Registration Dossier

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Diss Factsheets

Administrative data

Endpoint:
developmental toxicity
Remarks:
Combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start Date (First Day of Dose Administration) November 10, 2016 Experimental Termination Date (Last Animal Removed From Study) January 19, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Soybean oil, maleated, ester with triethanolamine
EC Number:
285-107-2
EC Name:
Soybean oil, maleated, ester with triethanolamine
Cas Number:
85029-82-9
Molecular formula:
C63H113NO12 to C67H123NO12
IUPAC Name:
Soybean oil, maleated, ester with triethanolamine
Test material form:
other: Clear amber viscous liquid
Details on test material:
100% product
Physical state/Appearance: Clear amber viscous liquid
Expiry Date: 24 June 2018
Storage Conditions: Room temperature in the dark

Test animals

Species:
rat
Strain:
other: CD® [Crl:CD®(SD)]
Details on test animals or test system and environmental conditions:
The rat is the usual rodent model used for evaluating the toxicity of various classes of chemicals and for which there is a large historical database.
Male and Femail animals used
A total of 69 male and 69 female CD® [Crl:CD®(SD)] rats (approximately 11 weeks of age at arrival) were received from Charles River Laboratories, Raleigh, North Carolina, on November 1, 2016 and October 20, 2016, respectively. During the 9- (males) or 21-day (females) acclimation period, the animals were observed twice daily with respect to general health and any signs of disease.

Randomization, Assignment to Study, and Maintenance
Using a standard, by weight, randomization procedure, 60 male and 60 female animals (weighing 354 to 394 g and 236 to 301 g, respectively, at randomization) were assigned to the control and treatment groups identified in following table.

Group Assignments
Group Number Dose Level (mg/kg/day) Number of Animals
Male Female
1 0 15a 15a
2 200 10 10
3 400 10 10
4 600 10 10
5 1000 15a 15a
a 5 animals were maintained for a 14-day recovery period.

Animals assigned to study had body weights that fell within +/-20% of the mean body weight for each sex. One extra female obtained, but not placed on study was found dead during the study. The remaining, surviving extra animals obtained, but not placed on study, were euthanized via carbon dioxide inhalation followed by an SOP approved method to ensure death. The carcasses were discarded.

Each animal was assigned an animal number to be used in the Provantis™ data collection system and was implanted with a microchip bearing a unique identification number. Pups were identified by tattoo on approximately LD 0. The individual animal number, implant number, and study number comprised a unique identification for each animal. Each cage was identified by the animal number, study number, group number, and sex.

On Day 1, one female at 400 mg/kg/day (animal number 316) was replaced due to a physical abnormality. This animal was replaced with an animal received but not previously assigned to study. The data recorded for the replaced animal are not reported in the data tables but are maintained in the study file.

The animals were individually housed in solid bottom cages with nonaromatic bedding in an environmentally controlled room, except during pairing, near parturition, and during lactation. During pairing, the rats were cohabited (one male and one female from the same treatment group) in the cage of the male. Animal enrichment was provided according to SOP. Fluorescent lighting was provided for approximately 12 hours per day. The dark cycle was interrupted intermittently due to study related activities. Temperature and humidity were continuously monitored, recorded, and maintained to the maximum extent possible within the ranges of 68 to 79°F and 30 to 70%, respectively. The actual temperature and humidity findings are not reported but are maintained in the study file.

Block Lab Diet (Certified Rodent Diet 5002, PMI Nutrition International, Inc.) was available ad libitum. The lot number from each diet lot used for this study was recorded. Certification analysis of each diet lot was performed by the manufacturer. Tap water was available ad libitum via an automatic watering system. The water supply is monitored for specified contaminants at periodic intervals according to SOP. The results of food and water analyses are retained in the Archives. The Study Director is not aware of any potential contaminants likely to be present in the diet or water that would have interfered with the results of the study. Therefore, no analyses other than those stated above were conducted.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Fresh vehicle, deionized water, was dispensed for use on study weekly and was stored refrigerated at 2 to 8°C.

The test article, was used as received from the Sponsor. No adjustment was made for purity when preparing the test article formulations. Formulations of the test article were prepared by mixing the appropriate amount of vehicle with the appropriate amount of test article at nominal concentrations of 40, 80, 120, and 200 mg/mL. Formulations were prepared weekly and were stored refrigerated at 2 to 8°C.
Analytical verification of doses or concentrations:
yes
Remarks:
HPLC
Details on analytical verification of doses or concentrations:
HPLC
All analytical work was conducted by MPI Research, using an analytical method developed and validated under MPI Research Study Number 1928-019. A deviation from the analytical method is presented in the attached Appendix B.
For full details please see the attached Appendix B Formulation Analysis Report
Details on mating procedure:
On Day 15, each female designated for terminal necropsy was housed in the cage of a male from the same treatment group. Positive evidence of copulation was established by daily inspection for a copulatory plug in the vagina and/or the presence of sperm in the vaginal lavage. The day on which positive evidence of copulation was observed was considered
GD 0. After evidence of mating was observed, the female was returned to an individual cage for the remainder of the study. The maximum pairing period was 14 days, at the end of which any females with no confirmed evidence of mating were returned to individual cages until scheduled euthanasia.

