days after the actual mating date.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability analyses performed previously in conjunction with the method development and validation study demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.
Concentration and homogeneity analyses were performed by using a validated analytical procedure.
Duplicate sets of samples (approximately 500 mg) for each sampling time point were collected. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ≤ 10%.
Samples were taken in week 1 for concentration determination (all groups) and homogeneity (low and high dose groups, the homogeneity results obtained from the top, middle and bottom for the low and
high dose group preparations were averaged and utilized as the concentration results.
Duration of treatment / exposure:
Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days).
Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during postcoitum, and at least 14-16 days of lactation (for 50-54 days). Females that failed to deliver pups were treated for 41 days, with the exception of females in the 1000 mg/kg bw/day dose group that were euthanized or found dead (one female) at the end of the premating period or during the post-coitum period due to adverse clinical observations.
Frequency of treatment:
Once daily, 7 days per week
Doses / concentrationsopen allclose all
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:
Mid dose group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
A 10-days dose range finder was conducted to select dose levels for the main study, and to determine the peak effect of occurrence of clinical signs after dosing. No guidelines were applicable as this study was intended for dose level selection purposes only. The test item and vehicle were administered to 3 females/group by single daily oral gavage for 10 days. Clinical Observations were done at least daily from Days 1-10, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. Body Weights: On Day 1 prior to dosing and on Days 5 and 10. Food Consumption: Over Days 1-5 and 5-10. All animals were subjected to an external, thoracic and abdominal examination on Day 10 after the last observation of clinical signs (scheduled necropsy). Animals were not deprived of food prior to necropsy. Terminal body weight, kidney and liver weight were determined at scheduled necropsy. No organs were fixed and histopathological examination was not performed. No signs of toxicity were noted at any dose level. Based on these results, selected dose levels for the main study were 100, 300 and 1000 mg/kg bw/day.

Positive control:
No.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
Time schedule: Twice daily. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: Once daily
During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals.

BODY WEIGHT: Yes
Time schedule for examinations: Prior to first dosing, and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13. A fasted weight was recorded on the day of necropsy.

FOOD CONSUMPTION:
Yes. Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

FOOD EFFICIENCY: No

WATER CONSUMPTION:
Yes. Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOLOGIC OBSERVATIONS
No

HEMATOLOGY
Time schedule for collection of blood: on the day of scheduled necropsy.
- Anaesthetic used for blood collection: Yes (isoflurane).
- Animals fasted: Males (with a maximum of 24 hours). Females were not fasted. Water was provided.
- How many animals: 5 animals/sex/group.
- Parameters checked were: According to test guidelines.

CLINICAL CHEMISTRY
Time schedule for collection of blood: on the day of scheduled necropsy.
- Animals fasted: Yes (with a maximum of 20 hours). Water was provided.
- How many animals: 5 animals/sex/group.
- Parameters checked were: According to test guidelines.
- Blood samples were processed for serum, and serum was analyzed for total Thyroxine (T4).

URIANALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (LND 6-13).
- Tests were performed after completion of clinical observations (including arena observation).
- Dose groups that were examined: all (5 animals/sex/group)
- Battery of functions tested: According to test guidelines. Hearing ability, pupillary reflex, static right ing reflex , fore- and hindlimb grip strength (recorded as the mean of three measurements per animal) and locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system). Total movements and ambulations were reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

GENERAL REPRODUCTION DATA:
From the mating period onwards, the following parameters were recorded for each female: male number paired with, mating date, confirmation of pregnancy and delivery day. Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.
Oestrous cyclicity (parental animals):
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
Daily vaginal lavage was performed for all females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of
copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous. This was done for all females, except for females that had to be euthanized in extremis or died spontaneously.
Sperm parameters (parental animals):
For the testes of all selected males (n=5) of the control and the high dose group, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On PND 4, the surplus pups (> 8 pups per litter) were euthanized by decapitation.

