long chain alkyl thio carbamide metal complex

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Sept 2009 - 15 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Sept 2009 - 15 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: U.S. EPA OPPTS Guideline 870.3550

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

Each rat was uniquely identified by a Monel® metal ear tag displaying the animal number.

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: males (day 0) 351 g to 443 g, females (day 0) 220 g to 281 g.
- 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 at least 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 (Bed O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The nesting material is periodically analyzed by the manufacturer for contaminants. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research Laboratories, LLC. The dams and their litters were housed in these cages until euthanasia on lactation day 4. Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until post-cohabitation or post-mating day 25. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The animal facilities at WIL Research Laboratories, LLC are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).
- Diet: Provided ad libitum. PMI Nutrition International, LLC Certified Rodent LabDiet® 5002. Feeders were changed and sanitized once per week.
- Water: Provided ad libitum. Reverse osmosis-purified (on site) drinking water, delivered by an automatic watering system.
- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C +/- 3°C (target); 21.1°C to 21.4°C (actual temps)
- Humidity: 50% +/- 20% (target); 37.7% to 57.1% (actual temps)
- Air changes: 10/hr minimum
- Photoperiod: 12-hour light (0600 hours to 1800 hours)/12-hour dark

IN-LIFE DATES: From: 22 September 2009 To: 6 January 2010

Route of administration:

oral: gavage

Vehicle:

other: Mineral Oil USP

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance (45% [w/w] in 55% [w/w] mineral oil) can become non-homogeneous upon stationary storage; therefore, the bulk test substance was warmed to approximately 60ºC and inverted approximately 20 times prior to preparing the formulations. The test substance formulations were prepared daily during 13-18 October 2009 and approximately weekly thereafter as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature. The test substance formulations were stirred continuously throughout the dose administration procedures. The vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan) once daily.

VEHICLE
- Lot/batch no. (if required): (lot nos. XX1110 and YG1036, exp dates: 15 July 2010 and 18 September 2011.
- Supplier: Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ and Gardena, CA.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyzed dosing formulations were within the WIL Research Laboratories, LLC’s standard operating procedure range for suspensions (85% to 115%), were homogeneous, and were stable for 10 days at room temperature. Based on these results, the protocol-specified dosages of test substance were administered to the animals. The test substance was not detected in the vehicle formulation that was administered to the control group (Group 1).

Prior to the initiation of dose administration, quadruplicate samples for homogeneity and concentration determination were collected from the top, middle, and bottom strata of the 20 and 200 mg/mL non-dosing formulations. For the assessment of 5- and 10-day stability and aliquot resuspension homogeneity, aliquots approximately equivalent in volume to a daily dispensation aliquot, were transferred from the pre-initiation batch into the same type of storage container that was used for the daily aliquots for dosing. After storage at room temperature for 5 and 10 days, each aliquot was resuspended for a minimum of 30 minutes by warming the test substance formulations to 60ºC (while stirring), and quadruplicate samples were withdrawn from the top and bottom strata of each aliquot for assessment of 5- and 10-day stability and aliquot resuspension homogeneity. Samples for homogeneity and concentration analyses were collected from the middle stratum of the control formulation and the top, middle, and bottom strata of each batch of test substance dosing formulations. Samples from the first and last dosing batch were analyzed for homogeneity and concentration. All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, LLC using a validated high performance liquid chromatography method using ultraviolet absorbance detection.

Details on mating procedure:

The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. 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 and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. 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.

Duration of treatment / exposure:

Males: Dosed during study days 0–27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses.
Females: Dosed from study day 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-45 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post cohabitation day 25 or post-mating day 25) for a total of 39-42 doses.

Frequency of treatment:

Once daily at approximately the same time each day

Duration of test:

Premating through PND 4

No. of animals per sex per dose:

12/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of a previous study.
- Rationale for animal assignment (if not random): body weight stratification in a block design using a computer randomization procedure

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were recorded weekly (prior to test substance administration during the treatment period) and on the day of necropsy. Each male and female was also observed for signs of toxicity approximately 1-2 and 3 hours following dose administration. The absence or presence of findings was recorded for individual animals. In addition, findings at the time of dose administration were recorded for individual animals. Females expected to deliver were observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHT: Yes
- Time schedule for examinations: Males: recorded weekly throughout the study and prior to the scheduled euthanasia. Females: recorded weekly until evidence of copulation was observed. 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 0 (when possible), 1, and 4.

