Thiophene, tetrahydro-, 1,1-dioxide, 3-(C9-11-isoalkyloxy) derivs., C10-rich

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fertility: NOAEL 600 mg/kg bw/day; study performed in line with OECD Guideline 421; Knapp (2006)

Systemic toxicity: NOAEL 175 mg/kg bw/day; study performed in line with OECD Guideline 421; Knapp (2006)

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 October 2005 to 7 September 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMAL RECEIPT AND ACCLIMATION

Fifty-five male and 55 female Crl:CD(SD) rats were received in good health from a reputable supplier on 22 November 2005. The animals were 59 days old upon receipt. Each animal was examined by a qualified technician on the day of receipt and weighed the following day. Each rat was uniquely identified by a Monel® metal eartag displaying the animal number. The animals were housed for an acclimation period of 13 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.

ANIMAL HOUSING

Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed 3 times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding. The nesting material is periodically analysed by the manufacturer for contaminants. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The dams and their litters were housed in these cages until euthanasia on lactation day 4. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).

DIET, DRINKING WATER AND MAINTENANCE

The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, is a certified feed with appropriate analyses performed by the manufacturer. Feed lots used during the study are documented in the study records. Feeders were changed and sanitized once per week. Municipal water supplying the facility is sampled for contaminants according to standard operating procedures. The results of the diet and water analyses are maintained at WIL Research Laboratories, LLC. 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.

ENVIRONMENTAL CONDITIONS

All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain daily averages of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20% relative humidity. Room temperature and relative humidity were controlled and monitored using the Metasys® DDC Electronic Environmental control system. These data were recorded approximately hourly and are summarised in attached Appendix D. Actual mean daily temperature ranged from 70.3°F to 71.2°F (21.3°C to 21.8°C) and mean daily relative humidity ranged from 30.2% to 43.2% during the study. Light timers were calibrated to provide a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

corn oil

Details on exposure:

The vehicle and test article formulations were administered orally by gastric intubation, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan) once daily. The males were dosed during study days 0-55 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 56 doses. The females were dosed from study day 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-53 doses. The female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. The dosage 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.

Details on mating procedure:

BREEDING PROCEDURES
The animals 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 prepared. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages. 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.

PARTURITION
All females were allowed to deliver naturally and rear their young to PND 4. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the control group (Group 1), a sufficient amount of corn oil was added to a glass container. The vehicle was stirred continuously throughout sampling and dispensation Aliquots of the vehicle were prepared for daily dispensation and stored at room temperature. The vehicle was stirred continuously throughout dose administration. The appropriate amount of the test article for each group was weighed into a tared, calibrated container. Vehicle was added to each container to bring the formulations nearly to the calibration mark. The formulations were mixed until uniform using a magnetic stirrer. Vehicle was added to each container to bring the formulations to the calibration mark, and the formulations were stirred until uniform using a magnetic stirrer. The test article formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored at room temperature. The test article formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.

SAMPLING AND ANALYSES
Prior to the initiation of dose administration, duplicate samples (1 mL each) for homogeneity determination were collected on 21 November 2005 from the top, middle and bottom strata of each dosing formulation, including the vehicle for the control group. In addition, duplicate samples (1 mL each) for stability and resuspension homogeneity determinations were collected on 1 December 2005 from the top and bottom strata of aliquots of these same dosing suspensions following storage at room temperature for 10 days; the aliquots were mixed using a magnetic stirrer for a minimum of 10 minutes prior to sample collection. Samples (1 mL each) for concentration analysis were collected weekly from the middle stratum of each dosing formulation (including the vehicle for the control group); only the samples from the first, fourth and last weekly formulations were analyzed. All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, LLC. The methodology and results of these analyses are presented in attached Appendix B

Duration of treatment / exposure:

approximately 8 weeks

Frequency of treatment:

daily

Details on study schedule:

ANIMAL RECEIPT AND ACCLIMATION
Fifty-five male and 55 female Crl:CD(SD) rats were received in good health from a reputable supplier on 22 November 2005. The animals were 59 days old upon receipt. Each animal was examined by a qualified technician on the day of receipt and weighed the following day. Each rat was uniquely identified by a Monel® metal eartag displaying the animal number. The animals were housed for an acclimation period of 13 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.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
Near the end 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 and meeting acceptable body weight requirements (300 g to 500 g for males and 200 g to 300 g for females) was selected for use in the computerized randomization procedure. At that time, the individual body weights and corresponding animal identification numbers were entered into the WIL Toxicology Data Management System (WTDMS™). A printout containing the animal numbers, corresponding body weights and individual group assignments was generated using a computer program which randomised the animals based on stratification of body weights in a block design. The animals then were arranged into groups according to the printout. Individual body weights at randomization were within ± 20% of the mean for each sex. The experimental design for WIL-186044 consisted of 3 test article-treated groups and 1 control group composed of 12 rats/sex/group. At the initiation of dose administration (study day 0), the males and females were approximately 10 weeks old. Body weights ranged from 334 g to 399 g for males and from 215 g to 271 g for females on study day 0. The animals were approximately 12 weeks old when paired on study day 13; female body weights ranged from 230 g to 301 g on gestation day 0.

Remarks:

Doses / Concentrations:
0 mg/kg/day (0 mg/ml)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
50 mg/kg/day (10 mg/ml)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
175 mg/kg/day (35 mg/ml)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
600 mg/kg/day (120 mg/ml)
Basis:
actual ingested

No. of animals per sex per dose:

12 animals per sex per dose level including control

Control animals:

yes, concurrent vehicle

Details on study design:

Dosage levels were selected based on the results of previous studies and were provided by the sponsor representative after consultation with the study director.

Positive control:

Not applicable

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual detailed clinical observations were recorded weekly (prior to test article administration during the treatment period). Each male and female was also observed for signs of toxicity at the time of dose administration and approximately 1-2 hours following dose administration. All significant findings were recorded.

BODY WEIGHTS
Individual male body weights were recorded weekly throughout the study and prior to the scheduled euthanasia. Individual female body weights were recorded weekly until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating period (males and females) and for the entire generation (males only). Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4. Mean gestation body weights and corresponding mean body weight changes are presented for these intervals and for the overall gestation interval (days 0-20). For individual data, the time periods a given animal was not weighed were designated as “NA” (Not Applicable).

FOOD CONSUMPTION
Individual food consumption was recorded weekly until pairing. Food intake was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4. Following mating, food consumption for all males was measured on a weekly basis until the scheduled euthanasia. Food consumption was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. Calculation of the comprehensive intervals excludes all erroneous values such as total
food spillage. When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, obvious erroneous value, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” (Not Applicable) on the individual report tables.

Oestrous cyclicity (parental animals):

No Data

Sperm parameters (parental animals):

PAS and hematoxylin staining were used for the right and left testes and epididymides. The testes were fixed in Bouin’s solution and embedded in paraffin. Sections of 2 to 4 microns were made for the testis (transverse) and the epididymis (longitudinal). Special emphasis was placed on the stages of spermatogenesis and histopathology of interstitial testicular cell structure.

Litter observations:

F1 LITTER PARAMETERS

LITTER VIABILITY AND DEATHS
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. Standardisation of litter size was not performed because the pups were euthanized on PND 4. Intact offspring dying were necropsied using a fresh dissection technique including the heart and major vessels (Stuckhardt and Poppe, 1984). The carcass of each pup was then discarded.

CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nursing behavior were recorded.

BODY WEIGHTS
Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dosage group.

SEX DETERMINATION
Pups were individually sexed on PND 0 and 4.

Postmortem examinations (parental animals):

MACROSCOPIC EXAMINATIONS

UNSCHEDULED DEATHS
A complete necropsy was performed on the male that was found dead. This included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain and the thoracic, abdominal and pelvic cavities, including viscera.

SCHEDULED EUTHANASIA
All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the female necropsies. Females that delivered were euthanized on lactation day 4; the number of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on post-mating day 25 (females with evidence of mating). Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). Necropsy included examination of the external surface, all orifices and the cranial cavity, external surfaces of the brain, and the thoracic, abdominal and pelvic cavities, including viscera. At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted):

Cervix
Testes with epididymides (a) and vas deferens (2)
Coagulating glands
Mammary gland
Thyroids [with parathyroids if present (2)]
Ovaries and oviducts (2)
Pituitary gland
Uterus (b) with vagina
Prostate gland
All gross lesions (c)
Seminal vesicles (2)

(a) = Testes and epididymides fixed in Bouin’s solution.
(b) = Not retained if placed in ammonium sulfide solution.
(c) = Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible.

ORGAN WEIGHTS
The following organs were weighed from all F0 animals at the scheduled necropsies:
Brain Ovaries with oviducts
Epididymides (a)
Pituitary gland
Kidneys
Testes (a)
Liver
Thyroids with parathyroids (b)

(a) = These paired organs were weighed separately.
(b) = Fixed in 10% neutral-buffered formalin prior to weighing
Except as noted, paired organs were weighed together. Absolute weights, organ to final body weight and organ to brain weight ratios were reported.

MICROSCOPIC EXAMINATIONS
After fixation, protocol-specified tissues were trimmed according to standard operating procedures and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides and stained with hematoxylin and eosin, with the following exceptions. PAS and hematoxylin staining were used for the right and left testes and epididymides. The testes were fixed in Bouin’s solution and embedded in paraffin. Sections of 2 to 4 microns were made for the testis (transverse) and the epididymis (longitudinal). Special emphasis was placed on the stages of spermatogenesis and histopathology of interstitial testicular cell structure. Microscopic examination was performed on all tissues from the male that was found dead and all animals in the control and 600 mg/kg/day groups at the scheduled necropsies; gross lesions from all dosage groups were also examined. Because of test article-related findings in the 600 mg/kg/day group males, the thyroid glands were also examined from all males in the 50 and 175 mg/kg/day groups. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section or other reasons as appropriate.

Postmortem examinations (offspring):

SCHEDULED EUTHANASIA
On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital. Gross lesions and malformations (if any) observed at the detailed physical examinations on PND 4 were preserved in the appropriate fixative for possible future examination.

Statistics:

STATISTICAL ANALYSES
All statistical tests were performed using appropriate computing devices or programs. For full details please see 'Any other information on materials and methods incl. tables'.

Reproductive indices:

Mating, fertility and copulation/conception indices were calculated as follows:

Male (female) mating index (%) = (No. of Males (Females) with Evidence of Mating Male (Female) (or Confirmed Pregnancy) / Total No. of Males (Females) Used for Mating) x 100

Male Fertility Index (%) = (No. of Males Siring a Litter / Total No. of Males Used for Mating) x100

Male Copulation Index (%) = (No. of Males Siring a Litter / No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy)) x 100

Female Fertility Index (%) = (No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating) x 100

Female Conception Index (%) = (No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or Confirmed Pregnancy)) x 100

Offspring viability indices:

Litter parameters were defined as follows:

Mean Live Litter Size = Total No. of Viable Pups on PND 0 / No. of Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = Σ ((Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter) / No. of Litters Per Group) x100

Postnatal Survival for All
Other Intervals (% Per Litter) = Σ ((Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N) / No. of Litters Per Group) x 100

Where N = PND 0-1 and 1-4

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

F0 GENERATION

MALES

CLINICAL OBSERVATIONS AND SURVIVAL
Summary Data: see attached Tables 1, 2, 3
Individual Data: see attached Tables 49, 50, 51

Test article-related clinical findings were noted in the 175 and 600 mg/kg/day group males at the time of and 1-2 hours following dose administration. Male no. 11517 in the 600 mg/kg/day group was found dead on study day 32. During the week prior to death, this male had an unkempt appearance, rales and red material around the eyes, nose and mouth, lower body weight gain (relative to its gain during the first week of dose administration) during study days 7-21 and a body weight loss during study days 21-27. Fractured nasal bones were noted at necropsy for this animal. The death of
male no. 11517 was attributed to complications from the fractures (presumed to be the result of a mechanical injury) and not to the test article. All other males survived to the scheduled necropsy. No test article-related clinical findings were observed in the males at the weekly detailed physical examinations. Test article-related increased incidences of salivation or evidence thereof (clear material around the mouth) were noted in the 175 and 600 mg/kg/day males at the time of and/or 1-2 hours following dose administration; the severity of these findings was primarily slight or moderate. Salivation was noted in a total of 9 and 12 males in the 175 and 600 mg/kg/day groups, respectively; the onset of this finding occurred as early as study day 15 in the 175 mg/kg/day group and study day 2 in the 600 mg/kg/day group. Clear material around the mouth was first observed on study
day 15 and study day 3 in a total of 10 and 12 males in the same respective groups. These findings were generally noted throughout the study. However, because the onset of salivation and clear material around the mouth occurred immediately following daily dose administration, these findings were not considered indicative of systemic toxicity. Red material around the mouth (slight or moderate) was also noted for 6 males in the 600 mg/kg/day group 1-2 hours following dose administration beginning on study day 15 and continued sporadically throughout the study. This finding was considered test article-related. Other clinical findings, including hair loss, red material and/or yellow material on various body surfaces, were observed infrequently, were noted similarly in the control group and/or did not occur in a dose-related manner.