PARAMETERS EXAMINED
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities. Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

GROSS NECROPSY
- All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, nongravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea was recorded in addition. Necropsy procedures were performed by qualified personnel with appropriate training and experience in animal anatomy and gross pathology.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in the guidelines were prepared for microscopic examination and weighed, respectively.
For males that failed to sire and females that failed to deliver pups histopathological examination of cervix, epididymis, coagulation gland, prostate gland, seminal vesicles, ovaries, testes, uterus and vagina was performed.
All tissues were examined by a board-certified toxicological pathologist with training and experience in laboratory animal pathology.
For the testes of all selected males of the control and the high dose group, and all males that failed to sire, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Postmortem examinations (offspring):
From two surplus pups per litter, blood was collected, if possible. All remaining pups were euthanized on PND 14-16. Sex was determined both externally and internally. Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. In addition, blood was collected from two pups per litter and the thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. The pups selected for blood sampling were the same pups as selected for thyroid preservation.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations. The following pairwise comparisons were made: low dose group vs. control group; mid dose group vs. control group; high dose group vs. control group. Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). The motor activity data set was compared using an overall Kruskal-Wallis. The Wilcoxon Rank-Sum test was applied to compare the treated groups to the control group. An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: (Number of females mated/Number of females paired) x 100
- Fertility index: (Number of pregnant females/Number of females paired) x 100
- Gestation index: (Number of females bearing live pups/Number of pregnant females) x 100
- Precoital time: Number of days between initiation of cohabitation and confirmation of mating
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
- Post-implantation survival index (%): (Total number of offspring born/ Total number of uterine implantation sites) x 100
- Live birth index: (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100
- Percentage live males at First Litter Check: (Number of live male pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Percentage live females at First Litter Check: (Number of live female pups at First Litter Check/Number of live pups at First Litter Check) x 100
- Viability index: (Number of live pups on Day 4 of lactation / Number of pups born alive) x 100
- Lactation index: (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males: Salivation occurred in most males in the 1000 mg/kg bw/day dose group throughout the treatment period. One male showed flat posture, red discoloration on the nose and laboured respiration and rales shortly before early sacrifice on day 20. The clinical signs among the surviving males comprised occasionally laboured breathing, rales, piloerection and red discolouration or discharge on the nose, generally during the reproductive period only. Salivation occurred from time to time during the premating and reproductive periods in several males in the 300 mg/kg bw/day dose group. Single males had severe rales, piloerection and a red discoloration on the nose sporadically throughout the treatment periods (premating and reproductive). In the 100 mg/kg bw/day dose group, piloerection, a scab on the tail and salivation occurred in single males in this dose group only on the first day one of dosing (salivation) or during the reproductive period (piloerection, scab).
Females: Salivation occurred in most females in the 1000 mg/kg bw/day dose group throughout the treatment period. Four females died prematurely. The clinical signs among the surviving females comprised occasional rales and piloerection during the treatment period. Salivation occurred from time to time during the premating and reproductive periods (post coitum, lactation) in some females in the 300 mg/kg bw/day dose group. Single females had hunched posture and laboured respiration once during the study. One to two females had piloerection, alopecia, scabs (cervical region) and a wound (cervical region) during the reproductive period. In the 100 mg/kg bw/day dose group, salivation occurred sporadically in one or two females during the treatment period.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were five animals (one male and four females) treated at 1000 mg/kg bw/day that were found dead or were sacrificed in extremis. The male was sacrificed in extremis on study day 20. Prior to euthanasia, this male had a flat posture, red discoloration on the nose and laboured respiration, rales and salivation. It gained weight prior to death. There were no relevant macroscopic findings but microscopic findings included slight necrosis of the tracheal epithelial, which may be suggestive for a gavage-related procedural incident. However, it cannot be excluded that like in the females discussed below, the morbidity may have been caused by regurgitation secondary to the test item. All other males survived to scheduled euthanasia. Three females treated at 1000 mg/kg bw/day were prematurely euthanized, due to respiratory clinical signs and one was found dead (3 females before mating at day 12-16 and 1 female at day 26). One female was euthanized in extremis on study day 12. Prior to euthanasia this female had slight hunched posture, severe laboured respiration and rales, piloerection, moderate salivation and was severely pale. The female lost weight prior to death. One female was found dead on study day 15. This female showed rales on day 1 and salivation during treatment, but no other clinical signs prior to death. One female was euthanized in extremis on study day 26. Prior to euthanasia this female had piloerection and severe laboured respiration, rales, salivation and ptosis. One female was euthanized in extremis on study day 16. Prior to euthanasia this female had slight quick breathing, piloerection and severe rales, shallow respiration, salivation and ptosis. The female lost weight prior to death. Macroscopically the lungs were not collapsed which microscopically could be explained by trachea and lung lesions (up to marked degree) such as: acute inflammation (trachea and lung), ulceration/erosions of bronchial epithelium and trachea, and bronchial fibrosis and/or hyperplasia. In one female, foreign material was found within the tracheal lumen.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Body weights and body weight gain of treated males remained in the same range as controls over the treatment period in the 100 and 300 mg/kg bw/day dose groups. In the 1000 mg/kg bw/day dose group male body weights and body weight gain were reduced, achieving levels of statistical significance on Day 15 and 29 of treatment (Day 1 and 15 of mating, respectively), resulting in a 9% lower mean body weight at the end of treatment compared to concurrent controls. It was noted that one male at 1000 mg/kg bw/day lost weight over the first week of treatment, but recovered during the second week. In the absence of any clinical sign during the first week, no toxicological significance was attached to this finding.