FOOD CONSUMPTION: Yes
- Individual food consumption was recorded on the corresponding weekly body weight days 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.

POST-MORTEM EXAMINATIONS: Yes
- F0 males were sacrificed following completion of the mating period; F0 females were sacrificed on post-mating day or post-cohabitation day #25, or on lactation day 4.

GROSS PATHOLOGY: Yes, all animals were euthanized using carbon dioxide inhalation.
- Organs examined (F0): Necropsy included examination of the external surface, all orifices, the external surface of the brain, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.
- Organs weighed (F0): brain, epididymides, kidneys, liver, ovaries with oviducts, pituitary, testes

HISTOPATHOLOGY: Yes (control and high-dose groups)
Tissues examined (F0): Coagulating glands, mammary gland (female only), ovaries and oviducts, pituitary gland, prostate gland, seminal vesicles, testes with epididymides and vas deferens (fixed in Bouin's solution), uterus with cervix and vagina, all gross lesions. After fixation, protocol-specified tissues were trimmed according to WIL SOP's 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.

Ovaries and uterine content:

Organ weights: Ovaries with oviducts

Fetal examinations:

Evaluations of offspring were conducted postnatally. See other information below.

Statistics:

Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
Parental mating, fertility, conception, and copulation indices were analyzed using the Chi square test with Yates’ correction factor (Hollander and Wolfe, 1999). Histopathological findings were analyzed by the Fisher’s Exact Test (Steel, 1980). Mean parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of corpora lutea and former implantation sites, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, and pre coital interval values were subjected to a parametric one way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.

Indices:

Mean Live Litter Size, Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter), and Postnatal Survival Between PND 0-1 and PND 1-4 (% Per Litter) were calculated.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, adverse behaviour/CNS-related findings of intermittent tremors, repetitive movement of mouth and jaws, excessive pawing and/or licking of the cage floor and/or walls (females only, beginning on study day 13), and/or head twitch were noted in the 1000 mg/kg/day group males and females at 1-2 and/or 3 hours following dose administration beginning on study day 12 and continuing sporadically throughout the remainder of treatment. Test substance-related, non-adverse, salivation or evidence thereof (clear material around the mouth) was noted in the 1000 mg/kg/day group males and females generally throughout the treatment period prior to dosing and 1 2 and/or 3 hours following dose administration. In addition, clinical findings of orange, orangish-red, and/or red material on various body surfaces were noted for males and females in all test substance-treated groups at all observation time points generally throughout the treatment period and were considered to be associated with the dark red color of the dosing formulations and were not considered adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Females in the top dose group (1000 mg/kg/day) had a higher mean liver weight when compared with the control group

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

One female from this group had yellow areas on the liver at the time of necropsy, which correlated histologically to areas of liquefactive necrosis. The affected rat also had the highest liver weight. The relationship between administration of the test substance and the hepatic necrosis is uncertain, as the liver was examined microscopically only for the animal with gross necropsy findings in the liver. There were no other test substance-related histologic changes.

Other effects:

no effects observed

Description (incidence and severity):

The following indices were calculated: Male Mating Index (%), Female Mating Index (%), Male Fertility Index (%), Female Fertility Index (%), Male Copulation Index (%), Female Conception Index (%), Pre-Coital Interval (days). No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance treated groups. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Test substance-related, adverse behavior/CNS-related findings of intermittent tremors, repetitive movement of mouth and jaws, excessive pawing and/or licking of the cage floor and/or walls (females only, beginning on study day 13), and/or head twitch were noted in the 1000 mg/kg/day group males and females at 1-2 and/or 3 hours following dose administration beginning on study day 12 and continuing sporadically throughout the remainder of treatment. Female rats in the top dose group (1000 mg/kg/day) had a higher mean liver weight when compared with the control group.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The mean number of pups born, live litter size, and the percentage of males at birth in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values. Postnatal survival between birth and PND 0 (relative to number born), PND 0-1 and 1-4, and from birth to PND 4 in the 100, 300, and 1000 mg/kg/day groups was unaffected by parental test substance administration. No statistically significant differences from the control group were noted.
Mean male and female pup body weights and body weight changes in the 100, 300, and 1000 mg/kg/day groups were unaffected by test substance administration during PND 1-4. No statistically significant differences from the control group were noted.