Animals designated for the recovery period were not cohabitated.
Duration of treatment / exposure:
The vehicle, deionized water, or test article, was administered to all CD® [Crl:CD®(SD)] rats once daily via oral gavage. Dosing of the males began 2 weeks prior to pairing, continued through mating, and for an additional 28 days following completion of the mating period. Dosing of the females began 2 weeks prior to pairing and continued through mating to Lactation Day
Frequency of treatment:
The vehicle, deionized water, or test article, was administered to all CD® [Crl:CD®(SD)] rats once daily via oral gavage. Dosing of the males began 2 weeks prior to pairing, continued through mating, and for an additional 28 days following completion of the mating period. Dosing of the females began 2 weeks prior to pairing and continued through mating to Lactation Day
Duration of test:
Dosing of the males began 2 weeks prior to pairing, continued through mating, and for an additional 28 days following completion of the mating period. Dosing of the females began 2 weeks prior to pairing and continued through mating to Lactation Day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
200 mg/kg bw/day
Dose / conc.:
400 mg/kg bw/day
Dose / conc.:
600 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
The low- and mid-dose groups each consisted of 10 rats/sex and the high-dose group consisted of 15 rats/sex. Concurrent control group of 15 rats/sex
Control animals:
yes, concurrent vehicle
Details on study design:
This study was based on the Current International Council on Harmonisation (ICH) Harmonised Tripartite Guidelines and generally accepted procedures for the testing of pharmaceutical compounds1 and the Draft Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, the Organization of Economic Cooperation and Development (OECD) Guideline for Testing of Chemicals, revised January 1994, and in accordance with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, National Academy Press, Washington, D.C., 2011. The protocol and amendments are presented following the report text.

Examinations

Maternal examinations:
Cage side Observations
All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily.
Detailed Clinical Observations
A detailed clinical examination of each animals was performed, at receipt, prior to randomization, and daily during the treatment period (75 minutes ±15 minutes postdose) and weekly during the recovery period. The observations recorded at receipt and prior to randomization are not reported but are maintained in the study file. On occasion, clinical observations were recorded at unscheduled intervals. The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration.


Veterinary Observations
On occasion, veterinary observations were conducted during the course of the study. All treatments and observations were recorded. The medical treatments and observations are not reported but are maintained in the study file.

Functional Observational Battery Observations
FOB evaluations were conducted on 5 male animals/group at termination and following 14 days of recovery, 5 lactating females/group on LD 10, and 5 recovery females/group following the completion of dosing and at the end of the recovery period. FOB evaluations included those conducted in the home-cage, during handling, in the open-field, and others.
During open-field evaluations, each animal was placed in a black plexiglass box and observed for a minimum of 3 minutes. The parameters evaluated in the FOB were based on those outlined in Moser, et al.7, 8. The observations included, but were not limited to, evaluation of activity and arousal, posture, rearing, bizarre behavior, clonic and tonic movements, gait, mobility, stereotypy, righting reflex, response to stimulus (approach, click, tail pinch, and touch), palpebral closure, pupil response, piloerection, exophthalmus, lacrimation, salivation, and respiration. Qualitative and/or quantitative measures of defecation and urination were also recorded. Forelimb and hind limb grip strength was measured using the procedure described by Meyer, et al. 9, and hind limb splay was quantitatively measured as described by Edwards and Parker10. Pain perception was assessed by measuring the latency of response to a nociceptive (thermal) stimulus when each animal was placed on a hot plate apparatus set to 52C (±1°C) as described by Ankier11. Body weight and temperature were also measured. A glossary of FOB terms used in this study is included in this report.

Motor Activity
Locomotor activity evaluations were conducted on the same animals used for FOB evaluations (5 male animals/group at termination and following 14 days of recovery,
5 lactating females/group on LD 10, and 5 recovery females/group following the completion of dosing and at the end of the recovery period). Each animal was placed into the assigned Hamilton-Kinder enclosure for monitoring. The duration of monitoring was 60 minutes with the data summarized into 5 minute segments. A range of different activities were assessed in a three dimensional array and were recorded. Only basic movement, fine movement, rearing, and distance (cm) were used in comparisons between treated and control animals as the most representative activity parameters.

Body Weights and Body Weight Changes
Body weights for all animals were measured and recorded at receipt, prior to randomization, and at scheduled necropsy. Body weights of males were recorded weekly during treatment, and on the days that FOB and locomotor activity evaluations were performed. Body weights of females were recorded weekly beginning on Week -2 when the pretest estrous evaluations began. Mated females with a confirmed GD 0 were weighed on GD 0, 3, 6, 10, 14, 17,
and 20; PND 0, 4, 7, 10, and 13; and on the days that FOB and locomotor activity evaluations were performed. After cohabitation, females with no evidence of mating were weighed weekly until euthanasia. All recovery animals were weighed weekly during the treatment and



recovery periods, and on the days that FOB and locomotor activity evaluations were performed. Any animals that were fasted had body weights recorded prior to and after the fasting period.

Body weight changes were calculated for the males and females between each weighing interval and over the entire premating and recovery periods and for the females on GD 0-3, 3-6, 6-10, 10-14, 14-17, 17-20, 0-20 and PND 0-4, 4-7, 7-10, 10-13, and 0-13.

The body weights recorded at receipt and prior to randomization are not reported but are maintained in the study file.