Body weights and body weight gain of treated females remained in the same range as controls over the treatment period in the 100 and 300 mg/kg bw/day dose groups and in the 1000 mg/kg bw/day dose group until the early termination.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for the males and females. A low food consumption was recorded for one cage, containing five high dose males over the first week of treatment. One of the males in this cage had lost weight during this period and it was considered likely that this was accompanied by a lower food consumption which consequently affected the food consumption value for this cage.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In the 1000 mg/kg bw/day dose group males, a decreased number of reticulocytes was observed (28,2%), achieving a level of statistical significance when compared to controls. Furthermore, a slightly higher mean corpuscular haemoglobin concentration (MCHC) value (4.3%) was calculated from the red blood cell parameters, also achieving a level of statistical significance when compared to the MCHC value in controls. No toxicologically relevant changes were noted in the other haematological parameters in males at 1000 mg/kg bw/day and in all haematology parameters in males at 100 and 300 mg/kg bw/day. The haematology results in females should be interpreted with caution, because for the females at 1000 mg/kg bw/day the blood levels were representative for Day 14 post-coitum (the day of their early sacrifice) and those for the females of the other groups (including controls) for PND 14-16 (at lactation). The slightly increased values for red blood cell, reticulocyte and platelet counts, and corresponding changes in Red Blood Cell Distribution Width and red blood cell derived indices Mean corpuscular haemoglobin and Mean corpuscular volume, in 1000 mg/kg bw/day dose group females in comparison with controls, were likely due to the difference in their physiological status, rather than indicative of a treatment-related effect. Although historical control data representative for Day 14 post-coitum were not available, the results obtained in females at 1000 mg/kg bw/day in this study were all within the normal range and it was concluded that there were no (marked) changes in any of the haematology parameters that indicated a treatment-related effect at sacrifice on Day 14 postcoitum. No toxicologically relevant changes were noted in haematological parameters in females treated at 100 and 300 mg/kg bw/day.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Coagulation: High mean values for Prothrombin Time and Activated Partial Thromboplastin Time were observed in control males in comparison with the historical control data. Since the mean values in treated males were similar to the historical control data and they showed no dose-response relationship, no toxicological significance was attached to the statistical significances apparent for PT in the treated groups.
Clinical biochemistry parameters: Statistically significant reductions in Aspartate aminotransferase in the 100 and 300 mg/kg bw/day dose groups were considered to have occurred by chance. Since no dose-response was present and a decrease in this enzyme level in plasma is of no biological relevance, no toxicological significance was attached to this finding. The clinical biochemistry results in females should be interpreted with caution, because for the females at 1000 mg/kg bw/day the blood levels were representative for Day 14 post-coitum (the day of their early sacrifice after 4 weeks of treatment) and those for the females of the other groups (including controls) for PND 14-16 (at lactation, after 7-8 weeks of treatment in this type of studies). In the six early sacrificed females at 1000 mg/kg bw/day, the mean values for several parameters showed a statistically significant difference when compared to controls, comprising; Alanine aminotransferase, Alkaline Phosphatase, total protein, albumin, urea, cholesterol, potassium, calcium and inorganic phosphate. Although the available historical control ranges for the blood-value of these parameters were applicable for lactating females, the absolute blood-values in the early sacrificed, pregnant females at 1000 mg/kg bw/day were still within the historical control range of the laboratory. Therefore the changes were considered minimal and likely due to the difference in their physiological status, rather than indicative of a treatment-related effect. This assumption might also be supported by the fact that no treatment-related changes in the clinical biochemistry parameters were observed in males at 100 mg/kg bw/day after a similar treatment period of 4 weeks. In lactating females at 300 mg/kg bw/day statistically significantly lower levels for total protein and albumin were observed on PND 14-16. The mean values for these parameters were at the lower limit of the normal range of this laboratory. No treatment-related changes were observed in the other clinical biochemistry parameters in females at 300 mg/kg bw/day and in all clinical biochemistry parameters in females at 100 mg/kg bw/day. Serum levels of T4 in F0 males were considered not to be affected by treatment.Serum levels of T4 in F0 females was not measured.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observation parameters were considered not to be affected by treatment in the males up to 1000 mg/kg bw/day and in the females up to 300 mg/kg bw/day. No functional tests were performed in females at 1000 mg/kg bw/day, because of their early sacrifice. A large variation in motor activity, i.e. mean total movements and ambulations, was observed between the males the four dose groups. In the absence of a clear dose response relationship and since all activity was within the normal range (Historical control data period 2015-2017: Total movements males: mean: 3341, P5-P95:1734-5284, n=200. Ambulations: mean: 883, P5-P95: 293-1143, n=200) the differences between groups were considered not indicative of a relation to treatment. In tested females, the motor activity was similar between treated and control groups. Males and females of all tested groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related microscopic observations in females up to 1000 mg/kg bw/day. Test item-related microscopic findings after treatment with Dehyton® DC were limited to the thyroid gland of the 300 and 1000 mg/kg bw/day group males: An increased incidence and severity in follicular cell hypertrophy in the thyroid gland of males was recorded at 300 mg/kg bw/day group (3/5 at minimal degree and 1/5 at slight degree compared to 1/5 at minimal degree in the controls) and at 1000 mg/kg bw/day group (2/5 at minimal degree and 2/5 males at slight degree, compared to 1/5 at minimal degree.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment. All females, except one 300 mg/kg bw/day dose group female, had regular cycles of 4 to 5 days. An irregular cycle was noted for one female at 300 mg/kg bw/day (with normal litter). Given the incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
The stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There was 1/10 couples of the controls, 2/10 couples treated at 100 mg/kg bw/day and 1/10 couples treated at 300 mg/kg bw/day that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain this finding.
In all females treated at 1000 mg/kg bw/day that were euthanized at Day 27-28, early placental/fetal development sites (development around day 9-12) were observed.
Mating index was considered not to be affected by treatment. Precoital time was considered not to be affected by treatment. All females showed evidence of mating within 5 days. Number of implantation sites was considered not to be affected by treatment.
Fertility index was considered not to be affected by treatment. A fertility index of 90%, 80%, 100% and 100% was observed for the controls and 100, 300 and 1000 mg/kg bw/day treated females respectively. It should be noted that a fertility index could be calculated for only 7/10 females at 1000 mg/kg bw/day. Three females of this dose group were dead before mating could have occurred.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Parental
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect seen up to 300 mg/kg bw/day
Remarks on result:
other: No high dose group (1000 mg/kg bw/day) could be assessed related to mortality seen at highest dose tested due to regurgitation of the formulations (secondary effect)
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects seen up to and including the highest dose tested (1000 mg/kg bw/day)

