bis(branched-alkyl) 2-{[(1-phenylethyl)amino]methyl}alkanedioate

Developmental toxicity / teratogenicity

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-Oct-2015 to 20-May-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

The protocol was designed in general accordance with the OECD Guideline for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, 28-Jul-2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Details on test material:

- Physical state: Pale amber liquid
- Analytical purity: 100 %
- Expiration date of the lot/batch: 08 January 2017
- Storage condition of test material: Approximately 4 °C in the dark

Test animals

Species:

rat

Strain:

other: Wistar Han [Crl:WI(Han)]

Details on test animals or test system and environmental conditions:

Sexually mature male and virgin female Wistar Han [Crl:WI(Han)] rats were used as the test system on this study. The animal model, the Crl:WI(Han) rat, is recognized as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, WIL Research has reproductive historical control data in the Crl:WI(Han) rat. The number of animals selected for this study 15 [Groups 1, 5, and 6] or 10 rats/sex/group [Groups 2-4]) was based on the OECD Guideline for the Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, 28-Jul-2015, which recommends evaluation of at least 8 pregnant females per group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination. The extra females were necessary to ensure a sufficient number of females were available for assignment to study following the pretest estrous cycle evaluations. In addition, 5 animals/sex in the control, high- dose test item group (Group 5), were necessary to assess the recovery, persistence, or progression of any toxic effects following the cessation of dosing.
Crl:WI(Han) rats (82 males and 98 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC or Kingston, NY on 13-Oct-2015. The animals were approximately 56 days old upon receipt. Each animal was examined by a qualified biologist on the day of receipt. The day following receipt, all animals were weighed and clinical observations were recorded. Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period. The animals were housed for an acclimation period of 34 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior, the testes were palpated at least once for all males, and daily vaginal lavages were performed on all females for a minimum of 2 weeks prior to randomization to determine the stage of estrous.


Following receipt and until pairing, all F0 animals were housed 2-3 per cage by sex in clean, solid-bottom cages with bedding material (Bed-O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The bedding material is periodically analyzed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research. Assignment of individual animals to social groups is presented in Appendix E. During cohabitation, breeding phase rats (10/sex/group) were paired in a solid-bottom cage with bedding material. Following the breeding period, the males were individually housed in solid-bottom cages until the scheduled necropsy. Following positive evidence of mating, the females were individually housed in plastic maternity cages with bedding material. The dams and their litters were housed in these cages until pup euthanasia on PND 13 and dam euthanasia on lactation day
14. Females that failed to deliver were housed in plastic maternity cages until post-mating day
25. The 5 rats/sex in the control, high-dose test item that were assigned to the recovery phase were not paired for mating and remained housed in groups of 2-3 in clean solid-bottom cages until euthanasia.
Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at WIL Research are accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Feed lots used during the study were documented in the study records. Feeders were changed and sanitized once per week. Municipal water supplying the facility was sampled for contaminants according to WIL Research SOPs. The results of the diet and water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study. Reverse osmosis-

purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study, with the following exception. Animals selected for clinical pathology assessments were fasted prior to clinical pathology blood collection when food, but not water, was withheld.

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively. Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. These data are summarized in Appendix F. Actual mean daily temperature ranged from 70.9°F to 72.0°F (21.6°C to 22.2°C) and mean daily relative humidity ranged from 34.0% to 50.3% during the study. Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

At the conclusion of the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health, meeting acceptable body weight requirements, and exhibiting normal 4-5 day estrous cycles (females) was selected for use in the computerized randomization procedure based on body weight stratification in a block design. At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMS™. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated. The animals then were arranged into groups according to the printout. Animals not assigned to study were transferred to the WIL Research colony.
The experimental design consisted of 4 test substance-treated groups (Groups 2-5), and 1 control group (Group 1) composed of 10 rats/sex/group. An additional 5 rats/sex in the control, high-dose test item group (Group 5), and were selected to be evaluated following a 15-day recovery period; animals assigned to the recovery period were not evaluated for reproductive toxicity. At the initiation of dose administration (study day 0), the males and females were approximately 13 weeks old. Male body weights ranged from 272 g to 377 g and female body weights ranged from 180 g to 236 g on study day 0. The animals were approximately 15 weeks old when paired on study day 13; female body weights ranged from 195 g to 248 g on gestation day 0.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

The vehicle used in preparation of the test substance formulations and for administration to the control group was arachis (peanut) oil, NF (lot nos. 2EA0347 and 2EH0290, exp. dates: 31-Jan-2016 and 31-Jul-2016, respectively).