Food Consumption
Food consumption was recorded weekly prior to pairing for mating. During the 14 days of the pairing period, food consumption was not recorded for any animals. Following the pairing period, the males were placed back on measured food consumption weekly until euthanasia. Food consumption was recorded on the corresponding gestation and lactation body weight days for mated females. Food consumption was recorded weekly during the treatment and recovery periods for the recovery animals.


Breeding Procedures
On Day 15, each female designated for terminal necropsy was housed in the cage of a male from the same treatment group. Positive evidence of copulation was established by daily inspection for a copulatory plug in the vagina and/or the presence of sperm in the vaginal lavage. The day on which positive evidence of copulation was observed was considered
GD 0. After evidence of mating was observed, the female was returned to an individual cage for the remainder of the study. The maximum pairing period was 14 days, at the end of which any females with no confirmed evidence of mating were returned to individual cages until scheduled euthanasia.

Animals designated for the recovery period were not cohabitated.

Disposition of Females with No Evidence of Mating
All females with no confirmed mating date that appeared to be nonpregnant on the basis of body weight and shape were euthanized 25 days after the last scheduled pairing day and examined as described in Section. 8.4, Postmortem Study Evaluations. Females that appeared to be pregnant on the basis of body weight and shape were allowed to deliver.



P Females that Failed to Deliver
On GD 25, a necropsy of all P females that failed to deliver was performed. Uteri from females that appeared nongravid were opened and placed in 10% ammonium sulfide solution for detection of implantations12. If no foci were detected, the female was considered to be nonpregnant.

P Parturition and F1 Litter Observations
Toward the end of the gestation period, P females were examined twice daily for signs of parturition. The mated females were allowed to give birth (F1). The duration of gestation was calculated, and any difficulties occurring at parturition were recorded. The day on which all pups were delivered was designated as LD 0. The litters were examined as soon as possible after delivery. Litter size, number of stillborn and liveborn pups, number of males and females, individual body weights, and gross abnormalities of the pups were recorded for each litter.

On LD 0, all live pups from each litter were tattooed to maintain individual identity, and eight pups of equal sex distribution (four males and four females), when possible, were randomly selected. After weighing on LD 4, each litter was reduced to the eight randomly selected pups from LD 0. The culled pups were euthanized by intraperitoneal injection of sodium pentobarbital solution and examined for external abnormalities. Pups with abnormalities were saved in 10% neutral buffered formalin for possible future examination, and the remaining carcasses were discarded. Additionally, blood was collected for hormone analysis from two culled pups per litter (pooled) on LD 4.

Litters were housed with the dams for 13 days after birth (LD 0 to 13). The dams and litters were observed daily for survival and behavioral alterations in nesting and nursing, and the presence of dead pups was recorded. Any intact, dead pups were necropsied, examined for anomalies, and pups with abnormalities were saved in 10% neutral buffered formalin for possible future examination. In addition, the hearts of these pups were dissected using a modified method described by Stuckhardt and Poppe13. Pups found dead on LD 0 had their lungs removed and placed in tap water to determine if they floated (i.e., liveborn) or sank (i.e., stillborn). In the event stillborn status could not be determined, the pup was considered liveborn for reporting purposes. Cannibalized pups were discarded without necropsy.

Pups were individually weighed and examined externally on LD 0, 4, 7, and 13. The dam and litter remained together until LD 13. Anogenital distance was recorded for all pups on LD 4 (post-cull) and the number of nipples was counted for male pups on LD 12.



Clinical Pathology and Hormone Analysis
Clinical pathology evaluations were conducted on 5 parental animals/sex/group at terminal necropsy (males), on LD 13 (females), and at recovery necropsy (males and females).
Additionally, blood was collected from 2 pups per litter on LD 4, 5 parental females/group with their litter at termination on LD 13, 5 parental males/group at terminal necropsy, and
5 animals/sex/group at recovery necropsy for TSH and T4 hormone analysis. When possible, blood samples for hormone analysis were collected from animals not utilized for clinical pathology evaluations. The methods and materials are described in the attached Appendix Q.


Ovaries and uterine content:
Estrous Cycle Determination
Beginning two weeks prior to dosing and continuing until evidence of copulation was observed or the cohabitation period ended, females were examined daily by vaginal lavage to establish estrous cyclicity. Estrous evaluations were also performed on LD 13 prior to terminal necropsy. Vaginal lavage of the females was completed daily by 10:00 A.M.
Fetal examinations:
Pups were individually weighed and examined externally on LD 0, 4, 7, and 13. The dam and litter remained together until LD 13. Anogenital distance was recorded for all pups on LD 4 (post-cull) and the number of nipples was counted for male pups on LD 12.
Statistics:
Table D defines the set of comparisons used in the statistical analyses described in this section.

Table D. Statistical Comparisons
Control Group Treatment Groups
1 2, 3, 4, 5

The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint by sex and group. For each endpoint, treatment groups were compared to the control group using the analysis outlined in Table E. Data for some endpoints, as indicated, were transformed by log or arcsin-square root transformation prior to conducting the specified analysis.
Indices:
Please see any other information section

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related clinical findings were noted in the treated males and females during the study (pre-mating, pairing, post mating, gestation, lactation, and recovery).