BODY WEIGHTS
Summary Data: see attached Tables 4, 5
Individual Data: see attached Tables 52, 53

Transient, test article-related lower mean body weight gains were noted in the 600 mg/kg/day group males relative to the control group. The reductions were not of sufficient magnitude to be considered adverse. Test article-related lower mean body weight gain was noted in the 600 mg/kg/day group males during the pre-mating period (study days 0-13) due to lower mean body weight gain observed study days 7-13; the difference from the control group was statistically significant (p<0.05) during study days 7-13. Mean body weight gain in this group was similar to that in the control group during the mating and post-mating periods, with the following exception. Lower (not statistically significant) mean body weight gain in this
group was noted during study days 21-27 compared to the control group value. The reductions observed in the 600 mg/kg/day group resulted in a slightly lower mean body weight gain when the entire generation (study days 0-56) was evaluated; the difference from the control group was not statistically significant. However, because the reductions were transient and not of sufficient magnitude to result in substantially lower mean body weights, the lower mean body weight gains in this group were not considered adverse. Mean body weights and body weight gains in the 50 and 175 mg/kg/day group males were unaffected by test article administration. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION
Summary Data: see attached Tables 6, 7
Individual Data: see attached Tables 54, 55

Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 175 and 600 mg/kg/day group males was unaffected by test article administration. The values in the test article-treated groups were generally similar to the control group values throughout the treatment period. None of the differences from the control group were statistically significant.

ANATOMIC PATHOLOGY
For organ weight results please see 'Any other information on results incl. tables'.

MACROSCOPIC EXAMINATIONS
Summary Data: see attached Tables 11, 12
Individual Data: see attached Tables 59, 60

Male no. 11517 in the 600 mg/kg/day group was found dead on study day 32. Macroscopic findings for this male consisted of fractured nasal bones and dark red contents in the stomach, duodenum, jejunum, ileum and cecum. This death was attributed to complications from the fractures (presumed to be a mechanical injury) and not to the test article. At the scheduled F0 male necropsies, no test article-related internal findings were
observed at any dosage level. Macroscopic findings observed in the test article-treated groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.

MICROSCOPIC EXAMINATIONS
Summary Data: see attached Tables 13, 14
Individual Data: see attached Tables 59, 60
Pathology Report: see attached Appendix E

Male no. 11517 in the 600 mg/kg/day group was found dead on study day 32. No test article-related microscopic findings were noted in the organs that were examined for this male. Microscopic findings in the bone, related to the macroscopic finding (fracture), consisted of fracture, hemorrhage, inflammation and fibrosis. A cause of death could not be determined. Test article-related microscopic findings were observed in the thyroid glands of the 600 mg/kg/day group males. Follicular cell hypertrophy was noted in 9 of 11 males in the 600 mg/kg/day group compared to no males in the control group; the difference was statistically significant (p<0.05). This finding was characterized by an increase in cell size, cytoplasm and basophilia. Occasionally, follicular cell hyperplasia was observed as well, and colloid depletion was identified in a single male in this group. These findings correlated with higher mean thyroid gland weights in the 600 mg/kg/day group males. Thyroid gland alterations were below the threshold of detection by routine light microscopy in the 50 and 175 mg/kg/day group males. The only other statistically significant (p<0.05) difference from the control group was an increase in the incidence of ultimobranchial cysts in the thyroid gland in the 600 mg/kg/day group males (10/11 males affected versus 2/12 males in the control group). However, ultimobranchial cysts are common congenital anomalies of the thyroid gland arising during early development of the organ (Hardisty and Boorman, 1990). Because the rats in this study were adults at the time of test article exposure, this finding was considered to be spurious rather than test article-related. In addition, there were no ultimobranchial cysts noted in the thyroid glands from the 50 and 175 mg/kg/day group males. No other test article-related findings were noted in the males in the 50, 175 and 600 mg/kg/day group. Remaining histologic changes were considered to be incidental findings, manifestations of spontaneous disease or related to some aspect of experimental manipulation other than exposure to the test article. There were no test article-related alterations in the incidence, severity or histologic character of these incidental and spontaneous tissue alterations.

REPRODUCTIVE PERFORMANCE
Summary Data: see attached Table 15
Individual Data: see attached Table 85
Historical Control Data: see attached Appendix F

No test article-related effects on F0 reproductive performance were observed at any dosage level. Male mating indices were 100.0% in all groups. Male fertility and copulation indices were 100.0%, 91.7%, 100.0% and 100.0% in the control, 50, 175 and 600 mg/kg/day groups, respectively. No statistically significant differences were noted between the control and test article-treated groups. One male in the 50 mg/kg/day group had evidence of mating but did not sire a litter.

FEMALES

CLINICAL OBSERVATIONS AND SURVIVAL
Summary Data: see attached Tables 16, 17, 18
Individual Data: see attached Tables 61, 62, 63

Test article-related clinical findings were noted in the 175 and 600 mg/kg/day group females. However, the findings were not considered adverse.
No test article-related clinical findings were observed in the females at the weekly detailed physical examinations. Test article-related increased incidences of salivation or evidence thereof (clear material around the mouth) were noted in the 175 and 600 mg/kg/day group females at the time of and/or 1-2 hours following dose administration. Salivation was noted in a total of 4 and 11 females, and clear material around the mouth was observed in a total of 8 and 12 females in the 175 and 600 mg/kg/day groups, respectively. The severity of these findings was primarily slight or moderate. A slight increase in the incidence of red material around the mouth (slight) was observed in a total of 8 females in the 600 mg/kg/day group 1 hour following dose administration. This finding was noted as early as study day 4, and was considered test article-related based on the occurrence in the 600 mg/kg/day group males. Salivation was first noted on study day 16 and study day 7 in the 175 and 600 mg/kg/day groups, respectively, and clear material around the mouth was noted beginning on study day 12 and study day 1 in the same respective groups. However, because the onset of these findings was immediately following dose administration, salivation and clear material around the mouth were not considered indicative of systemic toxicity. Other clinical findings, including hair loss on various body surfaces and/or red material around the nose, were observed infrequently, were noted similarly in the control group and/or did not occur in a dose-related manner.

PRE-MATING PERIOD

BODY WEIGHTS
Summary Data: see attached Tables 19, 20
Individual Data: see attached Tables 64, 65

There were no test-article-related effects on mean female body weights or body weight gains at any dosage level during the pre-mating period. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION
Summary Data: see attached Tables 21, 22
Individual Data: see attached Tables 66, 67

Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 175 and 600 mg/kg/day group females was unaffected by test article administration during the pre-mating period. None of the differences from the control group were statistically significant.

GESTATION PERIOD

BODY WEIGHTS
Summary Data: see attached Tables 23, 24
Individual Data: see attached Tables 68, 69

No test article-related effects on mean maternal body weights or body weight gains were noted during gestation at any dosage level. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION
Summary Data: see attached Tables 25, 26
Individual Data: see attached Tables 70, 71

Higher mean maternal food consumption, (primarily g/animal/day) was noted in the 600 mg/kg/day group females during gestation days 0-4, 4-7 and 11-14, resulting in higher mean food consumption when the entire gestation period (gestation days 0-20) was evaluated. The differences from the control group were statistically significant (p<0.05 or p<0.01). The increases in food consumption in this group were considered test article-related, but not adverse, because the increases were slight (2-3 g/animal/day) compared to the control group, and increases in food consumption of this magnitude are not considered adverse. No test article-related effects on food consumption were noted in the 50 and 175 mg/kg/day groups during gestation. No statistically significant differences from the control group were observed.

LACTATION PERIOD

BODY WEIGHTS
Summary Data: see attached Tables 27, 28
Individual Data: see attached Tables 72, 73

Mean body weights and body weight gains in the 50, 175 and 600 mg/kg/day groups were similar to the control group values during lactation days 1-4. No statistically significant differences were noted.

FOOD CONSUMPTION
Summary Data: see attached Tables 29, 30
Individual Data: see attached Tables 74, 75

Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 175 and 600 mg/kg/day group females was unaffected by test article administration during the early lactation period. The values in the test article-treated groups were similar to the control group values throughout the lactation period. No statistically significant differences were observed.

ANATOMIC PATHOLOGY
For organ weight results please see 'Any other information on results incl. tables'.

MACROSCOPIC EXAMINATIONS
Summary Data: see attached Tables 37, 38, 39
Individual Data: see attached Tables 82, 83, 84

At the scheduled F0 female necropsies, no test article-related internal findings were observed at any dosage level. Macroscopic findings observed in the test article-treated groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.
At the lactation day 4 necropsy, no test article-related effects were observed on the numbers of former implantation sites and unaccounted-for sites. The differences between the control and test article-treated groups were slight and not statistically significant.

MICROSCOPIC EXAMINATIONS
Summary Data: see attached Table 40
Individual Data: see attached Table 83
Pathology Report: see attached Appendix E

No test article-related findings were noted in the 600 mg/kg/day group females. Histologic changes were considered to be incidental findings, manifestations of spontaneous disease or related to some aspect of experimental manipulation other than exposure to the test article. Ultimobranchial cysts were noted in the thyroid gland of 6/12 and 4/12 females in the control and 600 mg/kg/day groups, respectively, which was the reverse of the trend observed in the males; there were no ultimobranchial cysts noted in the thyroid glands from the 50 and 175 mg/kg/day group males. There were no test article-related alterations in the incidence, severity or histologic character of these incidental and spontaneous tissue alterations.

REPRODUCTIVE PERFORMANCE
Summary Data: see attached Table 41
Individual Data: see attached Table 85
Historical Control Data: see attached Appendix F

No test article-related effects on F0 reproductive performance were observed at any dosage level. Female mating indices were 100.0% in all groups. Female fertility and conception indices were 100.0%, 91.7%, 100.0% and 100.0% in the control, 50, 175 and 600 mg/kg/day groups, respectively. No statistically significant differences were noted between the control and test article-treated groups. One female in the 50 mg/kg/day group had evidence of mating but failed to produce a litter. The mean numbers of days between pairing and coitus in the test article-treated groups were similar to the mean number in the concurrent control group. None of the differences were statistically significant, and the values in the test article-treated groups
were within the range of the WIL historical control data for reproductive studies.