Details on exposure:

The vehicle was dispensed approximately weekly for administration to the control group (Group
1) and for preparation of the test substance formulations; aliquots were prepared for daily

dispensation to the control group and stored at room temperature, protected from light. The vehicle was mixed throughout the sampling and dose administration procedures.
Dosing formulations were prepared at the test substance concentrations indicated in the following table:


Group Number
Treatment Dosage Level (mg/kg/day) Test Substance Concentrationa (mg/mL)
1 Vehicle control 0 0
2 Test item 200 40
3 Test item 300 60
4 Test item 400 80
5 Test item 500 1 00

a = The dosing formulations were not adjusted for purity.

The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
The first test substance dosing formulations were visually inspected by the Study Director’s designee and were found to be visibly homogeneous and acceptable for administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSES OF DOSING FORMULATIONS
Analyses of Dosing Formulations Report:

The analyzed dosing formulations were within WIL Research SOP range for suspensions (85% to 115%) and were homogeneous. The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).
Results of the analyses of dosing formulations are summarized below.

Text Table 1. Results of Homogeneity and Concentration Analyses

Group 2 Group 3 Group 4 Group 5
(40 mg/mL (60 mg/mL (80 mg/mL (100 mg/mL

Homogeneity Assessment of the 13-Nov-2015 Formulations

Mean Concentration (mg/mL) 41.2 61.1 83.2 103
RSD (%) 0.86 1.3 1.9 1.3
Mean % of Target 103 102 104 103
Homogeneity Assessment of the 05-Jan-2016 Formulations
Mean Concentration (mg/mL) 37.3 57.8 76.1 92.8
RSD (%) 1.6 2.2 2.5 3.3
Mean % of Target 93.3 96.3 95.1 92.8

Details on mating procedure:

The 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. Each female was cohabitated with 1 male in a solid-bottom cage containing bedding material. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0.
For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.
Mating, fertility, and copulation/conception indices were calculated as follows:

Mating, fertility, and copulation/conception indices were calculated as follows:
Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant) divided by the total No. of Males (Females) Used for Mating x 100
Male Fertility Index (%) = No of males siring a litter divided by Total number of males used for mating x 100
Male Copulation Index (%) = No. of Males Siring a Litter divided by No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy) x 100
Female Fertility Index (%) = No of males siring a litter divided by no of males with evidence of mating (or females with confirmed pregnancy) x 100
Female Conception Index (%) = No. of Females with Confirmed Pregnancy divided by No. of Females with Evidence of Mating (or Confirmed Pregnancy) x 100

The vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily. The males selected for pairing were dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females selected for pairing were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 13) for a total of 49-61 doses. Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. The extra 5 rats/sex in the control, high-dose (Group 5), were not used for mating, but were treated on a comparable regimen beginning on study day 0; following 28 doses for males and 49 doses for females, these animals remained on study for a 15-day recovery period. The dose volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
The following table presents the study group assignment:


Group Number Treatment Dosage Level Dose Volume Number of Males Number of Females
(mg/kg/day) (mL/kg)

1 Vehicle control 0 5 15a 15a
2 Test item 200 5 10 10
3 Test item 300 5 10 10
4 Test item 400 5 10 10
5 Test item 500 5 15a 15a

a = 5 animals/sex in Groups 1, 5, were necropsied following a 15-day recovery period. Recovery animals were not evaluated for reproductive performance.

The dosage levels were selected based on the results of a previous 28-day toxicity study (Rashid, 2015) in male and female rats. In the previous study, Wistar rats were dosed with the test substance at dosage levels of 100, 300, 400, and 1000 mg/kg/day. There was no treatment-related mortality noted in the previous study. Slight reductions in mean body weight gains were noted in all dose groups; however, mean body weights were not affected. The NOAEL in the previous study was considered to be 400 mg/kg/day. As a result, 500 mg/kg/day was chosen for the high-dosage level in the current study and was not expected to produce excessive toxicity.
The selected route of administration for this study was oral (gavage) because this a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Duration of treatment / exposure:

The males selected for pairing were dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females selected for pairing were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 13) for a total of 49-61 doses

Frequency of treatment:

The vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily

Duration of test:

he vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily. The males selected for pairing were dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females selected for pairing were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 13) for a total of 49-61 doses. Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. The extra 5 rats/sex in the control, high-dose (Group 5), were not used for mating, but were treated on a comparable regimen beginning on study day 0; following 28 doses for males and 49 doses for females, these animals remained on study for a 15-day recovery period. The dose volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Group Treatment Dosage Level Dose Volume Number of Number of
Number (mg/kg/day) (mL/kg) Males Females
1 Vehicle control 0 5 15a 15a
2 Test item 200 5 10 10
3 Test item 300 5 10 10
4 Test item 400 5 10 10
5 Test item 500 5 15a 15a

Control animals:

yes, concurrent vehicle

Details on study design:

At the conclusion of the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health, meeting acceptable body weight requirements, and exhibiting normal 4-5 day estrous cycles (females) was selected for use in the computerized randomization procedure based on body weight stratification in a block design. At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMS™. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated. The animals then were arranged into groups according to the printout. Animals not assigned to study were transferred to the WIL Research colony.

Examinations

Maternal examinations:

All rats were observed twice daily, once in the morning and once in the afternoon, for appearance, behavior, moribundity, mortality, and signs of overt toxicity. Individual detailed physical examinations were recorded weekly (prior to dose administration during the treatment period). Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties. Due to social housing, some observations could not be attributed to a single animal; however, no social housing observations were noted.

Fetal examinations:

Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring that were found dead were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984). The carcass of each pup was then discarded.

To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4. All selections were performed by computerized randomization. Blood samples for possible future thyroid hormone analysis were collected from 1 culled pup/sex/litter (pooled by litter), when possible, on PND 4 pups were euthanized by decapitation following blood collection and discarded. Remaining culled pups (not used for blood collection) were weighed, euthanized by an intraperitoneal injection of sodium pentobarbital on PND 4, and discarded.

Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a clinical examination on PND 1, 4, 7, 10, and 13. Any abnormalities in nesting and nursing behavior were recorded. The anogenital distance of all F1 pups was measured on PND 1; the absolute distance and absolute distance relative to the cube root of body weight were reported for each pup. Anogenital distance was defined as the distance from the caudal margin of the anus to the caudal margin of the genital tubercle.

Pups were individually weighed on PND 1, 4, 7, 10, and 13. Mean pup weights were presented by sex for each litter and by dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods a given pup was not weighed were left blank or designated as “NA” on the individual report tables.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was 2 or less. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield

minor variations from those listed in the report data tables. Statistical analyses were not conducted on F0 weekly female body weight data after 1 or more animals had entered the gestation phase. Where applicable, the litter was used as the experimental unit.
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.

Indices:

Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, estrous cycle lengths, pre-coital intervals, gestation length, numbers of former implantation sites, and unaccounted-for sites, number of pups born, live litter size on PND 0, absolute and relative organ weights, clinical pathology values, thyroid hormone values, anogential distance (absolute and relative to the cube root of body weight), and number of nipples/areolae values were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All F0 males and females survived to the scheduled necropsies. Test item related clinical findings of clear material around the mouth were generally noted in a dose-related manner for males and females in the 200, 300, 400, and 500 mg/kg/day groups at approximately 1 hour following dose administration throughout the treatment period. These findings were not observed during the weekly detailed physical examinations performed prior to dosing and were not considered adverse. No other test item related clinical findings were noted; findings noted in the test substance-treated groups, including hair loss on various body surfaces,

occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item related effects on body weights of body weight gains were noted for F0 males in the test substance-treated groups throughout the study. A significantly (p<0.01) higher mean body weight gain was noted for males in the 200 mg/kg/day group during study days 21-27; however, this was transient and did not occur in a dose-related manner. No other statistically significant differences from the control group were noted.

No test item related effects were noted on body weights or body weight gains for F0 females in the test substance-treated groups throughout the study. Significantly (p<0.01 or p<0.05) higher mean body weight gains were noted for females in the 500 mg/kg/day group during study days 27-35 and when the entire treatment period (study days 0-48) was evaluated, resulting in mean body weights that were up to 7.0% higher (not statistically significant) than the control group during the treatment period. These results were primarily due to periods of mean body weight loss noted for control group females (study days 27-35 and 35-42) and were considered incidental. A significantly (p<0.05) lower mean body weight gain was noted for females in the 500 mg/kg/day group when the entire recovery period was evaluated (study days 49-62); however, the direction of change was opposite that previously observed and the results were considered incidental. Mean body weights in this group were similar to the control group by the end of the recovery period (study day 62).
No test item related effects were noted on body weights or body weight gains for F0 females in the test substance-treated groups during gestation. Significantly (p<0.05 or p<0.01) higher mean body weight gains were noted for females in the 200, 300, and 500 mg/kg/day groups during gestation days 4-7; however, these results were transient, there was not a dose-response, and mean body weights were unaffected. No other differences from the control group were statistically significant.