The few findings observed among the treated males and females occurred at low incidence or with similar frequency in controls and were considered unrelated to treatment with the test item.
Mortality:
no mortality observed
Description (incidence):
All animals survived to scheduled necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related effects on mean body weights or body weight change were observed in the males or females during the treatment period (pre-mating, pairing, post-mating, gestation and lactation period) or during the recovery period. There were two intervals (Days 43-50 [treated recovery males] and Days 8-15 [treated recovery females]) at 1000 mg/kg/day in which body weight change was statistically higher than controls. These differences were considered incidental and unrelated to treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test article-related effects on mean food consumption were observed in the males or females during the treatment period (pre-mating, post-mating, gestation and lactation period) or during the recovery period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related effects among hematology parameters in either sex at any dose level. All mean and individual hematology values were considered within expected ranges for biological and/or procedure-related variation despite occasional mean values that reached statistical significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related effects among clinical chemistry parameters in either sex at any dose level. All mean and individual values were considered within expected ranges for biological and/or procedure-related variation.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No test article-related FOB findings were observed during the treatment or recovery period in the treated males and females.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related organ weight changes present at terminal or recovery necropsy in either sex.

All organ weight fluctuations (compared to controls) were considered incidental regardless of statistical significance, because of their small magnitude, lack of dose response in either sex, differences in sexual maturity, and/or individual animal variability, and were considered not to be test article related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related macroscopic pathologic findings in terminal or recovery animals in either sex.

One female at 200 mg/kg/day (animal number 215) had a subcutaneous mass that was microscopically correlated to mammary adenocarcinoma. Spontaneous mammary adenocarcinoma, although rare, does occur in animals less than 1 year of age, and was reported to occur in the test species with low incidence as early as 12 weeks of age. This finding was considered to be unrelated to the test article due to the low occurrence and absence of similar lesions in test article-treated animals at the higher dose.

All macroscopic findings were considered incidental because of their small magnitude, lack of microscopic correlates or lack of dose dependence, and/or recognition as a background finding in the test species.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test article-related microscopic findings present at the terminal necropsy in either sex.

All reproductive organs in test article-treated males and females were within normal limits. The mild hemorrhage in the uterus of one female at 600 mg/kg/day (animal number 420) is considered to be an incidental finding, possibly related to recent parturition, and unrelated to the test article.

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
All other microscopic findings were considered spontaneous/incidental due to the lack of dose dependence and recognition of similar findings as a background finding in the test species.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
In-life Examinations
Mortality
A record of animal fate and disposition is presented in the attached Appendix C. All animals survived to scheduled necropsy.
Detailed Clinical Observations
Detailed clinical observations are summarized in the attached Tables 1 (males), 2 (females – premating/mating), 3 (gestation), 4 (lactation), and 5 (females – recovery). Individual detailed clinical observations are presented in Appendix D.

No test article-related clinical findings were noted in the treated males and females during the study (pre-mating, pairing, post mating, gestation, lactation, and recovery).

The few findings observed among the treated males and females occurred at low incidence or with similar frequency in controls and were considered unrelated to treatment with the test item.

Functional Observational Battery Evaluations
FOB data are summarized in the attached Table 6. The codes for individual FOB values and individual FOB data are presented in the attached Appendix E.

No test article-related FOB findings were observed during the treatment or recovery period in the treated males and females.

Activity/Arousal Measurements
Activity and arousal values (continuous and categorical) in the treated groups (males and females) were comparable to control values and unaffected by treatment with the test item . A few values were statistically significant in both males and females either during the treatment period or recovery period when compared to controls, but were either not
dose-responsive or slight in magnitude of change and considered incidental and unrelated to treatment.

Neuromuscular Measurements
Neuromuscular values (continuous and categorical) in the treated groups (males and females) were comparable to control values and unaffected by treatment with the test item. A few values were statistically significant in males either during the treatment period or recovery period when compared to controls, but were either not dose-responsive or slight in magnitude of change and considered incidental and unrelated to treatment.



Sensorimotor Measurements
Sensorimotor values (continuous and categorical) in the treated groups (males and females) were comparable to control values and unaffected by treatment with the test item. A few values were statistically significant in females either during the treatment period or recovery period when compared to controls, but were either not dose-responsive or slight in magnitude of change and considered incidental and unrelated to treatment.

Autonomic Measurements
Autonomic values (continuous and categorical) in the treated groups (males and females) were comparable to control values and unaffected by treatment with the test item.

Physiological Measurements
Physiological values (continuous and categorical) in the treated groups (males and females) were comparable to control values and unaffected by treatment with the test item.

Locomotor Activity
Locomotor activity data are summarized in the attached Table 7. The individual locomotor activity data are presented in the attached Appendix F.

Locomotor activity (basic movement, fine movement, rearing, and total distance travelled) in the treated groups (males and females) were comparable to control values and unaffected by treatment with the test item. A few values were statistically significant in both males and females either during the treatment period or recovery period when compared to controls, but were either not dose-responsive or slight in magnitude of change and considered incidental and unrelated to treatment.

Body Weights and Body Weight Changes
Body weight data are summarized in the attached Tables 8 (males), 9 (females – premating), 10 (gestation), 11 (lactation), and 12 (females – recovery), and body weight change data are summarized in the attached Tables 13 (males), 14 (females – premating), 15 (gestation), 16 (lactation), and 17 (females – recovery). Individual body weight and body weight change data are presented in the attached Appendices G and H, respectively.

No test article-related effects on mean body weights or body weight change were observed in the males or females during the treatment period (pre-mating, pairing, post-mating, gestation and lactation period) or during the recovery period. There were two intervals (Days 43-50 [treated recovery males] and Days 8-15 [treated recovery females]) at 1000 mg/kg/day in which body weight change was statistically higher than controls. These differences were considered incidental and unrelated to treatment.