GESTATION LENGTH AND PARTURITION
Summary Data: see attached Table 42
Individual Data: see attached Table 86
Historical Control Data: see attached Appendix F

Mean gestation lengths in the 50, 175 and 600 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Increased organ weights and microscopic changes in 600 mg/kg/day males

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Increased liver weights in 600 mg/kg/day females

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

F1 LITTER DATA

PND 0 LITTER DATA AND POSTNATAL SURVIVAL
Summary Data: see attached Tables 43, 44
Individual Data: see attached Tables 87, 88
Historical Control Data: see attached Appendix F

The mean number of pups born and the percentage of males at birth in the 50, 175 and 600 mg/kg/day groups were similar to the concurrent control group values; no statistically significant differences were noted. The mean live litter size in the 50 mg/kg/day group was also similar to the control group value. Although mean live litter sizes on PND 0 in the 175 and 600 mg/kg/day groups (14.5 and 14.2 pups per dam, respectively) were lower than the concurrent control group value (16.0 pups per dam), the concurrent control group value was higher than the mean value in the WIL historical control data (14.1 pups per dam). Therefore, the differences in mean live litter size on PND 0 between the control group and the 175 and 600 mg/kg/day groups were not attributed to the test article. Postnatal survival from birth to PND 4 in the test article-treated groups was unaffected by test article administration.

GENERAL PHYSICAL CONDITION AND MORTALITIES
Summary Data: see attached Table 45
Individual Data: see attached Table 89

The numbers of F1 pups found dead and/or missing, as well as the general physical condition of all F1 pups in this study were unaffected by test article administration. Pups (litters) that were found dead numbered 1(1), 2(2), 3(3) and 4(3) in the control, 50, 175 and 600 mg/kg/day groups, respectively. Two, 0, 3 and 1 pups in the same respective groups were missing and presumed to have been cannibalized.

OFFSPRING BODY WEIGHTS
Summary Data: see attached Tables 46, 47
Individual Data: see attached Tables 90, 91
Historical Control Data: see attached Appendix F

Mean male and female pup body weights and body weight changes in the 50, 175 and 600 mg/kg/day groups were unaffected by test article administration during PND 1-4. No statistically significant differences from the control group were noted.

NECROPSIES OF PUPS FOUND DEAD
Summary Data: see attached Table 48
Individual Data: see attached Table 92

The numbers of pups (litters) found dead from PND 0 through PND 4 numbered 1(1), 2(2), 3(3) and 4(3) in the control, 50, 175 and 600 mg/kg/day groups, respectively. No internal findings that could be attributed to F0 maternal administration of the test article were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Dose descriptor:

NOAEL

Remarks:

Neonatal toxicity

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Lack of effects on live litter size, postnatal survival and F1 body weights at any dosage level

Critical effects observed:

no

Reproductive effects observed:

no

ANATOMIC PATHOLOGY

ORGAN WEIGHTS

Males

Summary Data: see attached Tables 8, 9, 10

Individual Data: see attached Tables 56, 57, 58

Test article-related effects on organ weights were noted in the 50, 175 and 600 mg/kg/day group males and are summarised in the following table:

Text Table 4: Test Article-Related Effects on Organ Weights in Male Rats

	50 mg/kg/day	175 mg/kg/day	600 mg/kg/day
Thyroid gland	Increased (27.7%)(1)	Increased (30.8%)(1)	Increased (71.3%)(1)
Liver	NC	Increased (17.5%)(1)	Increased (27.8%)(1)
Kidney	NC	NC	Increased (15.3%)(1)

(1) = Change occurred for absolute, relative to final body weight and relative to brain weight;

% difference from control group is shown for absolute weight.

NC = No change

Test article-related higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights occurred in a dose-related manner in the 50, 175 and 600 mg/kg/day group males when compared to the control group; the differences were statistically significant (p<0.05 or p<0.01). In addition, test article-related higher mean absolute and relative (to final body weight and to brain weight) liver weights occurred in a dose-related manner in the 175 and 600 mg/kg/day group males, and test article-related higher mean absolute and relative (to final body weight and to brain weight) kidney weights occurred in the 600 mg/kg/day group males. The differences from the control group values were statistically significant (p<0.05 or p<0.01). These increases were considered test article-related. Based on the lack of correlating microscopic findings and the lack of toxicological effects on male reproduction, which suggested that the alterations in the glandular weights were not adverse, the increase in mean thyroid gland weights in the 175 mg/kg/day group males was not considered adverse.

No other test article-related differences in organ weights were noted. Statistically significantly (p<0.05) higher mean absolute and/or relative (to final body weight) right epididymis weights were observed in the 50 and 600 mg/kg/day group males. These increases were not attributed to the test article because there was no dose-response relationship, and a similar increase was not noted in the contralateral organ in these groups.

ORGAN WEIGHTS

Females

Summary Data: see attached Tables 31, 32, 33, 34, 35, 36

Individual Data: see attached Tables 76, 77, 78, 79, 80, 81

Test article-related effects on organ weights were noted in the 175 and 600 mg/kg/day group females. The findings are summarised in the following table:

Text Table 5: Test Article-Related Effects on Organ Weights in Female Rats

	50 mg/kg/day	175 mg/kg/day	600 mg/kg/day
Liver	NC	NC	Increased (34.4%)(1)
Kidney	NC	Increased (15.0%)(1)	Increased (21.4%)(1)

(1) = Change occurred for absolute, relative to final body weight and relative to brain weight;

% difference from control group is shown for absolute weight.

NC = No change

Conclusions:

CONCLUSIONS
Based on the results of this study, a dosage level of 600 mg/kg/day (the highest dosage level tested) appeared to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of thiophene when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this study, the NOAEL for male systemic toxicity was considered to be 175 mg/kg/day based on increased organ weights and microscopic findings in the 600 mg/kg/day group but lack of microscopic findings in the 175 mg/kg/day group. The NOAEL for female systemic toxicity was considered to be 175 mg/kg/day based on increased liver weight in the 600 mg/kg/day group. Based on the lack of effects on live litter size, postnatal survival and F1 body weights at any dosage level, the NOAEL for F1 neonatal toxicity was considered to be at least 600 mg/kg/day.

Executive summary:

Objective

This study was designed to investigate the potential adverse effects of the test article on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behaviour, conception, parturition and lactation of the F0 generation and the development of offspring from conception through day 4 of postnatal life.

Study design

The test article, thiophene, [3-(decyloxy) tetrahydro-1,1-dioxide], in the vehicle, corn oil,was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats, each group consisting of 12 males and 12 females. Dosage levels were 50, 175 and 600 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 12 rats/sex received the vehicle on a comparable regimen. Males and females received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia (for a total of 56 doses). Females were dosed through lactation day 3 for a total of 39-53 doses; the female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. 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 were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on postnatal days (PND) 1 and 4. Pups were euthanized and discarded without further examination on PND 4. F0 females were euthanized on lactation day 4. F0 males were euthanized following completion of the F0 female necropsies. 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 and high-dosage groups; thyroid glands were also examined from the F0 males in the 50 and 175 mg/kg/day groups.

Results

One male in the 600 mg/kg/day group was found dead on study day 32 due to complications from fractured nasal bones; the death was not attributed to the test article. All other animals survived to the scheduled necropsy. Salivation and clear material around the mouth were noted in the 175 and 600 mg/kg/day group males and females at the time of and/or 1-2 hours following dose administration. Salivation was observed on at least 1 occasion each in 9 and 4 males and females, respectively, in the 175 mg/kg/day group and in 12 and 11 males and females, respectively, in the 600 mg/kg/day group. Clear material around the mouth was also observed on at least 1 occasion each in 10 and 8 males and females, respectively, in the 175 mg/kg/day group and in all males and females in the 600 mg/kg/day group. The severity of salivation and clear material around the mouth was primarily slight or moderate in both groups. These findings were attributed to the test article, but were not considered adverse because the onset occurred immediately following dose administration; thus these findings were not considered indicative of systemic toxicity. Red material around the mouth (slight or moderate in severity) was also noted 1-2 hours following dose administration in 6 and 10 males and females, respectively, in the 600 mg/kg/day group. Slightly lower mean body weight gains were noted in the 600 mg/kg/day group males during the pre-mating period and when the entire treatment period was evaluated. The reductions in the males in this group were due to transient lower mean body weight gains during study days 7-13 and 21-27. However, the reductions in mean body weight gain were not of sufficient magnitude to result in substantially lower mean body weights in the 600 mg/kg/day group males; therefore, the lower mean body weight gains observed in these males was not considered adverse. No test article-related effects were observed on mean body weights or body weight gains during the pre-mating, gestation or lactation periods in the 50, 175 or 600 mg/kg/day group females. Food consumption in the males and females was not adversely affected by test article administration at any dosage level. Higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights were noted in the 50, 175 and 600 mg/kg/day group males. Potentially test article-related higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights were noted the 175 and 600 mg/kg/day group females. The increases occurred in a dose-related manner and were more pronounced in the males. However, the increases in the females and the 175 mg/kg/day group males were not considered adverse based on the lack of correlating microscopic findings in the 175 mg/kg/day group males and the 600 mg/kg/day group females. Mean absolute and relative (to final body weight and to brain weight) liver weights were higher in the 175 mg/kg/day group males and the 600 mg/kg/day group males and females. Mean absolute and relative (to final body weight and to brain weight) kidney weights were also higher in the 600 mg/kg/day group males. There were no test article-related macroscopic findings in the males and females at the scheduled necropsy. The mean numbers of former implantation sites and unaccounted-for sites in the test article-treated group females were similar to those in the control group. At the microscopic evaluation, follicular cell hypertrophy of the thyroid gland in the 600 mg/kg/day group males correlated with higher mean thyroid gland weights. No test article-related microscopic findings were noted in the thyroid glands of the 50 and 175 mg/kg/day group males. There were no test article-related microscopic findings observed in the 600 mg/kg/day group females. There were no test article-related effects on male and female reproductive performance. The numbers of days between pairing and coitus in the test article-treated groups were affected by test article administration. Mean gestation lengths in the 50, 175 and 600 mg/kg/day groups were similar to the control group value; there were no signs of dystocia in this study. The mean numbers of pups born, live litter size on PND 0, the percentage of males at birth and postnatal survival in the test article-treated groups were similar to those in the control group. The general physical condition of the F1 pups and mean F1 body weights were not affected by test article administration to the F0 parental animals. There were no macroscopic findings in the F1 pups that were found dead that could be attributed to F0 treatment with the test article.

Conclusion

Based on the results of this study, a dosage level of 600 mg/kg/day (the highest dosage level tested) appeared to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of thiophene when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this study, the NOAEL for male systemic toxicity was considered to be 175 mg/kg/day based on increased organ weights and microscopic findings in the 600 mg/kg/day group but lack of microscopic findings in the 175 mg/kg/day group. The NOAEL for female systemic toxicity was considered to be 175 mg/kg/day based on increased liver weight in the 600 mg/kg/day group. Based on the lack of effects on live litter size, postnatal survival and F1 body weights at any dosage level, the NOAEL for F1 neonatal toxicity was considered to be at least 600 mg/kg/day.

Reference 2

Endpoint:

two-generation reproductive toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Species:

rat

Quality of whole database:

One reliable study available, therefore quality of whole database is sufficient.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The test substance was administered orally by gavage once daily to three groups of Crl:CD(SD) each of 12 male and 12 female rats at 50, 175 and 600 mg/kg/day. A concurrent control group of 12 rats/sex received the vehicle, corn oil, on a comparable regimen. Males and females received 14 doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia (for a total of 56 doses). Females were dosed through lactation day 3 for a total of 39-53 doses; the female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. 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 were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on postnatal days (PND) 1 and 4. Pups were euthanized and discarded without further examination on PND 4. F0 females were euthanized on lactation day 4. F0 males were euthanized following completion of the F0 female necropsies. 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 and high-dosage groups; thyroid glands were also examined from the F0 males in the 50 and 175 mg/kg/day groups.