No test item related effects were noted on body weights or body weight gains for F0 females in the test substance-treated groups during lactation. None of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption, evaluated as g/animal/day, in test item treated F0 males was similar to that in the control group throughout the study. No statistically significant differences were observed.

Mean food consumption, evaluated as g/animal/day, in the test item treated F0 females was unaffected by test substance administration during the pre-mating period (breeding and recovery phase females) or throughout the study (recovery phase females). None of the differences from the control group were statistically significant.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related alterations in hematology and coagulation parameters at the scheduled necropsies. However, some statistically significant differences were observed when the recovery necropsy control and test substance-treated groups were compared. Lower mean corpuscular hemoglobin and higher hemoglobin distribution width values were noted in the 500 mg/kg/day group males, and higher hematocrit values and absolute neutrophil and monocyte counts were noted in the 500 mg/kg/day group females. Differences in erythrocytic parameters were not considered to be test substance-related because measured values were similar to the control groups and all erythrocytic values at the primary necropsy were similar to control groups. Changes in leukocyte values were not considered test substance-related because total white blood cell counts were similar to the control group, all leukocyte parameters at the primary necropsy were similar to the control group, there were no histologic correlates, and values were of minimal magnitude difference. Statistically significant findings that involved percentage leukocyte differential counts were not itemized above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.

SERUM CHEMISTRY
There were no test substance-related alterations in serum chemistry parameters at the scheduled necropsies. However, some statistically significant differences were observed when the control and test substance-treated groups were compared. Higher total serum protein, albumin, and calcium values were noted in the 500 mg/kg/day group females, Globulin and albumin/globulin ratio values were similar to controls and the magnitude difference was minimal. Higher serum calcium values related to higher albumin values and were of minimal magnitude difference. The difference in serum creatinine values was in a direction of no known toxicologic significance.
At recovery, higher serum alkaline phosphatase values were noted in the 500 mg/kg/day group males, Values were similar to the control groups at the primary necropsy, and were of minimal magnitude difference at recovery.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related alterations in final body weights at the scheduled necropsies. Test substance-related higher absolute and relative (to body and brain weight) liver (200, 300, 400, and 500 mg/kg/day groups) and kidney (400 and 500 mg/kg/day group males) weights were noted at the primary necropsy.
Higher absolute and/or relative (to body and brain weight) liver weights were noted in the 200, 300, 400, and 500 mg/kg/day group males and the 400 and 500 mg/kg/day group females. Higher absolute and/or relative (to body and brain) kidney weights were noted in the 400 and 500 mg/kg/day group males (statistically significant in all except absolute kidney weight in the 400 mg/kg/day group males). Liver and kidney weights were similar to the control groups at the recovery necropsy. In the absence of any histopathologic correlates, these changes were considered adaptive and non-adverse.
There were no other test substance-related effects on organ weights.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Review of the gross necropsy observations revealed no observations that were considered to be associated with administration of the test substance. However, 1 macroscopic observation deserves mention: small thyroid glands were noted in two 500 mg/kg/day group males at the primary necropsy. Individual thyroid gland weights were within the range of the control group; thus, the finding was not considered to be test substance-related.
The mean numbers of unaccounted-for sites and implantation sites in the groups were similar to the control group values.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related histologic changes at the primary necropsy. Histologic changes noted were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Maternal developmental toxicity

Number of abortions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

The mean number of pups born, live litter size, and the percentage of males at birth in the treated groups were similar to the control group values. Postnatal survival in the treated groups was unaffected by test substance administration.

Changes in pregnancy duration:

not specified

Description (incidence and severity):

Mean gestation lengths in the test item treated groups were similar to that in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.
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): Mean gestation lengths in the test item treated groups were similar to that in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

Details on maternal toxic effects:

Test substance-related lower total thyroxine (T4) values were noted in the 300 (-7.1%), 400 (-13.3%), and 500 (-10.4%) mg/kg/day group males at the primary necropsy (statistically significant in only the 400 mg/kg/day group). There was a general dose-response relationship, although the 400 mg/kg/day group males had a lower group mean value than the 500 mg/kg/day group males, and 4 individuals in the 400 mg/kg/day group had values minimally lower than the range of the concurrent control group compared to only two 500 mg/kg/day group

individual animals with values lower than the concurrent control group. Lower T4 values did not correlate with normal thyroid/parathyroid gland weights, but were considered to correlate with higher liver weights noted in test item treated males at the primary necropsy, via the mechanism of test substance-related hepatic enzyme induction (Zabka et al., 2011). Values were similar to the control group at the recovery necropsy.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: Under the conditions of this screening study, based on the lack of any adverse test item related effects at any dosage level