Food Consumption
Food consumption data are summarized in the attached Tables 18 (males), 19 (females – premating), 20 (gestation), 21 (lactation), and 22 (females – recovery). Individual food consumption data are presented in the attached Appendix I.


No test article-related effects on mean food consumption were observed in the males or females during the treatment period (pre-mating, post-mating, gestation and lactation period) or during the recovery period.

Reproductive Performance
Estrous Cyclicity
Estrous cyclicity data are summarized in the attached Table 23 and presented individually in the attached Appendix J.

No effect of the the test itemwas observed on estrous cycle parameters in the treated females. Mean cycle length and mean number of cycles over the 2-week premating evaluation period in the treated groups were comparable to controls. Mean cycle length in the treated groups ranged from 4.1 to 4.6 days and was comparable to controls at 4.5 days. Likewise, mean number of cycles ranged from 2.6 to 2.7 cycles in comparison to 2.5 cycles in controls.

Reproductive and Fertility Indices
Reproductive and fertility indices are summarized in the attached Table 24. Individual mating records are presented in the attached Appendix K.

No effect on reproductive performance of the males or females was observed in the TEST ITEM-treated groups. The mating index in the treated groups ranged from 90% to 100% and was comparable to the control group (100%). Fertility (ratio of pregnant to number paired) and fecundity indices (ratio of pregnant to number mated) ranged from 80% to 100% in the treated groups and were comparable to the control group (90%).

The mean Copulatory Interval (i.e., mean number-of-days to mating) in the test item-treated groups ranged from 2.5 to 4.6 days and was comparable to the 1.9 days in the controls.

Clinical Pathology
The Clinical Pathology Report is presented in the attached Appendix Q.

Summary
There were no test article-related effects among clinical pathology parameters in rats of either sex following repeated daily oral administration of the the test itemat doses of 200, 400, 600, or 1000 mg/kg/day. All mean and individual values were considered within expected ranges for biological and/or procedure-related variation despite occasional mean values that reached statistical significance.

Hormone Analysis
There were no test article-related effects on T4 or TSH concentrations in either sex at any dose level. All mean and individual values were considered within expected ranges for biological and/or procedure-related variation.



Post-mortem Study Evaluations
The Pathology Report is presented in the attached Appendix R.

No effects on treated males and females were observed on, estrous cycle parameters, reproductive performance

No effects on treated males and females were observed on reproductive performance

Maternal developmental toxicity

Number of abortions:
not examined
Description (incidence and severity):
No effect of the test item at the dose levels evaluated was observed on parturition data in the treated females. Pregnancy rates were 90%, 80%, 90%, 100% and 90% in the control, 200,
400, 600, and 1000 mg/kg/day groups, respectively. There were 9, 8, 9, 10, and 9 litters delivered with viable pups in the control, 200, 400, 600, and 1000 mg/kg/day dose groups, respectively. Parturition parameters (gestation length, number of pups at birth, number of stillborn pups at birth, gestation index, and stillborn index) in the test item-treated groups were comparable to controls. There was a statistically increased number of stillborn pups at 400 mg/kg/day in comparison to controls (0.3 vs. 0.0). However without a dose response this increase in number of stillborn pups at 400 mg/kg/day was considered incidental and unrelated to treatment.
Pre- and post-implantation loss:
not specified
Description (incidence and severity):
No effect of the test item at the dose levels evaluated was observed on parturition data in the treated females. Pregnancy rates were 90%, 80%, 90%, 100% and 90% in the control, 200,
400, 600, and 1000 mg/kg/day groups, respectively. There were 9, 8, 9, 10, and 9 litters delivered with viable pups in the control, 200, 400, 600, and 1000 mg/kg/day dose groups, respectively. Parturition parameters (gestation length, number of pups at birth, number of stillborn pups at birth, gestation index, and stillborn index) in the test item-treated groups were comparable to controls. There was a statistically increased number of stillborn pups at 400 mg/kg/day in comparison to controls (0.3 vs. 0.0). However without a dose response this increase in number of stillborn pups at 400 mg/kg/day was considered incidental and unrelated to treatment.
Total litter losses by resorption:
not specified
Description (incidence and severity):
Litter size in the treated groups on LD 4 pre- and post-cull, and on LD 7 and 13 was comparable to controls and unaffected by treatment with the test item.

F1 Pup Survival
Pup survival over LD 0 to 4 pre-cull (Viability Index) and LD 4 post-cull to 13 are summarized in the attached Table 25, and individual litter survival data are presented in the attached Appendix L.