One 600 mg/kg/day male was found dead on study day 32 due to complications from fractured nasal bones; the death was not attributed to the test substance. All other animals survived to the scheduled necropsy. Salivation and clear material around the mouth were noted in 175 and 600 mg/kg/day males and females at the time of and/or 1-2 hours following dose administration. Salivation was observed on at least 1 occasion each in 9 males and 4 females in the 175 mg/kg/day group and in 12 males and 11 females in the 600 mg/kg/day group. Clear material around the mouth was also observed on at least 1 occasion each in 10 males and 8 females in the 175 mg/kg/day group and in all 600 mg/kg/day males and females. The severity of salivation and clear material around the mouth was primarily slight or moderate in both groups. These findings were attributed to the test substance, but were not considered adverse because the onset occurred immediately following dose administration; thus these findings were not considered indicative of systemic toxicity. Red material around the mouth (slight or moderate in severity) was also noted 1-2 hours following dose administration in 6 males and 10 females in the 600 mg/kg/day group. Slightly lower mean body weight gains were noted in the 600 mg/kg/day males during the pre-mating period and when the entire treatment period was evaluated. The reductions in the males in this group were due to transient lower mean body weight gains during study days 7-13 and 21-27. However, the reductions in mean body weight gain were not of sufficient magnitude to result in substantially lower mean body weights in 600 mg/kg/day males; therefore, the lower mean body weight gains observed in these males was not considered adverse. No treatment-related effects were observed on mean body weights or body weight gains during the pre-mating, gestation or lactation periods in 50, 175 or 600 mg/kg/day females. Food consumption in the males and females was not adversely affected by the test substance at any dosage level. Higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights were noted in 50, 175 and 600 mg/kg/day males. Potentially test substance-related higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights were noted in 175 and 600 mg/kg/day females. The increases occurred in a dose-related manner and were more pronounced in the males. However, the increases in females and 50 mg/kg/day males were not considered adverse based on the lack of correlating microscopic findings in 50 mg/kg/day males and 600 mg/kg/day females and no statistical significance. Mean absolute and relative (to final body weight and to brain weight) liver weights were higher in 175 mg/kg/day males and 600 mg/kg/day males and females. Mean absolute and relative (to final body weight and to brain weight) kidney weights were also higher in 600 mg/kg/day males. There were no treatment-related macroscopic findings in males and females at the scheduled necropsy. The mean numbers of former implantation sites and unaccounted for sites in the treated females were similar to those in the control group. At the microscopic evaluation, follicular cell hypertrophy of the thyroid gland in 600 mg/kg/day males correlated with higher mean thyroid gland weights. No treatment-related microscopic findings were noted in the thyroid glands of 50 and 175 mg/kg/day males. There were no treatment-related microscopic findings observed in the 600 mg/kg/day group females. There were no treatment-related effects on male and female reproductive performance. The numbers of days between pairing and coitus in the treated groups were affected by test substance administration. Mean gestation lengths in 50, 175 and 600 mg/kg/day groups were similar to the control group value and there were no signs of dystocia.

Based on the results of this study, a dosage level of 600 mg/kg/day appeared to be the NOAEL for reproductive toxicity of the test substance. Under the conditions of this study, the NOAEL for male systemic toxicity was considered to be 50 mg/kg/day based on increased organ weights in the 175 and 600 mg/kg/day groups and microscopic findings in the 600 mg/kg/day group (Appendix 1). The NOAEL for female systemic toxicity was considered to be 175 mg/kg/day based on increased liver weight in the 600 mg/kg/day group.

Short description of key information:
Fertility: NOAEL 600 mg/kg bw/day; study performed in line with OECD Guideline 421; Knapp (2006)

Systemic toxicity: NOAEL 175 mg/kg bw/day; study performed in line with OECD Guideline 421; Knapp (2006)

Effects on developmental toxicity

Description of key information

Key study

Developmental toxicity: NOAEL 1000 mg/kg bw/day; study performed in line with OECD Guideline 414 and OPPTS 870.3700; Herberth (2017)

Systemic toxicity: NOAEL 600 mg/kg bw/day; study performed in line with OECD Guideline 414 and OPPTS 870.3700; Herberth (2017)

Supporting study

Developmental toxicity: NOAEL 600 mg/kg bw/day; study performed in line with OECD Guideline 421; Knapp (2006)
Systemic toxicity: NOAEL 175 mg/kg bw/day; study performed in line with OECD Guideline 421; Knapp (2006)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

3 October 2005 to 7 September 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the studywas conducted under GLP conditions.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

ANIMAL RECEIPT AND ACCLIMATION
Fifty-five male and 55 female Crl:CD(SD) rats were received in good health from a reputable supplier on 22 November 2005. The animals were 59 days old upon receipt. Each animal was examined by a qualified technician on the day of receipt and weighed the following day. Each rat was uniquely identified by a Monel® metal eartag displaying the animal number. The animals were housed for an acclimation period of 13 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.

ANIMAL HOUSING
Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed 3 times per week. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding. The nesting material is periodically analysed by the manufacturer for contaminants. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The dams and their litters were housed in these cages until euthanasia on lactation day 4. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996).

DIET, DRINKING WATER AND MAINTENANCE
The basal diet used in this study, PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002, is a certified feed with appropriate analyses performed by the manufacturer. Feed lots used during the study are documented in the study records. Feeders were changed and sanitized once per week. Municipal water supplying the facility is sampled for contaminants according to standard operating procedures. The results of the diet and water analyses are maintained at WIL Research Laboratories, LLC. 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.

ENVIRONMENTAL CONDITIONS
All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and humidity controls were set to maintain daily averages of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20% relative humidity. Room temperature and relative humidity were controlled and monitored using the Metasys® DDC Electronic Environmental control system. These data were recorded approximately hourly and are summarised in attached Appendix D. Actual mean daily temperature ranged from 70.3°F to 71.2°F (21.3°C to 21.8°C) and mean daily relative humidity ranged from 30.2% to 43.2% during the study. Light timers were calibrated to provide a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Route of administration:

oral: gavage

Type of inhalation exposure (if applicable):

other: Not applicable

Vehicle:

corn oil

Details on exposure:

The vehicle and test article formulations were administered orally by gastric intubation, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan) once daily. The males were dosed during study days 0-55 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 56 doses. The females were dosed from study day 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-53 doses. The female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. The dosage 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the control group (Group 1), a sufficient amount of corn oil was added to a glass container. The vehicle was stirred continuously throughout sampling and dispensation Aliquots of the vehicle were prepared for daily dispensation and stored at room temperature. The vehicle was stirred continuously throughout dose administration. The appropriate amount of the test article for each group was weighed into a tared, calibrated container. Vehicle was added to each container to bring the formulations nearly to the calibration mark. The formulations were mixed until uniform using a magnetic stirrer. Vehicle was added to each container to bring the formulations to the calibration mark, and the formulations were stirred until uniform using a magnetic stirrer. The test article formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation and stored at room temperature. The test article formulations were stirred continuously throughout the preparation, sampling and dose administration procedures.

SAMPLING AND ANALYSES

Prior to the initiation of dose administration, duplicate samples (1 mL each) for homogeneity determination were collected on 21 November 2005 from the top, middle and bottom strata of each dosing formulation, including the vehicle for the control group. In addition, duplicate samples (1 mL each) for stability and resuspension homogeneity determinations were collected on 1 December 2005 from the top and bottom strata of aliquots of these same dosing suspensions following storage at room temperature for 10 days; the aliquots were mixed using a magnetic stirrer for a minimum of 10 minutes prior to sample collection. Samples (1 mL each) for concentration analysis were collected weekly from the middle stratum of each dosing formulation (including the vehicle for the control group); only the samples from the first, fourth and last weekly formulations were analyzed. All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, LLC. The methodology and results of these analyses are presented in attached Appendix B

Details on mating procedure:

BREEDING PROCEDURES
The animals 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 prepared. Each female was housed in the home cage of the male. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages. 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.

PARTURITION
All females were allowed to deliver naturally and rear their young to PND 4. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.

Duration of treatment / exposure:

approximately 8 weeks

Frequency of treatment:

Daily

Duration of test:

approximately 8 weeks

No. of animals per sex per dose:

12 animals per sex per dose level including control

Control animals:

yes, concurrent vehicle

Details on study design:

Dosage levels were selected based on the results of previous studies and were provided by the sponsor representative after consultation with the study director.

Maternal examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual detailed clinical observations were recorded weekly (prior to test article administration during the treatment period). Each male and female was also observed for signs of toxicity at the time of dose administration and approximately 1-2 hours following dose administration. All significant findings were recorded.

BODY WEIGHTS
Individual male body weights were recorded weekly throughout the study and prior to the scheduled euthanasia. Individual female body weights were recorded weekly until evidence of copulation was observed. Mean weekly body weights and body weight changes are presented for each interval. In addition, cumulative mean body weight changes are presented for the pre-mating period (males and females) and for the entire generation (males only). Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4. Mean gestation body weights and corresponding mean body weight changes are presented for these intervals and for the overall gestation interval (days 0-20). For individual data, the time periods a given animal was not weighed were designated as “NA” (Not Applicable).

FOOD CONSUMPTION
Individual food consumption was recorded weekly until pairing. Food intake was not recorded during the mating period. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 1 and 4. Following mating, food consumption for all males was measured on a weekly basis until the scheduled euthanasia. Food consumption was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. Calculation of the comprehensive intervals excludes all erroneous values such as total
food spillage. When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, obvious erroneous value, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were designated as “NA” (Not Applicable) on the individual report tables.

MACROSCOPIC EXAMINATIONS

UNSCHEDULED DEATHS
A complete necropsy was performed on the male that was found dead. This included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain and the thoracic, abdominal and pelvic cavities, including viscera.

SCHEDULED EUTHANASIA
All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the female necropsies. Females that delivered were euthanized on lactation day 4; the number of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on post-mating day 25 (females with evidence of mating). Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964). Necropsy included examination of the external surface, all orifices and the cranial cavity, external surfaces of the brain, and the thoracic, abdominal and pelvic cavities, including viscera. At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin (except as noted):

Cervix
Testes with epididymides (a) and vas deferens (2)
Coagulating glands
Mammary gland
Thyroids [with parathyroids if present (2)]
Ovaries and oviducts (2)
Pituitary gland
Uterus (b) with vagina
Prostate gland
All gross lesions (c)
Seminal vesicles (2)

(a) = Testes and epididymides fixed in Bouin’s solution.
(b) = Not retained if placed in ammonium sulfide solution.
(c) = Representative sections of corresponding organs from a sufficient number of controls were retained for comparison, if possible.

ORGAN WEIGHTS
The following organs were weighed from all F0 animals at the scheduled necropsies:
Brain Ovaries with oviducts
Epididymides (a)
Pituitary gland
Kidneys
Testes (a)
Liver
Thyroids with parathyroids (b)

(a) = These paired organs were weighed separately.
(b) = Fixed in 10% neutral-buffered formalin prior to weighing
Except as noted, paired organs were weighed together. Absolute weights, organ to final body weight and organ to brain weight ratios were reported.

MICROSCOPIC EXAMINATIONS
After fixation, protocol-specified tissues were trimmed according to standard operating procedures and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides and stained with hematoxylin and eosin, with the following exceptions. PAS and hematoxylin staining were used for the right and left testes and epididymides. The testes were fixed in Bouin’s solution and embedded in paraffin. Sections of 2 to 4 microns were made for the testis (transverse) and the epididymis (longitudinal). Special emphasis was placed on the stages of spermatogenesis and histopathology of interstitial testicular cell structure. Microscopic examination was performed on all tissues from the male that was found dead and all animals in the control and 600 mg/kg/day groups at the scheduled necropsies; gross lesions from all dosage groups were also examined. Because of test article-related findings in the 600 mg/kg/day group males, the thyroid glands were also examined from all males in the 50 and 175 mg/kg/day groups. Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section or other reasons as appropriate.

Ovaries and uterine content:

Females that delivered were euthanized on lactation day 4; the number of former implantation sites and corpora lutea were recorded. Females that failed to deliver were euthanized on post-mating day 25 (females with evidence of mating). Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).

Fetal examinations:

F1 LITTER PARAMETERS

LITTER VIABILITY AND DEATHS
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. Standardisation of litter size was not performed because the pups were euthanized on PND 4. Intact offspring dying were necropsied using a fresh dissection technique including the heart and major vessels (Stuckhardt and Poppe, 1984). The carcass of each pup was then discarded.