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

The mean number of pups born, live litter size, and the percentage of males at birth in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values. Postnatal survival between birth and PND 0 (relative to number born), PND 0-1 and 1-4, and from birth to PND 4 in the 100, 300, and 1000 mg/kg/day groups was unaffected by parental test substance administration. No statistically significant differences from the control group were noted.
Pups (litters) that were found dead numbered 1(1), 2(2), 2(1), and 6(4) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. The numbers of pups (litters) found dead during PND 0-4 numbered 1(1), 2(2), 2(1), and
6(4) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Key result

Developmental effects observed:

no

PND 0 LITTER DATA AND POSTNATAL SURVIVAL

The mean number of pups born, live litter size, and the percentage of males at birth in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values. Postnatal survival between birth and PND 0 (relative to number born), PND 0-1 and 1-4, and from birth to PND 4 in the 100, 300, and 1000 mg/kg/day groups was unaffected by parental test substance administration. No statistically significant differences from the control group were noted.

GENERAL PHYSICAL CONDITION

The general physical condition of all F1 pups in this study were unaffected by test substance administration.

OFFSPRING BODY WEIGHTS

Mean male and female pup body weights and body weight changes in the 100, 300, and 1000 mg/kg/day groups were unaffected by test substance administration during PND 1 4. No statistically significant differences from the control group were noted. NECROPSIES OF PUPS FOUND DEAD

The numbers of pups (litters) found dead during PND 0-4 numbered 1(1), 2(2), 2(1), and 6(4) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Conclusions:

The NOAEL for developmental/neonatal effects is 1000 mg/kg bw/day, the highest dose tested.

Executive summary:

EC# 457-320-2 has been tested in a guideline (OECD 421) reproductive/developmental toxicity GLP screening study. The test substance in the mineral oil vehicle 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 100, 300, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 12 rats/sex received the vehicle (mineral oil, USP) on a comparable regimen. Males and females were approximately 12 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39–45 doses; females with no evidence of mating or those that failed to deliver were dosed through the day prior to euthanasia (post-cohabitation day 25 or post-mating day 25) for a total of 39–42 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. Necropsies were performed on pups that died. All other F₁ pups were euthanized on PND 4 and discarded without further examination. F₀ males were euthanized following completion of the mating period and F₀ females were euthanized on post-mating day 25 (females with evidence of mating), post-cohabitation day 25 (females with no evidence of mating), or on lactation day 4. Complete necropsies were conducted on all F₀ animals, and selected organs were weighed. Selected tissues of the reproductive system were examined microscopically from all F₀ animals in the control and high-dose groups.

All male and female rats survived to the scheduled necropsy. Test substance-related, adverse behavior/CNS-related findings of intermittent tremors, repetitive movement of mouth and jaws, excessive pawing and/or licking of the cage floor and/or walls (females only, beginning on study day 13), and/or head twitch were noted in the 1000 mg/kg/day group males and females at 1-2 and/or 3 hours following dose administration beginning on study day 12 and continuing sporadically throughout the remainder of treatment. There were no test substance-related effects on mean body weights, body weight gains, or food consumption for males throughout the treatment period or for females during the pre-mating, gestation, or lactation treatment periods at any dosage level.

There were no test substance-related effects on male and female reproductive performance (mating, fertility, copulation, and conception indices and pre-coital intervals), mean gestation lengths, or the process of parturition at any dosage level.

Female rats in the top dose group (1000 mg/kg/day) also had an increased mean liver weight when compared with the control group. In reproductive tissues, no test substance-related changes in organ weights, gross necropsy observations, or histologic changes were detected. In addition, the mean numbers of unaccounted-for sites, former implantation sites, and corpora lutea were similar between the control and test substance-treated groups.

There were no effects on the numbers of pups born, live litter size, percentage of males at birth, pup survival, or on mean male and female pup body weights and body weight gains during PND 1–4. No test substance-related clinical findings were noted for pups at any dosage level; no test substance-related macroscopic findings were observed in pups that were found dead.

In the absence of test substance-related effects on reproductive performance, gestation lengths, parturition, live litter size, numbers of pups born, and reproductive organs, a dosage level of 1000 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats. Based on behavior/CNS-related findings and increased mean liver weights at 1000 mg/kg/day, the NOAEL for male and female systemic toxicity was considered to be 300 mg/kg/day. Based on the absence of effects on postnatal survival or pup body weights, the NOAEL for neonatal toxicity was determined to be 1000 mg/kg/day.

