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

Toxicity to reproduction

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

800-172-4

Cas Number:

398141-87-2

Molecular formula:

Complex UVCB substance

IUPAC Name:

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

Test material form:

liquid

Details on test material:

Sponsor's identification: Thiophene
Lot no. number: TS05023
Description: Yellow, viscous liquid
Purity: 100%
Date received: 12 September 2005
Expiry date: 1 August 2006
Storage conditions: Room temperature

Test animals

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.

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Remarks:

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

Doses / concentrations 2

Remarks:

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

Doses / concentrations 3

Remarks:

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

Doses / concentrations 4

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

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

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.

Effect levels (P0)

1

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

2

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

3

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

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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

Details on results (F1)

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.

Effect levels (F1)

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

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.