CLINICAL OBSERVATIONS
Litters were examined daily for survival and any adverse changes in appearance or behavior. Each pup received a detailed physical examination on PND 1 and 4. Any abnormalities in nursing behavior were recorded.

BODY WEIGHTS
Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dosage group.

SEX DETERMINATION
Pups were individually sexed on PND 0 and 4.

SCHEDULED EUTHANASIA
On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital. Gross lesions and malformations (if any) observed at the detailed physical examinations on PND 4 were preserved in the appropriate fixative for possible future examination.

Statistics:

STATISTICAL ANALYSES
All statistical tests were performed using appropriate computing devices or programs. For full details please see 'Any other information on materials and methods incl. tables'.

Indices:

Mating, fertility and copulation/conception indices were calculated as follows:

Male (female) mating index (%) = (No. of Males (Females) with Evidence of Mating Male (Female) (or Confirmed Pregnancy) / Total No. of Males (Females) Used for Mating) x 100

Male Fertility Index (%) = (No. of Males Siring a Litter / Total No. of Males Used for Mating) x100

Male Copulation Index (%) = (No. of Males Siring a Litter / No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy)) x 100

Female Fertility Index (%) = (No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating) x 100

Female Conception Index (%) = (No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or Confirmed Pregnancy)) x 100

Litter parameters were defined as follows:

Mean Live Litter Size = Total No. of Viable Pups on PND 0 / No. of Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter) = Σ ((Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter) / No. of Litters Per Group) x100

Postnatal Survival for All
Other Intervals (% Per Litter) = Σ ((Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N) / No. of Litters Per Group) x 100

Where N = PND 0-1 and 1-4

Historical control data:

Not applicable.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
F0 GENERATION

MALES

CLINICAL OBSERVATIONS AND SURVIVAL
Summary Data: see attached Tables 1, 2, 3
Individual Data: see attached Tables 49, 50, 51

Test article-related clinical findings were noted in the 175 and 600 mg/kg/day group males at the time of and 1-2 hours following dose administration. Male no. 11517 in the 600 mg/kg/day group was found dead on study day 32. During the week prior to death, this male had an unkempt appearance, rales and red material around the eyes, nose and mouth, lower body weight gain (relative to its gain during the first week of dose administration) during study days 7-21 and a body weight loss during study days 21-27. Fractured nasal bones were noted at necropsy for this animal. The death of male no. 11517 was attributed to complications from the fractures (presumed to be the result of a mechanical injury) and not to the test article. All other males survived to the scheduled necropsy. No test article-related clinical findings were observed in the males at the weekly detailed physical examinations. Test article-related increased incidences of salivation or evidence thereof (clear material around the mouth) were noted in the 175 and 600 mg/kg/day males at the time of and/or 1-2 hours following dose administration; the severity of these findings was primarily slight or moderate. Salivation was noted in a total of 9 and 12 males in the 175 and 600 mg/kg/day groups, respectively; the onset of this finding occurred as early as study day 15 in the 175 mg/kg/day group and study day 2 in the 600 mg/kg/day group. Clear material around the mouth was first observed on study day 15 and study day 3 in a total of 10 and 12 males in the same respective groups. These findings were generally noted throughout the study. However, because the onset of salivation and clear material around the mouth occurred immediately following daily dose administration, these findings were not considered indicative of systemic toxicity. Red material around the mouth (slight or moderate) was also noted for 6 males in the 600 mg/kg/day group 1-2 hours following dose administration beginning on study day 15 and continued sporadically throughout the study. This finding was considered test article-related. Other clinical findings, including hair loss, red material and/or yellow material on various body surfaces, were observed infrequently, were noted similarly in the control group and/or did not occur in a dose-related manner.

BODY WEIGHTS
Summary Data: see attached Tables 4, 5
Individual Data: see attached Tables 52, 53

Transient, test article-related lower mean body weight gains were noted in the 600 mg/kg/day group males relative to the control group. The reductions were not of sufficient magnitude to be considered adverse. Test article-related lower mean body weight gain was noted in the 600 mg/kg/day group males during the pre-mating period (study days 0-13) due to lower mean body weight gain observed study days 7-13; the difference from the control group was statistically significant (p<0.05) during study days 7-13. Mean body weight gain in this group was similar to that in the control group during the mating and post-mating periods, with the following exception. Lower (not statistically significant) mean body weight gain in this group was noted during study days 21-27 compared to the control group value. The reductions observed in the 600 mg/kg/day group resulted in a slightly lower mean body weight gain when the entire generation (study days 0-56) was evaluated; the difference from the control group was not statistically significant. However, because the reductions were transient and not of sufficient magnitude to result in substantially lower mean body weights, the lower mean body weight gains in this group were not considered adverse. Mean body weights and body weight gains in the 50 and 175 mg/kg/day group males were unaffected by test article administration. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION
Summary Data: see attached Tables 6, 7
Individual Data: see attached Tables 54, 55

Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 175 and 600 mg/kg/day group males was unaffected by test article administration. The values in the test article-treated groups were generally similar to the control group values throughout the treatment period. None of the differences from the control group were statistically significant.

ANATOMIC PATHOLOGY
For organ weight results please see 'Any other information on results incl. tables section'.

MACROSCOPIC EXAMINATIONS
Summary Data: see attached Tables 11, 12
Individual Data: see attached Tables 59, 60

Male no. 11517 in the 600 mg/kg/day group was found dead on study day 32. Macroscopic findings for this male consisted of fractured nasal bones and dark red contents in the stomach, duodenum, jejunum, ileum and cecum. This death was attributed to complications from the fractures (presumed to be a mechanical injury) and not to the test article. At the scheduled F0 male necropsies, no test article-related internal findings were observed at any dosage level. Macroscopic findings observed in the test article-treated groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.

MICROSCOPIC EXAMINATIONS
Summary Data: see attached Tables 13, 14
Individual Data: see attached Tables 59, 60
Pathology Report: see attached Appendix E

Male no. 11517 in the 600 mg/kg/day group was found dead on study day 32. No test article-related microscopic findings were noted in the organs that were examined for this male. Microscopic findings in the bone, related to the macroscopic finding (fracture), consisted of fracture, hemorrhage, inflammation and fibrosis. A cause of death could not be determined. Test article-related microscopic findings were observed in the thyroid glands of the 600 mg/kg/day group males. Follicular cell hypertrophy was noted in 9 of 11 males in the 600 mg/kg/day group compared to no males in the control group; the difference was statistically significant (p<0.05). This finding was characterized by an increase in cell size, cytoplasm and basophilia. Occasionally, follicular cell hyperplasia was observed as well, and colloid depletion was identified in a single male in this group. These findings correlated with higher mean thyroid gland weights in the 600 mg/kg/day group males. Thyroid gland alterations were below the threshold of detection by routine light microscopy in the 50 and 175 mg/kg/day group males. The only other statistically significant (p<0.05) difference from the control group was an increase in the incidence of ultimobranchial cysts in the thyroid gland in the 600 mg/kg/day group males (10/11 males affected versus 2/12 males in the control group). However, ultimobranchial cysts are common congenital anomalies of the thyroid gland arising during early development of the organ (Hardisty and Boorman, 1990). Because the rats in this study were adults at the time of test article exposure, this finding was considered to be spurious rather than test article-related. In addition, there were no ultimobranchial cysts noted in the thyroid glands from the 50 and 175 mg/kg/day group males. No other test article-related findings were noted in the males in the 50, 175 and 600 mg/kg/day group. Remaining histologic changes were considered to be incidental findings, manifestations of spontaneous disease or related to some aspect of experimental manipulation other than exposure to the test article. There were no test article-related alterations in the incidence, severity or histologic character of these incidental and spontaneous tissue alterations.

REPRODUCTIVE PERFORMANCE
Summary Data: see attached Table 15
Individual Data: see attached Table 85
Historical Control Data: see attached Appendix F

No test article-related effects on F0 reproductive performance were observed at any dosage level. Male mating indices were 100.0% in all groups. Male fertility and copulation indices were 100.0%, 91.7%, 100.0% and 100.0% in the control, 50, 175 and 600 mg/kg/day groups, respectively. No statistically significant differences were noted between the control and test article-treated groups. One male in the 50 mg/kg/day group had evidence of mating but did not sire a litter.

FEMALES

CLINICAL OBSERVATIONS AND SURVIVAL
Summary Data: see attached Tables 16, 17, 18
Individual Data: see attached Tables 61, 62, 63

Test article-related clinical findings were noted in the 175 and 600 mg/kg/day group females. However, the findings were not considered adverse.
No test article-related clinical findings were observed in the females at the weekly detailed physical examinations. Test article-related increased incidences of salivation or evidence thereof (clear material around the mouth) were noted in the 175 and 600 mg/kg/day group females at the time of and/or 1-2 hours following dose administration. Salivation was noted in a total of 4 and 11 females, and clear material around the mouth was observed in a total of 8 and 12 females in the 175 and 600 mg/kg/day groups, respectively. The severity of these findings was primarily slight or moderate. A slight increase in the incidence of red material around the mouth (slight) was observed in a total of 8 females in the 600 mg/kg/day group 1 hour following dose administration. This finding was noted as early as study day 4, and was considered test article-related based on the occurrence in the 600 mg/kg/day group males. Salivation was first noted on study day 16 and study day 7 in the 175 and 600 mg/kg/day groups, respectively, and clear material around the mouth was noted beginning on study day 12 and study day 1 in the same respective groups. However, because the onset of these findings was immediately following dose administration, salivation and clear material around the mouth were not considered indicative of systemic toxicity. Other clinical findings, including hair loss on various body surfaces and/or red material around the nose, were observed infrequently, were noted similarly in the control group and/or did not occur in a dose-related manner.

PRE-MATING PERIOD

BODY WEIGHTS
Summary Data: see attached Tables 19, 20
Individual Data: see attached Tables 64, 65

There were no test-article-related effects on mean female body weights or body weight gains at any dosage level during the pre-mating period. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION
Summary Data: see attached Tables 21, 22
Individual Data: see attached Tables 66, 67

Mean food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 175 and 600 mg/kg/day group females was unaffected by test article administration during the pre-mating period. None of the differences from the control group were statistically significant.

GESTATION PERIOD

BODY WEIGHTS
Summary Data: see attached Tables 23, 24
Individual Data: see attached Tables 68, 69

No test article-related effects on mean maternal body weights or body weight gains were noted during gestation at any dosage level. Differences from the control group were slight and not statistically significant.

FOOD CONSUMPTION
Summary Data: see attached Tables 25, 26
Individual Data: see attached Tables 70, 71

Higher mean maternal food consumption, (primarily g/animal/day) was noted in the 600 mg/kg/day group females during gestation days 0-4, 4-7 and 11-14, resulting in higher mean food consumption when the entire gestation period (gestation days 0-20) was evaluated. The differences from the control group were statistically significant (p<0.05 or p<0.01). The increases in food consumption in this group were considered test article-related, but not adverse, because the increases were slight (2-3 g/animal/day) compared to the control group, and increases in food consumption of this magnitude are not considered adverse. No test article-related effects on food consumption were noted in the 50 and 175 mg/kg/day groups during gestation. No statistically significant differences from the control group were observed.

LACTATION PERIOD

BODY WEIGHTS
Summary Data: see attached Tables 27, 28
Individual Data: see attached Tables 72, 73

Mean body weights and body weight gains in the 50, 175 and 600 mg/kg/day groups were similar to the control group values during lactation days 1-4. No statistically significant differences were noted.

FOOD CONSUMPTION
Summary Data: see attached Tables 29, 30
Individual Data: see attached Tables 74, 75

Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 50, 175 and 600 mg/kg/day group females was unaffected by test article administration during the early lactation period. The values in the test article-treated groups were similar to the control group values throughout the lactation period. No statistically significant differences were observed.