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Neuropathological findings:
not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment. Eight of 13 pups in one litter in the 300 mg/kg bw/day dose group had alopecia. As this finding was confined to a single litter it was considered not related to treatment. Alopecia is known to occur in pups and when it occurs within a litter is assumed to be genetic.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment. The number of dead pups at first litter check was 0, 0, and 9 from three litters for the control, 100, and 300 mg/kg bw/day dose groups, respectively, resulting in a live birth index of 100, 100 and 92%. The low value in the 300 mg/kg dose group was considered to have occurred by chance, because 7 out of 9 dead pups belonged to one litter. One additional pup in this litter was missing on Day 2 postpartum. This pup had been noted as having less milk and a scab on its snout. Two additional pups in this litter that survived to planned necropsy also had a scab on the snout. The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment. A viability index of 97%, 100% and 99% was observed for the controls and 100 and 300 mg/kg bw/day treated females, respectively. The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. A lactation index of 100%, 100% and 99% was observed for the controls and 100 and 300 mg/kg treated females, respectively.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment.
Sexual maturation:
no effects observed
Description (incidence and severity):
Sex ratio was considered not to be affected by treatment. Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment. Treatment up to 300 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
Development
Generation:
F1
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects seen at 300 mg/kg bw/day
Remarks on result:
other: No developmental data available for the 1000 mg/kg bw/day group, because of sacrifice of females due to secondary effect of test item before possible delivery.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Analysis of dose preparations: The concentrations analysed in the test item formulations were in agreement with target concentrations (i.e. mean accuracies between 97% and 103%). A small response at the retention time of the test item was observed in the chromatograms of the control group (vehicle) formulation was considered an analytical issue, rather than the presence of test item in the control formulation. In the worst case of a contribution of 4.8% to the low dose group formulation the accuracy of the formulations was still within acceptable limits. The formulations of the low and the high dose group were homogeneous (i.e. coefficient of variation 6.4%).