No effect of the test item on F1 pup survival over Postnatal Day (PND) 0 to 4 or PND 4 to 13 was observed in the treated groups in comparison to controls. Mean pup survival
PND 0 to 4 (pre-cull) ranged from 98.07% to 99.26% in the treated groups and was comparable to controls at 99.35%. Mean pup survival was 100% in the treated groups over PND 4 (post-cull) to PND 13 and was comparable to controls at 100%.
Early or late resorptions:
not specified
Dead fetuses:
effects observed, non-treatment-related
Description (incidence and severity):
There was a statistically increased number of stillborn pups at 400 mg/kg/day in comparison to controls (0.3 vs. 0.0). However without a dose response this increase in number of stillborn pups at 400 mg/kg/day was considered incidental and unrelated to treatment.
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Parturition parameters (gestation length, number of pups at birth, number of stillborn pups at birth, gestation index, and stillborn index) in the test item-treated groups were comparable to controls.
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Parturition parameters (gestation length, number of pups at birth, number of stillborn pups at birth, gestation index, and stillborn index) in the test item-treated groups were comparable to controls.
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
No effect of the test item at the dose levels evaluated was observed on parturition data in the treated females. Pregnancy rates were 90%, 80%, 90%, 100% and 90% in the control, 200, 400, 600, and 1000 mg/kg/day groups, respectively.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
F1 Pup Detailed Clinical Observations
Pup detailed clinical observations are summarized in the attached Table 26, and individual pup detailed clinical observations are presented in the attached Appendix M.

No effect of the test item in the treated groups was observed from the F1 pup detailed clinical examinations (PND 0-13). The few findings observed among the F1 pups in the treated groups occurred at low incidence and were considered unrelated to the test article.
Pup survival over LD 0 to 4 pre-cull (Viability Index) and LD 4 post-cull to 13 are summarized in the attached Table 25, and individual litter survival data are presented in the attached Appendix L.

No effect of the test item on F1 pup survival over Postnatal Day (PND) 0 to 4 or PND 4 to 13 was observed in the treated groups in comparison to controls. Mean pup survival
PND 0 to 4 (pre-cull) ranged from 98.07% to 99.26% in the treated groups and was comparable to controls at 99.35%. Mean pup survival was 100% in the treated groups over PND 4 (post-cull) to PND 13 and was comparable to controls at 100%.
Mean pup body weights are summarized in the attached Table 27 and presented individually in Appendix N.
F1 pup mean body weights on PND 0, 4 (pre- and post-cull), 7, and 13 in the treated groups were comparable to controls and unaffected by treatment with the test item.
Pup macroscopic observations are summarized in the attached Table 29 and individual findings are presented in the attached Appendix P.

No effect of the test item was observed from the macroscopic examination of stillborn pups, PND 4 culled pups (examined externally), pups found dead during lactation and PND 13 pups. The few findings observed among pups in the test item-treated groups occurred at low incidence and were considered unrelated to the test article.
There were no test article-related effects among clinical pathology parameters in rats of either sex following repeated daily oral administration of the test item at doses of 200, 400, 600, or 1000 mg/kg/day. All mean and individual values were considered within expected ranges for biological and/or procedure-related variation despite occasional mean values that reached statistical significance.
P parturition and F1 litter data are summarized in the attached Table 25 and individual parturition and litter data are presented in the attached Appendix L.

No effect of the test item at the dose levels evaluated was observed on parturition data in the treated females. Pregnancy rates were 90%, 80%, 90%, 100% and 90% in the control, 200, 400, 600, and 1000 mg/kg/day groups, respectively. There were 9, 8, 9, 10, and 9 litters delivered with viable pups in the control, 200, 400, 600, and 1000 mg/kg/day dose groups, respectively. Parturition parameters (gestation length, number of pups at birth, number of stillborn pups at birth, gestation index, and stillborn index) in the test item -treated groups were comparable to controls. There was a statistically increased number of stillborn pups at 400 mg/kg/day in comparison to controls (0.3 vs. 0.0). However without a dose response this increase in number of stillborn pups at 400 mg/kg/day was considered incidental and unrelated to treatment.

Litter size in the treated groups on LD 4 pre- and post-cull, and on LD 7 and 13 was comparable to controls and unaffected by treatment with the test item.

Pup sex ratios (% male pups/litter) are summarized in the attached Table 25. The number of pups per sex in individual litters throughout lactation is presented in the attached Appendix L, and the sex of individual pups within each litter is presented in the attached Appendix N.

No effect of the test item the dose levels evaluated was observed on F1 pup sex ratios. Mean pup sex ratios observed in the treated groups at birth, PND 4 (pre-and post-culling) and PND 13 were comparable to controls.

Mean pup anogenital distance is summarized in the attached Table 28. Mean pup anogenital distance and nipple counts are presented individually in the attached Appendix O.


F1 pup mean anogenital distance in the treated groups ranged from 4.3 to 4.9 mm (males) and
2.3 to 2.9 mm (females) and was comparable to controls (4.8 mm [males] and 2.7 mm [females]) and was unaffected by treatment with the test item.

No F1 male pups were found to have nipples in the tee-treated groups.

Clinical Pathology
The Clinical Pathology Report is presented in the attached Appendix Q.

Summary
There were no test article-related effects among clinical pathology parameters in rats of either sex following repeated daily oral administration of the test item at doses of 200, 400, 600, or 1000 mg/kg/day. All mean and individual values were considered within expected ranges for biological and/or procedure-related variation despite occasional mean values that reached statistical significance.
Hormone Analysis
There were no test article-related effects on T4 or TSH concentrations in either sex at any dose level. All mean and individual values were considered within expected ranges for biological and/or procedure-related variation.

Postmortem Study Evaluations
The Pathology Report is presented in the attached Appendix R.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEC
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: No tet item effects seen on treated males and females
Remarks on result:
other: No effects on treated males and females were observed
Remarks:
No effects on treated males and females were observed on survival, clinical observations, body weights and body weight change, food consumption, estrous cycle parameters, reproductive performance (mating, fertility, and fecundity), behavioral evaluations (functional observational battery and locomotor activity), clinical pathology parameters, organ weights, macroscopic observations, and microscopic observations. No test item-related effects were observed on F1 survival, clinical observations, body weights, and macroscopic findings.