ANATOMIC PATHOLOGY
For organ weight results please see 'Any other information on results incl. tables'.

MACROSCOPIC EXAMINATIONS
Summary Data: see attached Tables 37, 38, 39
Individual Data: see attached Tables 82, 83, 84

At the scheduled F0 female necropsies, no test article-related internal findings were observed at any dosage level. Macroscopic findings observed in the test article-treated groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.
At the lactation day 4 necropsy, no test article-related effects were observed on the numbers of former implantation sites and unaccounted-for sites. The differences between the control and test article-treated groups were slight and not statistically significant.

MICROSCOPIC EXAMINATIONS
Summary Data: see attached Table 40
Individual Data: see attached Table 83
Pathology Report: see attached Appendix E

No test article-related findings were noted in the 600 mg/kg/day group females. Histologic changes were considered to be incidental findings, manifestations of spontaneous disease or related to some aspect of experimental manipulation other than exposure to the test article. Ultimobranchial cysts were noted in the thyroid gland of 6/12 and 4/12 females in the control and 600 mg/kg/day groups, respectively, which was the reverse of the trend observed in the males; there were no ultimobranchial cysts noted in the thyroid glands from other female groups. There were no test article-related alterations in the incidence, severity or histologic character of these incidental and spontaneous tissue alterations.

REPRODUCTIVE PERFORMANCE
Summary Data: see attached Table 41
Individual Data: see attached Table 85
Historical Control Data: see attached Appendix F

No test article-related effects on F0 reproductive performance were observed at any dosage level. Female mating indices were 100.0% in all groups. Female fertility and conception indices were 100.0%, 91.7%, 100.0% and 100.0% in the control, 50, 175 and 600 mg/kg/day groups, respectively. No statistically significant differences were noted between the control and test article-treated groups. One female in the 50 mg/kg/day group had evidence of mating but failed to produce a litter. The mean numbers of days between pairing and coitus in the test article-treated groups were similar to the mean number in the concurrent control group. None of the differences were statistically significant, and the values in the test article-treated groups were within the range of the WIL historical control data for reproductive studies.

GESTATION LENGTH AND PARTURITION
Summary Data: see attached Table 42
Individual Data: see attached Table 86
Historical Control Data: see attached Appendix F

Mean gestation lengths in the 50, 175 and 600 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted in these groups.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: other:

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: other:

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: other:

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects. Remark: "(under the conditions of the test, number of corpora lutea, implantation rate, postimplantation loss, litter size at first littercheck, body weights of pups or results of external examination gave no indication of embryotoxic or teratogenic effects

Details on embryotoxic / teratogenic effects:
F1 LITTER DATA

PND 0 LITTER DATA AND POSTNATAL SURVIVAL
Summary Data: see attached Tables 43, 44
Individual Data: see attached Tables 87, 88
Historical Control Data: see attached Appendix F

The mean number of pups born and the percentage of males at birth in the 50, 175 and 600 mg/kg/day groups were similar to the concurrent control group values; no statistically significant differences were noted. The mean live litter size in the 50 mg/kg/day group was also similar to the control group value. Although mean live litter sizes on PND 0 in the 175 and 600 mg/kg/day groups (14.5 and 14.2 pups per dam, respectively) were lower than the concurrent control group value (16.0 pups per dam), the concurrent control group value was higher than the mean value in the WIL historical control data (14.1 pups per dam). Therefore, the differences in mean live litter size on PND 0 between the control group and the 175 and 600 mg/kg/day groups were not attributed to the test article. Postnatal survival from birth to PND 4 in the test article-treated groups was unaffected by test article administration.

GENERAL PHYSICAL CONDITION AND MORTALITIES
Summary Data: see attached Table 45
Individual Data: see attached Table 89

The numbers of F1 pups found dead and/or missing, as well as the general physical condition of all F1 pups in this study were unaffected by test article administration. Pups (litters) that were found dead numbered 1(1), 2(2), 3(3) and 4(3) in the control, 50, 175 and 600 mg/kg/day groups, respectively. Two, 0, 3 and 1 pups in the same respective groups were missing and presumed to have been cannibalized.

OFFSPRING BODY WEIGHTS
Summary Data: see attached Tables 46, 47
Individual Data: see attached Tables 90, 91
Historical Control Data: see attached Appendix F

Mean male and female pup body weights and body weight changes in the 50, 175 and 600 mg/kg/day groups were unaffected by test article administration during PND 1-4. No statistically significant differences from the control group were noted.

NECROPSIES OF PUPS FOUND DEAD
Summary Data: see attached Table 48
Individual Data: see attached Table 92

The numbers of pups (litters) found dead from PND 0 through PND 4 numbered 1(1), 2(2), 3(3) and 4(3) in the control, 50, 175 and 600 mg/kg/day groups, respectively. No internal findings that could be attributed to F0 maternal administration of the test article were noted at the necropsies of pups that were found dead. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Lack of effects on live litter size, postnatal survival and F1 body weights at any dosage level

Abnormalities:

not specified

Developmental effects observed:

no

Anatomic Pathology

Organ weights

Males

Summary Data: see attached Tables 8, 9, 10

Individual Data: see attached Tables 56, 57, 58

Test article-related effects on organ weights were noted in the 50, 175 and 600 mg/kg/day group males and are summarised in the following table:

Text Table 4: Test Article-Related Effects on Organ Weights in Male Rats

	50 mg/kg/day	175 mg/kg/day	600 mg/kg/day
Thyroid gland	Increased (27.7%)(1)	Increased (30.8%)(1)	Increased (71.3%)(1)
Liver	NC	Increased (17.5%)(1)	Increased (27.8%)(1)
Kidney	NC	NC	Increased (15.3%)(1)

(1) = Change occurred for absolute, relative to final body weight and relative to brain weight; % difference from control group is shown for absolute weight.

NC = No change

Test article-related higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights occurred in a dose-related manner in the 50, 175 and 600 mg/kg/day group males when compared to the control group; the differences were statistically significant (p<0.05 or p<0.01). In addition, test article-related higher mean absolute and relative (to final body weight and to brain weight) liver weights occurred in a dose-related manner in the 175 and 600 mg/kg/day group males, and test article-related higher mean absolute and relative (to final body weight and to brain weight) kidney weights occurred in the 600 mg/kg/day group males. The differences from the control group values were statistically significant (p<0.05 or p<0.01). These increases were considered test article-related. Based on the lack of correlating microscopic findings and statistical significance, the increase in mean thyroid gland weights in the 50 mg/kg/day group males was not considered adverse.

No other test article-related differences in organ weights were noted. Statistically significantly (p<0.05) higher mean absolute and/or relative (to final body weight) right epididymis weights were observed in the 50 and 600 mg/kg/day group males. These increases were not attributed to the test article because there was no dose-response relationship, and a similar increase was not noted in the contralateral organ in these groups.

Organ weights

Females

Summary Data: see attached Tables 31, 32, 33, 34, 35, 36

Individual Data: see attached Tables 76, 77, 78, 79, 80, 81

Test article-related effects on organ weights were noted in the 175 and 600 mg/kg/day group females. The findings are summarised in the following table:

Text Table 5: Test Article-Related Effects on Organ Weights in Female Rats

	50 mg/kg/day	175 mg/kg/day	600 mg/kg/day
Liver	NC	NC	Increased (34.4%)(1)
Kidney	NC	Increased (15.0%)(1)	Increased (21.4%)(1)

(1) = Change occurred for absolute, relative to final body weight and relative to brain weight; % difference from control group is shown for absolute weight.

NC = No change

Conclusions:

CONCLUSIONS
Based on the results of this study, a dosage level of 600 mg/kg/day (the highest dosage level tested) appeared to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of thiophene when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this study, the NOAEL for male systemic toxicity was considered to be 50 mg/kg/day based on increased organ weights in the 175 and 600 mg/kg/day groups and microscopic findings in the 600 mg/kg/day group. The NOAEL for female systemic toxicity was considered to be 175 mg/kg/day based on increased liver weight in the 600 mg/kg/day group. Based on the lack of effects on live litter size, postnatal survival and F1 body weights at any dosage level, the NOAEL for F1 neonatal toxicity was considered to be at least 600 mg/kg/day.

Executive summary:

Objective

This study was designed to investigate the potential adverse effects of the test article on male and female reproduction within the scope of a screening study. This encompassed gonadal function, mating behaviour, conception, parturition and lactation of the F0 generation and the development of offspring from conception through day 4 of postnatal life.

Study design

The test article, thiophene, [3-(decyloxy) tetrahydro-1, 1-dioxide], in the vehicle, corn oil was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats, each group consisting of 12 males and 12 females. Dosage levels were 50, 175 and 600 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 12 rats/sex received the vehicle on a comparable regimen. Males and females received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia (for a total of 56 doses). Females were dosed through lactation day 3 for a total of 39-53 doses; the female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. 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 were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on postnatal days (PND) 1 and 4. Pups were euthanized and discarded without further examination on PND 4. F0 females were euthanized on lactation day 4. F0 males were euthanized following completion of the F0 female necropsies. 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 and high-dosage groups; thyroid glands were also examined from the F0 males in the 50 and 175 mg/kg/day groups.

Results

One male in the 600 mg/kg/day group was found dead on study day 32 due to complications from fractured nasal bones; the death was not attributed to the test article. All other animals survived to the scheduled necropsy. Salivation and clear material around the mouth were noted in the 175 and 600 mg/kg/day group males and females at the time of and/or 1-2 hours following dose administration. Salivation was observed on at least 1 occasion each in 9 and 4 males and females, respectively, in the 175 mg/kg/day group and in 12 and 11 males and females, respectively, in the 600 mg/kg/day group. Clear material around the mouth was also observed on at least 1 occasion each in 10 and 8 males and females, respectively, in the 175 mg/kg/day group and in all males and females in the 600 mg/kg/day group. The severity of salivation and clear material around the mouth was primarily slight or moderate in both groups. These findings were attributed to the test article, but were not considered adverse because the onset occurred immediately following dose administration; thus these findings were not considered indicative of systemic toxicity. Red material around the mouth (slight or moderate in severity) was also noted 1-2 hours following dose administration in 6 and 10 males and females, respectively, in the 600 mg/kg/day group. Slightly lower mean body weight gains were noted in the 600 mg/kg/day group males during the pre-mating period and when the entire treatment period was evaluated. The reductions in the males in this group were due to transient lower mean body weight gains during study days 7-13 and 21-27. However, the reductions in mean body weight gain were not of sufficient magnitude to result in substantially lower mean body weights in the 600 mg/kg/day group males; therefore, the lower mean body weight gains observed in these males was not considered adverse. No test article-related effects were observed on mean body weights or body weight gains during the pre-mating, gestation or lactation periods in the 50, 175 or 600 mg/kg/day group females. Food consumption in the males and females was not adversely affected by test article administration at any dosage level. Higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights were noted in the 50, 175 and 600 mg/kg/day group males. Potentially test article-related higher mean absolute and relative (to final body weight and to brain weight) thyroid gland weights were noted the 175 and 600 mg/kg/day group females. The increases occurred in a dose-related manner and were more pronounced in the males. However, the increases in the females and the 50 mg/kg/day group males were not considered adverse based on the lack of correlating microscopic findings in the 50 mg/kg/day group males and the 600 mg/kg/day group females and no statistical significance. Mean absolute and relative (to final body weight and to brain weight) liver weights were higher in the 175 mg/kg/day group males and the 600 mg/kg/day group males and females. Mean absolute and relative (to final body weight and to brain weight) kidney weights were also higher in the 600 mg/kg/day group males. There were no test article-related macroscopic findings in the males and females at the scheduled necropsy. The mean numbers of former implantation sites and unaccounted-for sites in the test article-treated group females were similar to those in the control group. At the microscopic evaluation, follicular cell hypertrophy of the thyroid gland in the 600 mg/kg/day group males correlated with higher mean thyroid gland weights. No test article-related microscopic findings were noted in the thyroid glands of the 50 and 175 mg/kg/day group males. There were no test article-related microscopic findings observed in the 600 mg/kg/day group females. There were no test article-related effects on male and female reproductive performance. The numbers of days between pairing and coitus in the test article-treated groups were affected by test article administration. Mean gestation lengths in the 50, 175 and 600 mg/kg/day groups were similar to the control group value; there were no signs of dystocia in this study. The mean numbers of pups born, live litter size on PND 0, the percentage of males at birth and postnatal survival in the test article-treated groups were similar to those in the control group. The general physical condition of the F1 pups and mean F1 body weights were not affected by test article administration to the F0 parental animals. There were no macroscopic findings in the F1 pups that were found dead that could be attributed to F0 treatment with the test article.