Summary results dose range finding study: No mortality was observed. At both dose levels, slight salivation was noted (occasional salivation immediately after dosing was seen in 1/3 females at 500 mg/kg bw/day, and in 3/3 at 1000 mg/kg bw/day). Piloerection was noted at several occasions after dosing in all three high dose females. Diarrhoea was observed in 1/3 female on Day 10 at 500 mg/kg bw/day, this was not observed in the higher dose group. Body weight development was normal in all dosed rats and no abnormalities were seen at necropsy. Liver and kidney weights were considered to be normal for all females.

Applicant's summary and conclusion

Conclusions:
An oral Repeated Dose Toxicity Study combined with a Reproduction/Developmental Toxicity Screening was performed according to OECD/EC guidelines and GLP principles. Based on the mortality observed at the high level dose due to regurgitation of the test item (secondary effect), the parental no-observed-adverse-effect level (NOAEL) of Dehyton® DC was established at 300 mg/kg bw/day. No reproduction toxicity was observed up to the highest dose level tested, therefore the reproduction NOAEL was found to be 1000 mg/kg bw/day.
Executive summary:

An oral Repeated Dose Toxicity Study combined with a Reproduction/Developmental Toxicity Screening was performed according to OECD/EC guidelines and GLP principles. Wistar Han rats were treated with Dehyton®DC by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, water, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 14-16 days of lactation (for 50-54 days). Females that failed to deliver pups were treated for 41 days, with the exception of females in the 1000 mg/kg bw/day dose group that were euthanized or found dead (one female) at the end of the premating period or during the post-coitum period due to adverse clinical observations. Accuracy and homogeneity of formulations determined by chemical analyses confirmed accurate dosing. At 1000 mg/kg bw/day, there was a high mortality in the females (4/10) and one premature death in the males. These deaths were concluded to be related to regurgitation and thus secondary to the test item (possibly triggered by physical/chemical properties of the test-item solution in combination with the route of administration). The surviving females in this group were early terminated shortly after the fourth female died, i.e. on Day 14 post-coitum. These early sacrificed Group 4 females, were all pregnant of a normal number of foetuses, and did not show any direct test item-related morphological changes. There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. No reproduction toxicity was observed up to the highest dose level tested, although only 6/10 females at 1000 mg/kg could be examined. Based on these results, the no-observed-adverse-effect level (NOAEL) was found to be 300 mg/kg bw/day and the reproduction NOAEL was found to be 1000 mg/kg bw/day.