Maternal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
not specified

Results (fetuses)

Fetal body weight changes:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Mean pup body weights are summarized in the attached Table 27 and presented individually in Appendix N.
F1 pup mean body weights on PND 0, 4 (pre- and post-cull), 7, and 13 in the treated groups were comparable to controls and unaffected by treatment with the test item.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
F1 Pup Survival
Pup survival over LD 0 to 4 pre-cull (Viability Index) and LD 4 post-cull to 13 are summarized in the attached Table 25, and individual litter survival data are presented in the attached Appendix L.

No effect of the test item on F1 pup survival over Postnatal Day (PND) 0 to 4 or PND 4 to 13 was observed in the treated groups in comparison to controls. Mean pup survival
PND 0 to 4 (pre-cull) ranged from 98.07% to 99.26% in the treated groups and was comparable to controls at 99.35%. Mean pup survival was 100% in the treated groups over PND 4 (post-cull) to PND 13 and was comparable to controls at 100%.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Pup sex ratios (% male pups/litter) are summarized in the attached Table 25. The number of pups per sex in individual litters throughout lactation is presented in the attached Appendix L, and the sex of individual pups within each litter is presented in the attached Appendix N.
No effect of the test item at the dose levels evaluated was observed on F1 pup sex ratios. Mean pup sex ratios observed in the treated groups at birth, PND 4 (pre-and post-culling) and PND 13 were comparable to controls.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
No effect of the test item at the dose levels evaluated was observed on parturition data in the treated females. Pregnancy rates were 90%, 80%, 90%, 100% and 90% in the control, 200, 400, 600, and 1000 mg/kg/day groups, respectively. There were 9, 8, 9, 10, and 9 litters delivered with viable pups in the control, 200, 400, 600, and 1000 mg/kg/day dose groups, respectively. Parturition parameters (gestation length, number of pups at birth, number of stillborn pups at birth, gestation index, and stillborn index) in the test item-treated groups were comparable to controls. There was a statistically increased number of stillborn pups at 400 mg/kg/day in comparison to controls (0.3 vs. 0.0). However without a dose response this increase in number of stillborn pups at 400 mg/kg/day was considered incidental and unrelated to treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
F1 Pup Survival
Pup survival over LD 0 to 4 pre-cull (Viability Index) and LD 4 post-cull to 13 are summarized in the attached Table 25, and individual litter survival data are presented in Appendix L.

No effect of the test item on F1 pup survival over Postnatal Day (PND) 0 to 4 or PND 4 to 13 was observed in the treated groups in comparison to controls. Mean pup survival
PND 0 to 4 (pre-cull) ranged from 98.07% to 99.26% in the treated groups and was comparable to controls at 99.35%. Mean pup survival was 100% in the treated groups over PND 4 (post-cull) to PND 13 and was comparable to controls at 100%.
External malformations:
not specified
Description (incidence and severity):
F1 Pup Detailed Clinical Observations
Pup detailed clinical observations are summarized in the attached Table 26, and individual pup detailed clinical observations are presented in the attached Appendix M.

No effect of the test item in the treated groups was observed from the F1 pup detailed clinical examinations (PND 0-13). The few findings observed among the F1 pups in the treated groups occurred at low incidence and were considered unrelated to the test article.
Skeletal malformations:
not specified
Description (incidence and severity):
F1 Pup Macroscopic Examinations
Pup macroscopic observations are summarized in Table 29 and individual findings are presented in the attached Appendix P.

No effect of the test item was observed from the macroscopic examination of stillborn pups, PND 4 culled pups (examined externally), pups found dead during lactation and PND 13 pups. The few findings observed among pups in the test item-treated groups occurred at low incidence and were considered unrelated to the test article.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
F1 Pup Macroscopic Examinations
Pup macroscopic observations are summarized in Table 29 and individual findings are presented in the attached Appendix P.

No effect of the test item was observed from the macroscopic examination of stillborn pups, PND 4 culled pups (examined externally), pups found dead during lactation and PND 13 pups. The few findings observed among pups in the test item-treated groups occurred at low incidence and were considered unrelated to the test article.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEC
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No tet item effects seen on treated males and females
Remarks on result:
other: No tet item effects seen on treated males and females
Remarks:
No effects on treated males and females were observed on survival, clinical observations, body weights and body weight change, food consumption, estrous cycle parameters, reproductive performance (mating, fertility, and fecundity), behavioral evaluations (functional observational battery and locomotor activity), clinical pathology parameters, organ weights, macroscopic observations, and microscopic observations. No test item-related effects were observed on F1 survival, clinical observations, body weights, and macroscopic findings.

Fetal abnormalities

Key result
Abnormalities:
no effects observed
Localisation:
other: No effect of the test item was observed from the macroscopic examination of stillborn pups, PND 4 culled pups (examined externally), pups found dead during lactation and PND 13 pups.
Description (incidence and severity):
No effect of the test item was observed from the macroscopic examination of stillborn pups, PND 4 culled pups (examined externally), pups found dead during lactation and PND 13 pups. The few findings observed among pups in the test item-treated groups occurred at low incidence and were considered unrelated to the test article.