Remarks on result:

other: Under the conditions of this screening study, based on the lack of any adverse test item related effects at any dosage level

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup body weights and body weight changes during PND 1-13 in the treated groups were unaffected by parental administration of the test substance. No statistically significant differences from the control group were noted.
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 male and female pup body weights and body weight changes during PND 1-13 in the treated groups were unaffected by parental administration of the test substance. No statistically significant differences from the control group were noted.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of pups born, live litter size, and the percentage of males at birth in the treated groups were similar to the control group values. Postnatal survival in the treated groups was unaffected by test substance administration.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The mean number of pups born, live litter size, and the percentage of males at birth in the treated groups were similar to the control group values. Postnatal survival in the treated groups was unaffected by test substance administration.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean number of pups born, live litter size, and the percentage of males at birth in the treated groups were similar to the control group values. Postnatal survival in the treated groups was unaffected by test substance administration

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Postnatal survival in the treated groups was unaffected by test substance administration.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by parental administration of test item. Pups (litters) that were found dead numbered 1(1), 0(0), 1(1), 1(1), 3(3), and 2(1) in the control, 200, 300, 400, and 500 mg/kg/day groups, respectively. Two, 0, 5, 2, 3, and 2 pups in these same respective groups were missing and presumed to have been cannibalized.
The anogenital distances (absolute and relative to the cube root of pup body weight) in the treated groups were similar to the control group values. Differences from the control group were slight and not statistically significant.
Areolae/nipple anlagen in the F1 male pups was unaffected by parental test item administration when evaluated on PND 12. The test substance-treated group values were not statistically different from the control group values.
v

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Under the conditions of this screening study, based on the lack of any adverse test item related effects at any dosage level

Remarks on result:

other: Under the conditions of this screening study, based on the lack of any adverse test item related effects at any dosage level

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

The objectives of the study were to provide preliminary information on the potential adverse effects of the test substances on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the F0 generation and the development of offspring from conception through PND 13.

Test item related higher liver weights were noted in the 200, 300, 400, and 500 mg/kg/day group males and 400 and 500 mg/kg/day group females and higher kidney weights were noted in the 400 and 500 mg/kg/day group males at the primary necropsy. Higher weights were of minimal magnitude difference, had no microscopic or serum chemistry correlates, and were considered to be minimal, non-adverse adaptive changes related to microsomal enzyme induction (Khan et al., 2013; Hall et al., 2012). Weights were similar to the control groups at the recovery necropsy.

Test item related lower T4 values were noted in the 300, 400, and 500 mg/kg/day group males at the primary necropsy (statistically significant in the 400 mg/kg/day group males). Individual values were minimally lower than the control group, and the difference was attributed to enhanced T4 metabolism due to test item related hepatic enzyme induction (Zabka et al., 2011). There was no corresponding increase in thyroid/parathyroid gland weights and no microscopic correlates; the finding was considered to be adaptive and not adverse. Minimally lower T4 values were noted at PND 13 in the 300 and 400 mg/kg/day group males and females (statistically significant in the 400 mg/kg/day group males); there was no dose-response relationship, since values in the 500 mg/kg/day group animals were similar to the control group. The relationship of lower T4 values to administration of the test item in PND 13 pups is uncertain, but may be due to enhanced T4 metabolism secondary to test item related hepatic enzyme induction. Lower T4 values were considered to be adaptive and not adverse.

There were no test substance-related alterations in hematology, coagulation, or serum chemistry parameters and no test substance-related macroscopic or microscopic findings.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this screening study, based on the lack of any adverse the test item related effects at any dosage level, a dosage level of 500 mg/kg/day (the highest dosage level of test item evaluated) was considered to be the no-observed-adverse-effect level (NOAEL) for  F0 systemic toxicity, F0 reproductive toxicity, and F1 neonatal toxicity when administered orally by gavage to Crl:WI(Han) rats.

Executive summary:

The objectives of the study were to provide preliminary information on the potential adverse effects of the test substance on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behavior, conception, parturition, and lactation of the F0 generation and the development of offspring from conception through PND 13.