Conclusion

Based on the results of this study, a dosage level of 600 mg/kg/day (the highest dosage level tested) appeared to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of thiophene when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this study, the NOAEL for male systemic toxicity was considered to be 50 mg/kg/day based on increased organ weights in the 175 and 600 mg/kg/day groups and microscopic findings in the 600 mg/kg/day group. The NOAEL for female systemic toxicity was considered to be 175 mg/kg/day based on increased liver weight in the 600 mg/kg/day group. Based on the lack of effects on live litter size, postnatal survival and F1 body weights at any dosage level, the NOAEL for F1 neonatal toxicity was considered to be at least 600 mg/kg/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 November 2016 to 25 January 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

minor deviation to planned post-mortem and pathology procedure with no impact on study results or integrity (see below)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

minor deviation to planned post-mortem and pathology procedure with no impact on study results or integrity (see below)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION
- Sexually mature, virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. This species and strain of animal is recognized as appropriate for developmental toxicity studies. Charles River Ashland has historical control data on the background incidence of fetal malformations and developmental variations in the Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants.
- The number of animals selected for this study (25 females/group) was based on the United States EPA Health Effects Test Guidelines OPPTS 870.3700, Prenatal Development Toxicity Study, August 1998 and the OECD Guidelines for the Testing of Chemicals: Guideline 414, Prenatal Developmental Toxicity Study, January 2001, which recommend evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 20 at termination.
- Sexually mature, virgin female Crl:CD(SD) rats (150 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC, on 01 Nov 2016.
- The animals were approximately 69 days old upon receipt. Each female was examined by a qualified biologist on the day of receipt.
- On 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 a minimum of 14 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for mortality and changes in general appearance and behaviour.

ANIMAL HOUSING
- Upon arrival, all rats were housed 2 animals/cage in clean, solid-bottom cages with bedding material (Bed-O'Cobs; The Andersons, Cob Products Division, Maumee, OH). The bedding material is periodically analysed 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 Charles River.
- The rats were paired for mating in the home cage of the male.
- Following positive evidence of mating, the females were individually housed in clean, solid-bottom cages with bedding material. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals. The animal facilities at Charles River Ashland are fully 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.

DIET, DRINKING WATER AND MAINTENANCE
- 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 Charles River. 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 Charles River SOPs. The results of the diet and water analyses are maintained at Charles River.
- 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.

ENVIRONMENTAL CONDITIONS
- 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 68 °F to 78 °F (20 °C to 26 °C) and 30% to 70%, respectively.
- Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis.
- Actual mean daily temperature ranged from 71.7 °F to 73.3 °F (22.1 °C to 22.9 °C) and mean daily relative humidity ranged from 40.2% to 52.0% 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.

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

Lot number 1FE0684; Expiry date 31 May 2017

Details on exposure:

PREPARATION
- 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 preparation, sampling, and dose administration procedures.
- Dosing formulations were prepared at the test substance concentrations indicated in the table below.
- 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 and were found to be visibly homogeneous and acceptable for administration.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS AND BREEDING PROCEDURES
- At the conclusion of the acclimation period, all available 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 and meeting acceptable body weight requirements was placed in a solid-bottom cage with bedding material with a resident male from the same strain and source for breeding.
- Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females.
- A breeding record containing the male and female identification numbers and the dates of cohabitation was maintained.
- The selected females were approximately 12 weeks old when paired for breeding.
- Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist.
- Each mating pair was examined daily. The day on which evidence of mating was identified was termed Gestation Day 0 and the animals were separated.
- The experimental design consisted of 4 test substance-treated groups and 1 control group, composed of 25 rats/group.
- The bred females were assigned to groups using a WTDMS computer program, which randomized the animals based on stratification of the Gestation Day 0 body weights in a block design.
- Animals not assigned to study were transferred to the Charles River rat colony or euthanized by carbon dioxide inhalation and discarded.
- Body weight values of the selected females ranged from 223 g to 289 g on Gestation Day 0.

ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- The vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, disposable, plastic feeding tubes (Instech Solomon, Plymouth Meeting, PA), once daily during Gestation Days 6 to 19.
- 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.
- Study group assignments are shown in the table below.
- The dosage levels were selected based on the results of a previous dose range-finding study in rats.3 In that study, female rats were dosed from Gestation Day 6 through 19 at dosage levels of 0, 50, 175, 600, and 1000 mg/kg/day. There were no significant clinical findings noted in the previous study and mean body weights, body weight gains, and food consumption were unaffected by treatment. As a result, dosage levels of 175, 350, 600, and 1000 mg/kg/day were chosen for the current study.
- The selected route of administration for this study was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLING AND ANALYSIS
- Test substance homogeneity and stability following at least 8 days of room temperature storage was established to cover the range of concentrations used on this study. Therefore, stability assessments were not conducted at part of this study.
- Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first and last test substance dosing formulations and from the middle stratum of the first and last control dosing formulations.
- One set of samples from each collection was subjected to the appropriate analyses.
- All remaining samples were stored frozen at room temperature as back-up.
- All analyses were conducted by the Charles River Analytical Chemistry Department using a validated high performance liquid chromatography method with charged aerosol detection.

Duration of treatment / exposure:

Gestation Days 6 to 19

Frequency of treatment:

Daily

Duration of test:

20 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- A flow chart summarising the study design is attached.

Maternal examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
- All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily during Gestation Days 0 to 20 (prior to dose administration during the treatment period).
- Animals were also observed for signs of toxicity at approximately 1 hour following dose administration. The absence or presence of findings was recorded for all animals.

BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
- Individual maternal body weights were recorded on Gestation Days 0 and 6–20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for Gestation Days 6–9, 9–12, 12 15, 15–20, and 6–20.
- Gravid uterine weight was collected and net body weight (the Gestation Day 20 body weight exclusive of the weight of the uterus and contents) and net body weight change (the Gestation Day 0–20 body weight change exclusive of the weight of the uterus and contents) were calculated and presented for each gravid female at the scheduled laparohysterectomy

FOOD CONSUMPTION
- Individual food consumption was recorded on Gestation Days 0 and 6–20 (daily). Food intake was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.
- When food consumption could not be determined for an animal during a given interval (due to weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data.
- The time periods when food consumption values were unavailable for a given animal were designated as “NA” on the individual report tables.

Ovaries and uterine content:

GESTATION DAY 20 LAPAROHYSTERECTOMY
- The laparohysterectomies and macroscopic examinations were performed blind to treatment group. All rats were euthanised on Gestation Day 20 by carbon dioxide inhalation. The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised. The number of corpora lutea on each ovary was recorded (using magnification). The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined. The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
- Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.
- Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded. Intrauterine data were summarized using 2 methods of calculation.
- An example of each method of calculation is attached.

Fetal examinations:

MORPHOLOGICAL EXAMINATION
- Fetal examinations were performed blind to treatment group. Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the Charles River study number, the female number, and the fetus number. The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.
- Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels. The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development. Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique. The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.
- Following fixation in alcohol, each fetus was stained with Alizarin Red S and Alcian Blue. Fetuses were then examined for skeletal malformations and developmental variations.
- External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life). Representative photographs of all malformations, as appropriate, were included in the study records. Corresponding low magnification photographs, depicting both the malformed fetus and a comparison control fetus, or normal littermate, were also included in the study records as needed and as appropriate for comparison, when possible.
- The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis (see details of calculations, attached).

Statistics:

See below

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Test substance-related clinical findings of red and clear material around the mouth were noted in a dose-related manner in the 350, 600, and 1000 mg/kg/day groups approximately 1 hour following dose administration. These findings were generally noted throughout the treatment period, and were noted sparingly in the 350 mg/kg/day group. The observations generally did not persist to the daily clinical examinations and therefore, were not considered adverse.
- Other observations noted in the 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

Description (incidence):

- All females in the control, 175, 350, 600, and 1000 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 20.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MATERNAL BODY WEIGHTS AND GRAVID UTERINE WEIGHTS
- Test substance-related, significantly (p < 0.05 or p < 0.01) lower mean body weight gains were noted for females in the 600 and 1000 mg/kg/day group during Gestation Days 6–9. During Gestation Days 9–12, mean body weight gains in these groups were significantly (p < 0.01) higher than the control group; however, these results did not occur in a dose-related manner and the differences were attributed to a lower control group mean during this period. Mean body weight gains in these groups were similar to the control group during Gestation Days 12–15. During Gestation Days 15–20, a test substance-related lower (significant at p < 0.01) mean body weight gain was noted in the 1000 mg/kg/day group, while mean body weight gain in the 600 mg/kg/day group was similar to the control group. As a result, mean body weight gain in the 1000 mg/kg/day group was significantly (p < 0.01) lower than the control group when the entire treatment period (Gestation Days 6–20) was evaluated and mean body weights in the 1000 mg/kg/day group were significantly (p < 0.05) lower than the control group on Gestation Days 9 (4.5%) and 20 (5.0%). Mean body weight gain in the 600 mg/kg/day group was similar to the control group when the entire treatment period (Gestation Days 6–20) was evaluated and mean body weights in this groups were similar to the control group throughout the study. Therefore, the lower mean body weight gain observed in the 600 mg/kg/day group during Gestation Days 6-9 was not considered adverse. Mean gravid uterine weight in the 1000 mg/kg/day group was significantly (p < 0.01) lower than the control group. However, the value was within the range of values in the Charles River Ashland historical control data (Version 2016.03) and was attributed to the non-test article-related lower mean number of viable fetuses (see Section 6.6.). Mean gravid uterine weight in the 600 mg/kg/day groups and net body weights and net body weight gains in the 600 and 1000 mg/kg/day group were similar to the control group.
- Mean maternal body weights, body weight gains, net body weights, net body weight gains, and gravid uterine weights in the 175 and 350 mg/kg/day groups were unaffected by test substance administration. During Gestation Days 9–12, mean body weight gains in these groups were significantly (p < 0.05) higher than the control group; however, these results did not occur in a dose-related manner and the differences were attributed to a lower control group mean during this period. Other differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 1000 mg/kg/day group was significantly (p < 0.01) lower than the control group during Gestation Days 6–9. This was considered test substance-related and corresponded to the lower mean body weight gains noted for these females during this period. Mean food consumption in this group was similar to the control group throughout the remainder of the treatment period (Gestation Days 9–20).
- Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 175, 350, and 600 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- All females survived to the scheduled necropsy on Gestation Day 20.
- No test substance-related internal findings were observed at any dosage level.
- Macroscopic findings observed in the test substance-treated groups occurred infrequently and/or in a manner that was not dose-related.