Overall developmental toxicity

Key result
Developmental effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day
Treatment related:
no

Any other information on results incl. tables

Analysis of Dosing Formulations

The Formulation Analysis Report is presented in the attached Appendix B.

 

Homogeneity

Homogeneity analysis was conducted from the Week 1 preparation at 40 and 200 mg/mL and met acceptance criteria. Percent relative standard deviations were 0.998 and 1.121 at 40 and 200 mg/mL, respectively, and were within the acceptance criteria of ≤ 10%, confirming that dosing formulations were homogenous.

 

 

Table F.   Homogeneity

 

 

Dose Level (mg/kg/day)

 

Nominal Concentration (mg/mL)

Average Calculated Concentration (mg/mL)

 

 

Average

% Recoverya

 

% Relative Standard Deviation

200

1000

40.0

200

42.0492

213.0425

105.1

106.5

0.998

1.121

aAverage % recovery was calculated from the nominal concentration.

Concentration

Concentration analysis was conducted from the Week 1 and Week 8/9 preparations at 0, 40, 80, 120 and 200 mg/mL. No test item was observed in the samples from the control group (0 mg/mL). The average percent recovery was within the acceptance criteria (≤ 15%) and ranged from 101.8% to 109.2% in the test item -treated groups and confirmed that animals received the appropriate doses.

 

Table G. Concentration

 

Dose Level (mg/kg/day)

Nominal Concentration (mg/mL)

Average Calculated Concentrationa(mg/mL)

 

Average

% Recoverya, b

% Relative Standard Deviationa

0

200

400

600

1000

0.00

40.0

80.0

120

200

BLQ

40.7125 – 42.0492

84.1770 – 84.4547

123.3292 – 130.4690

213.0425 – 218.4317

NA

101.8 – 105.1

105.2 – 105.6

102.8 – 108.7

106.5 – 109.2

NA

0.998 – 4.684

0.375 – 0.747

0.150 – 0.647

1.121 – 1.182

aResults are the range of values determined during Weeks 1 and 8/9.

bAverage %recovery was calculated from the nominal concentration.

NA – Not Applicable

 

Applicant's summary and conclusion

Conclusions:
This study was conducted to evaluate for possible adverse effects of the test article following repeated dosing to male and female rats, including systemic toxicity, neurotoxic potential, reproductive performance (gonadal function, mating behavior, conception, development of the conceptus, and parturition), and F1 offspring growth and maturation up through LD 13.

No effects on treated males and females were observed on survival, clinical observations, body weights and body weight change, food consumption, estrous cycle parameters, reproductive performance (mating, fertility, and fecundity), behavioral evaluations (functional observational battery and locomotor activity), clinical pathology parameters, organ weights, macroscopic observations, and microscopic observations. No test item-related effects were observed on F1 survival, clinical observations, body weights, and macroscopic findings.

Based on these findings, 1000 mg/kg/day, the highest dose level evaluated, was considered the no-observed-adverse-effect level (NOAEL) for the test item in male and female rats and for F1 offspring growth (PND 0 to 13).
Executive summary:

This study was conducted for the study sponsor to evaluate the possible adverse effects of the test article following repeated exposure. This evaluation included systemic toxicity, male and female reproductive performance (gonadal function, mating behavior, conception, development of the conceptus, and parturition) and neurotoxic potential. The vehicle, deionized water, or test article, was administered to all CD® [Crl:CD®(SD)] rats once daily via oral gavage. Dosing of the males began 2 weeks prior to pairing, continued through mating, and for an additional 28 days following completion of the mating period. Dosing of the females began 2 weeks prior to pairing and continued through mating to Lactation Day (LD) 13, Gestation Day (GD) 25, or 13 days after cohabitation. Animals were assigned to the study as indicated in the following table.

 

Study Design

Table A.

Study Design

 

Group Number

 

Dose Level (mg/kg/day)

Dose Volume (mL/kg)

Dose Concentration (mg/mL)

Number of Animals

 

Male

 

Female

1

0

5

0

15a

15a

2

200

5

40

10

10

3

400

5

80

10

10

4

600

5

120

10

10

5

1000

5

200

15a

15a

a5 animals were maintained for a 14-day recovery period.

Assessment of toxicity was based on clinical observations, body weight, and food consumption; functional observational battery (FOB) evaluations and motor activity; clinical pathology including hormone analysis for thyroxine (T4) and thyroid-stimulating hormone (TSH) and anatomic pathology including an evaluation of spermatogenesis. Observations of the parental (P) females also included parturition and litter data and success in rearing offspring to LD 13. Observations of the offspring (F1) included survival, sex, body weights, gross abnormalities, anogenital distance, and nipple count (male pups only). Reproductive parameters of the parental animals (mating, fertility, and fecundity) and estrous cycle determination were also evaluated.

 

Homogeneity and concentration analyses confirmed that dose formulations were homogeneous (RSD 0.998% and 1.121%) and that animals received the targeted concentrations (101.8% to 109.2%).

 

No effects on treated males and females were observed on survival, clinical observations, body weights and body weight change, food consumption, estrous cycle parameters, reproductive performance (mating, fertility, and fecundity), behavioral evaluations (functional observational battery and locomotor activity), clinical pathology parameters, organ weights, macroscopic observations, and microscopic observations. No test item -related effects were observed on F1 survival, clinical observations, body weights, and macroscopic findings.