The test substance, in the vehicle (arachis [peanut] oil) were administered orally by gavage once daily to 4 groups Groups 2-5) of Crl:WI(Han) rats. The low- and mid-dose groups (Groups 2-4) each consisted of 10 rats/sex and the high-dose group (Group 5) of 15 rats/sex. Dosage levels were 200, 300, 400, 500, and 250 mg/kg/day in Groups 2-5, respectively. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Males and females were approximately 13 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 13 for a total of 49-61 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. The extra 5 males and 5 females in the control group, high-dose group (Group 5), that were not used for mating were treated beginning on study day 0; following 28 doses for males and 49 doses for females, these animals were assigned to a 15-day non-dosing recovery period.

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 13. F1 clinical observations, body weights, and sexes were recorded at appropriate intervals and anogenital distance was recorded on PND 1. To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4; blood samples for possible thyroid

 

hormone analysis were collected from the culled pups (1/sex/litter). All F1 male pups were evaluated for areolae/nipple anlagen on PND 12. Remaining F1 pups were euthanized on PND 13; blood samples for thyroid hormone analysis were analyzed and selected organs were weighed from 1 pup/sex/litter. Clinical pathology evaluations (hematology and serum chemistry)  were  performed  on  10  F0  animals/sex/group  at  the  primary  necropsy  and 5 animals/sex in the control group, high-dose group (Group 5), at the recovery necropsy. Blood samples for thyroid hormone analysis were collected from F0 males and females at the primary and recovery necropsies; only male samples were analyzed. F0 males were euthanized following completion of the mating period or 15-day recovery period and F0 females were euthanized on lactation day 14 for females that delivered, post-mating day 25 for females that failed to deliver, or following the 15-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control group, high-dose group (Group 5), at the primary necropsy.

All F0 males and females survived to the scheduled necropsies. Test item related clinical findings of clear material around the mouth were generally noted in a dose-related manner for males and females in the 200, 300, 400, and 500 mg/kg/day test item groups at approximately 1 hour following dose administration throughout the treatment period. These findings were not considered adverse. No other test item related clinical findings were noted.

No Test item related effects were noted on F0 male and female body weights, body weight gains, or food consumption throughout the study. F0 reproductive performance (mating, fertility, copulation, and conception), gestation length, and the process of parturition were unaffected by test item administration. No test item related effects were noted on F1 pup growth, viability, clinical condition, anogenital distance, or areolae/nipple anlagen (males only).

Test item related higher liver weights were noted in the 200, 300, 400, and 500 mg/kg/day test item group males and 400 and 500 mg/kg/day test item group females and higher kidney weights were noted in the 400 and 500 mg/kg/day test item group males at the primary necropsy. Higher weights were of minimal magnitude difference, had no microscopic or  serum

 

chemistry correlates, and were considered to be minimal, non-adverse adaptive changes related to microsomal enzyme induction (Khan et al., 2013; Hall et al., 2012). Weights were similar to the control groups at the recovery necropsy.

Test item related lower T4 values were noted in the 300, 400, and 500 mg/kg/day test item group males at the primary necropsy. Individual values were minimally lower than the control group, and the difference was attributed to enhanced T4 metabolism due to test item related hepatic enzyme induction (Zabka et al., 2011). There was no corresponding increase in thyroid/parathyroid gland weights and no microscopic correlates; the finding was considered to be adaptive and not adverse. Minimally lower T4 values were noted at PND 13 in the 300 and 400 mg/kg/day test item group males and females; there was no dose-response relationship, since values in the 500 mg/kg/day test item group animals were similar to the control group. The relationship of lower T4 values to administration of the test item in PND 13 pups is uncertain, but may be due to enhanced T4 metabolism secondary to test item related hepatic enzyme induction. Lower T4 values were considered to be adaptive and not adverse.

There were no test item related alterations in F0 hematology, coagulation, or serum chemistry parameters and no test item related macroscopic or microscopic findings for F0 animals. For F1 pups, no test item related organ weight alterations or macroscopic findings (for F1 pups found dead) were noted.

Under the conditions of this screening study, based on the lack of any adverse the test item related effects at any dosage level, a dosage level of 500 mg/kg/day (the highest dosage level of test item evaluated) was considered to be the no-observed-adverse-effect level (NOAEL) for  F0 systemic toxicity, F0 reproductive toxicity, and F1 neonatal toxicity when administered orally by gavage to Crl:WI(Han) rats.