 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: U.S. EPA OPPTS Guideline 870.3550

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid

Details on test material:

Dark opaque red liquid
Batch: 30848-47
Expiry: 15-Sept-2012

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Each rat was uniquely identified by a Monel® metal ear tag displaying the animal number.

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: males (day 0) 351 g to 443 g. females (day 0) 220 g to 281 g.
- 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 at least 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 (Bed O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The nesting material is periodically analyzed by the manufacturer for contaminants. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research Laboratories, LLC. The dams and their litters were housed in these cages until euthanasia on lactation day 4. Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until post-cohabitation or post-mating day 25. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). The animal facilities at WIL Research Laboratories, LLC are accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International).
- Diet: Provided ad libitum. PMI Nutrition International, LLC Certified Rodent LabDiet® 5002. Feeders were changed and sanitized once per week.
- Water: Provided ad libitum. Reverse osmosis-purified (on site) drinking water, delivered by an automatic watering system.
- Acclimation period: 21 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C +/- 3°C (target); 21.1°C to 21.4°C (actual temps)
- Humidity: 50% +/- 20% (target); 37.7% to 57.1% (actual temps)
- Air changes: 10/hr minimum
- Photoperiod: 12-hour light (0600 hours to 1800 hours)/12-hour dark

IN-LIFE DATES: From: 22 September 2009 To: 6 January 2010

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Mineral oil USP

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance (45% [w/w] in 55% [w/w] mineral oil) can become non-homogeneous upon stationary storage; therefore, the bulk test substance was warmed to approximately 60ºC and inverted approximately 20 times prior to preparing the formulations. The test substance formulations were prepared daily during 13-18 October 2009 and approximately weekly thereafter as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature. The test substance formulations were stirred continuously throughout the dose administration procedures. The vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan) once daily.

VEHICLE
- Lot/batch no. (if required): (lot nos. XX1110 and YG1036, exp dates: 15 July 2010 and 18 September 2011.
- Supplier: Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ and Gardena, CA,

Details on mating procedure:

The animals were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. 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 and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyzed dosing formulations were within the WIL Research Laboratories, LLC’s standard operating procedure range for suspensions (85% to 115%), were homogeneous, and were stable for 10 days at room temperature. Based on these results, the protocol-specified dosages of test substance were administered to the animals. The test substance was not detected in the vehicle formulation that was administered to the control group (Group 1).

Prior to the initiation of dose administration, quadruplicate samples for homogeneity and concentration determination were collected from the top, middle, and bottom strata of the 20 and 200 mg/mL non-dosing formulations. For the assessment of 5- and 10-day stability and aliquot resuspension homogeneity, aliquots approximately equivalent in volume to a daily dispensation aliquot, were transferred from the pre-initiation batch into the same type of storage container that was used for the daily aliquots for dosing. After storage at room temperature for 5- and 10-days, each aliquot was resuspended for a minimum of 30 minutes by warming the test substance formulations to 60ºC (while stirring), and quadruplicate samples were withdrawn from the top and bottom strata of each aliquot for assessment of 5- and 10-day stability and aliquot resuspension homogeneity. Samples for homogeneity and concentration analyses were collected from the middle stratum of the control formulation and the top, middle, and bottom strata of each batch of test substance dosing formulations. Samples from the first and last dosing batch were analyzed for homogeneity and concentration. All analyses were conducted by the Analytical Chemistry Department, WIL Research Laboratories, LLC using a validated high performance liquid chromatography method using ultraviolet absorbance detection.

Duration of treatment / exposure:

Males: Dosed during study days 0-27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses.
Females: Dosed from study day 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-45 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post cohabitation day 25 or post-mating day 25) for a total of 39-42 doses.