Other effects:

no effects observed

Description (incidence and severity):

GESTATION DAY 20 LAPAROHYSTERECTOMY
- Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 175, 350, 600, and 1000 mg/kg/day.
- A significantly (p < 0.05) lower mean number of viable fetuses was noted in the 1000 mg/kg/day group. However, this value was within the range of values in the Charles River Ashland historical control data.
- Parameters evaluated included postimplantation loss, live litter size, mean fetal body weights, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- Fetus No. 2402-01 in the 1000 mg/kg/day group had exencephaly with open eyelid (right); this finding was confirmed skeletally, consisting of absent frontal (bilateral), parietal (bilateral), interparietal, and supraoccipital bones.
- Fetus No. 2388-05 in the 175 mg/kg/day group had microphthalmia (right).
- These findings were noted in single fetuses. In addition, the mean litter proportions of these findings were within the ranges of values in the Charles River Ashland historical control data (Version 2016.03) and were not statistically significantly different than the concurrent control group. Therefore, these findings were not considered test substance-related.
- No external developmental variations were observed in fetuses in this study.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- No test substance-related skeletal malformations were noted. Fetus No. 2396-08 in the 1000 mg/kg/day group had sternoschisis (sternal bands not joined [entire length]), vertebral anomaly without associated rib anomaly (exxoccipital and cervical Arch No. 1 fused [bilateral]), and 6 cervical vertebrae (25 presacral vertebrae). In the 600 mg/kg/day group, Fetus No. 2403-10 had costal cartilage anomaly, consisting of right Costal Cartilage No. 1 and costal cartilage arising from 7th cervical rib fused and associate with sternum in normal No. 1 position. These findings occurred in single fetuses and the finding in the costal cartilage anomaly in the 600 mg/kg/day group did not occur in a dose-related manner. In addition, the mean litter proportions of these findings were within the ranges of values in the Charles River Ashland historical control data and were not statistically significantly different than the concurrent control group. Therefore, these findings were not considered test substance-related.
- No test substance-related skeletal developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the concurrent control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

- No test substance-related visceral malformations were noted. In the 1000 mg/kg/day group, Fetus No. 2310-06 had hydrocephaly (increased cavitation of the lateral [bilateral] and third ventricles). In the 600 mg/kg/day group, Fetus No. 2325-03 had situs inversus (trachea, esophagus, heart, great and major vessels, lungs, liver, stomach, pancreas, spleen, kidneys, adrenals, and intestine laterally transposed). In the 350 mg/kg/day group, Fetus No. 2370-06 had retroesophageal aortic arch (aortic arch coursed retroesophageal immediately following the left carotid and returned in normal position adjacent to the ductus arteriosus). Fetus No. 2345-06 in the 350 mg/kg/day group had transposition of the great vessels, interventricular septal defect (2 mm opening in the anterior portion of the septum), and lobular dysgenesis of the lungs (1 lobe present [bilateral]). Interventricular septal defect (< 1 mm opening in the anterior portion of the septum) was also noted for Fetus No. 2400-05 in the 350 mg/kg/day group. These findings were noted in single fetuses and/or were not noted in a dose-related manner. In addition, the mean litter proportions of these findings were within the ranges of values in the Charles River Ashland historical control data (Version 2016.03) and were not statistically significantly different than the concurrent control group. In addition, interventricular septal defect was also noted for 2 fetuses (No. 2426-03 and 2426-16) in the concurrent control group (< 1 mm opening in the anterior portion of the septum). Therefore, these findings were not considered test substance-related.
- No test substance-related visceral developmental variations were noted. Findings observed in the test substance-treated groups were noted infrequently, similarly in the concurrent control group, were not observed in a dose-related manner, the differences in the mean litter proportions were not statistically significant compared to the concurrent control group, and/or the values were within the ranges of the Charles River Ashland historical control data. Renal papilla(e) not fully developed was noted for Fetus No. 2296-03 in the 175 mg/kg/day group, Fetus No. 2337-16 in the 350 mg/kg/day group, and Fetus Nos. 2325-04, 2362-03, 2411-13 in the 600 mg/kg/day group. This finding was not classified as either a malformation or developmental variation, was not included on the summary tables, and was not considered to be test substance-related because it occurred infrequently and in a manner that was not dose-related.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

FETAL MORPHOLOGICAL DATA
- The numbers of fetuses (litters) available for morphological evaluation were 361(24), 347(25), 354(24), 366(24), and 340(25) in the control, 175, 350, 600, and 1000 mg/kg/day groups, respectively.
- Malformations were observed in 3(2), 1(1), 3(3), 2(2), and 3(3) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.

Details on embryotoxic / teratogenic effects:

SUMMARY OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
- The numbers of fetuses (litters) available for morphological evaluation were 361(24), 347(25), 354(24), 366(24), and 340(25) in the control, 175, 350, 600, and 1000 mg/kg/day groups, respectively.
- Malformations were observed in 3(2), 1(1), 3(3), 2(2), and 3(3) fetuses (litters) in these same respective dose groups and were considered spontaneous in origin.
- When the total malformations and developmental variations were evaluated on a proportional basis, no statistically significant differences from the concurrent control group were noted. Fetal malformations and developmental variations, when observed in the test substance-treated groups, occurred infrequently or at a frequency similar to that in the concurrent control group, did not occur in a dose-related manner, and/or were within the Charles River Ashland historical control data ranges.
- Based on these data, no fetal malformations or developmental variations were attributed to the test substance.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

other: based on lack of effects at any dosage level

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

ANALYSES OF DOSING FORMULATIONS

- The analysed dosing formulations were within Charles River SOP range for suspensions (85% to 115%) and were homogeneous.

- The test substance was not detected in the analysed vehicle formulation that was administered to the control group (Group 1).

- Results of the analyses of dosing formulations are summarised in the table attached.

Conclusions:

Based on the adverse effects on body weight, body weight gain, and food consumption at 1000 mg/kg/day, a dosage level of 600 mg/kg/day was considered to be the no-observedadverse-effect level (NOAEL) for maternal toxicity. Due to the lack of effects at any dosage level, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the NOAEL for embryo/fetal development when test item was administered orally by gavage to bred Crl:CD(SD) rats.

Executive summary:

GUIDELINE

The objectives of the study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity. The protocol was designed in general accordance with the United States EPA Health Effects Test Guidelines: OPPTS 870.3700, Prenatal Developmental Toxicity Study, Aug 1998, and the OECD Guideline for the Testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, 22 Jan 2001.

 

METHODS

Test substance in the vehicle (corn oil) was administered orally by gavage to 4 groups of 25 bred female Crl:CD(SD) rats once daily from Gestation Days 6–19. Dosage levels were 175, 350, 600, and 1000 mg/kg/day administered at a dose volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 13 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On

Gestation Day 20, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

 

RESULTS

All females in the control, 175, 350, 600, and 1000 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 20. Test substance-related clinical findings of red and clear material around the mouth were noted in a dose-related manner in the 350, 600, and 1000 mg/kg/day groups approximately 1 hour following dose administration; these findings were noted sparingly in the 350 mg/kg/day group. The observations generally did not persist to the daily clinical examinations and therefore, were not considered adverse. No other test substance-related clinical findings were noted.

 

Test substance-related lower mean body weight gains were noted in the 600 and 1000 mg/kg/day groups during Gestation Days 6–9 and in the 1000 mg/kg/day group during Gestation Days 15–20, resulting in lower mean body weights in the 1000 mg/kg/day group on Gestation Day 9 (4.5%) and 20 (5.0%). Corresponding effects on food consumption were noted for females in the 1000 mg/kg/day group during Gestation Days 6–9. Mean body weights in the 600 mg/kg/day group were similar to the control group throughout the study. Therefore, the lower mean body weight gain observed in the 600 mg/kg/day group during Gestation Days 6–9 was not considered adverse. No other test substance-related effects were noted on mean body weights, body weight gains, gravid uterine weights, net body weights, and net body weight gains. No test substance-related maternal macroscopic findings were noted at the scheduled necropsy on Gestation Day 20. Intrauterine growth and survival and fetal morphology were unaffected by maternal test substance administration.

 

CONCLUSION

Based on the adverse effects on body weight, body weight gain, and food consumption at 1000 mg/kg/day, a dosage level of 600 mg/kg/day was considered to be the no-observedadverse-effect level (NOAEL) for maternal toxicity. Due to the lack of effects at any dosage level, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the NOAEL for embryo/fetal development when test item was administered orally by gavage to bred Crl:CD(SD) rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

Two reliable studies available, therefore quality of whole database is sufficient.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study

The objectives of the study were to determine the potential of the test substance to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity. The protocol was designed in general accordance with the United States EPA Health Effects Test Guidelines: OPPTS 870.3700, Prenatal Developmental Toxicity Study, Aug 1998, and the OECD Guideline for the Testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, 22 Jan 2001.

Test substance in the vehicle (corn oil) was administered orally by gavage to 4 groups of 25 bred female Crl:CD(SD) rats once daily from Gestation Days 6–19. Dosage levels were 175, 350, 600, and 1000 mg/kg/day administered at a dose volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 13 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On Gestation Day 20, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females in the control, 175, 350, 600, and 1000 mg/kg/day groups survived to the scheduled necropsy on Gestation Day 20. Test substance-related clinical findings of red and clear material around the mouth were noted in a dose-related manner in the 350, 600, and 1000 mg/kg/day groups approximately 1 hour following dose administration; these findings were noted sparingly in the 350 mg/kg/day group. The observations generally did not persist to the daily clinical examinations and therefore, were not considered adverse. No other test substance-related clinical findings were noted.

 

Test substance-related lower mean body weight gains were noted in the 600 and 1000 mg/kg/day groups during Gestation Days 6–9 and in the 1000 mg/kg/day group during Gestation Days 15–20, resulting in lower mean body weights in the 1000 mg/kg/day group on Gestation Day 9 (4.5%) and 20 (5.0%). Corresponding effects on food consumption were noted for females in the 1000 mg/kg/day group during Gestation Days 6–9. Mean body weights in the 600 mg/kg/day group were similar to the control group throughout the study. Therefore, the lower mean body weight gain observed in the 600 mg/kg/day group during Gestation Days 6–9 was not considered adverse. No other test substance-related effects were noted on mean body weights, body weight gains, gravid uterine weights, net body weights, and net body weight gains. No test substance-related maternal macroscopic findings were noted at the scheduled necropsy on Gestation Day 20. Intrauterine growth and survival and fetal morphology were unaffected by maternal test substance administration.

Based on the adverse effects on body weight, body weight gain, and food consumption at 1000 mg/kg/day, a dosage level of 600 mg/kg/day was considered to be the no-observedadverse-effect level (NOAEL) for maternal toxicity. Due to the lack of effects at any dosage level, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the NOAEL for embryo/fetal development when test item was administered orally by gavage to bred Crl:CD(SD) rats.

Supporting study

The test substance was administered orally by gavage once daily to three groups of Crl:CD(SD) each of 12 male and 12 female rats at 50, 175 and 600 mg/kg/day. A concurrent control group of 12 rats/sex received the vehicle, corn oil, on a comparable regimen. Males and females received 14 doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia (for a total of 56 doses). Females were dosed through lactation day 3 for a total of 39-53 doses; the female that failed to deliver was dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. 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 were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on postnatal days (PND) 1 and 4. Pups were euthanized and discarded without further examination on PND 4. F0 females were euthanized on lactation day 4. F0 males were euthanized following completion of the F0 female necropsies. 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 and high-dosage groups; thyroid glands were also examined from the F0 males in the 50 and 175 mg/kg/day groups.

The mean numbers of pups born, live litter size on PND 0, the percentage of males at birth and postnatal survival in the treated groups were similar to those in the control group. The general physical condition of the F1 pups and mean F1 body weights were not affected by the test substance to the F0 parental animals. There were no macroscopic findings in the F1 pups that were found dead that could be attributed to F0 treatment with the test substance.

Under the conditions of this study, the NOAEL for male systemic toxicity was considered to be 175 mg/kg/day based on increased organ weights in the 175 and 600 mg/kg/day groups and microscopic findings in the 600 mg/kg/day group. The NOAEL for female systemic toxicity was considered to be 175 mg/kg/day based on increased liver weight in the 600 mg/kg/day group. Based on the lack of effects on live litter size, postnatal survival and F1 body weights at any dosage level, the NOAEL for F1 neonatal toxicity was considered to be at least 600 mg/kg/day.

Justification for classification or non-classification

The substance does not require classification in accordance with the criteria specified in Regulation 1272/2008 and subsequent amendments.