Frequency of treatment:

Once daily at approximately the same time each day

Details on study schedule:

The animals were approximately 14 weeks old when paired on study day 13.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
100, 300, or 1000 mg/kg/day administered at a dosage volume of 5 mL/kg.
Basis:
other: actual (gavage)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

0 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of a previous study.
- Rationale for animal assignment (if not random): body weight stratification in a block design using a computer randomization procedure

Positive control:

None

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for mortality and moribundity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were recorded weekly (prior to test substance administration during the treatment period) and on the day of necropsy. Each male and female was also observed for signs of toxicity approximately 1-2 and 3 hours following dose administration. The absence or presence of findings was recorded for individual animals. In addition, findings at the time of dose administration were recorded for individual animals. Females expected to deliver were observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHT: Yes
- Time schedule for examinations: Males: recorded weekly throughout the study and prior to the scheduled euthanasia. Females: recorded weekly until evidence of copulation was observed. 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 0 (when possible), 1, and 4.

FOOD CONSUMPTION: Yes
- Individual food consumption was recorded on the corresponding weekly body weight days 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.

Oestrous cyclicity (parental animals):

Not evaluated

Sperm parameters (parental animals):

Not evaluated

Litter observations:

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.

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. Intact offspring found dead were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. 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 dose group. When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods a given pup was not weighed were designated as “NA” (Not Applicable) on the individual report tables.

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

Postmortem examinations (parental animals):

POST-MORTEM EXAMINATIONS: Yes
- F0 males were sacrificed following completion of the mating period; F0 females were sacrificed on post-mating day or post-cohabitation day #25, or on lactation day 4.

GROSS PATHOLOGY: Yes, all animals were euthanized using carbon dioxide inhalation.
- Organs examined (F0): Necropsy included examination of the external surface, all orifices, the external surface of the brain, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.
- Organs weighed (F0): brain, epididymides, kidneys, liver, ovaries with oviducts, pituitary, testes

HISTOPATHOLOGY: Yes (control and high-dose groups)
Tissues examined (F0): Coagulating glands, mammary gland (female only), ovaries and oviducts, pituitary gland, prostate gland, seminal vesicles, testes with epididymides and vas deferens (fixed in Bouin's solution), uterus with cervix and vagina, all gross lesions. After fixation, protocol-specified tissues were trimmed according to WIL SOP's 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.

Postmortem examinations (offspring):

On PND 4, surviving F1 pups were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded without examination.

Statistics:

Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
Parental mating, fertility, conception, and copulation indices were analyzed using the Chi square test with Yates’ correction factor (Hollander and Wolfe, 1999). Histopathological findings were analyzed by the Fisher’s Exact Test (Steel, 1980). Mean parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, gestation length, numbers of corpora lutea and former implantation sites, number of pups born, live litter size on PND 0, unaccounted-for sites, absolute and relative organ weights, and pre coital interval values were subjected to a parametric one way analysis of variance (ANOVA) (Snedecor and Cochran, 1980) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences between the control and test substance-treated groups. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.

Reproductive indices:

Mating, fertility, and copulation/conception indices were calculated.

Offspring viability indices:

Mean Live Litter Size, Postnatal Survival Between Birth and PND 0 or PND 4 (% Per Litter), and Postnatal Survival Between PND 0-1 and PND 1-4 (% Per Litter) were calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related, adverse behaviour/CNS-related findings of intermittent tremors, repetitive movement of mouth and jaws, excessive pawing and/or licking of the cage floor and/or walls (females only, beginning on study day 13), and/or head twitch were noted in the 1000 mg/kg/day group males and females at 1-2 and/or 3 hours following dose administration beginning on study day 12 and continuing sporadically throughout the remainder of treatment. Test substance-related, non-adverse, salivation or evidence thereof (clear material around the mouth) was noted in the 1000 mg/kg/day group males and females generally throughout the treatment period prior to dosing and 1 2 and/or 3 hours following dose administration. In addition, clinical findings of orange, orangish-red, and/or red material on various body surfaces were noted for males and females in all test substance-treated groups at all observation time points generally throughout the treatment period and were considered to be associated with the dark red color of the dosing formulations and were not considered adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test substance-related effects on mean body weights or body weight gains.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no test substance-related effects on food consumption for males throughout the treatment period or for females during the pre-mating, gestation, or lactation treatment periods at any dosage level.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

The following indices were calculated: Male Mating Index (%), Female Mating Index (%), Male Fertility Index (%), Female Fertility Index (%), Male Copulation Index (%), Female Conception Index (%), Pre-Coital Interval (days). No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance treated groups. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value.

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
Test substance-related, adverse behavior/CNS-related findings of intermittent tremors, repetitive movement of mouth and jaws, excessive pawing and/or licking of the cage floor and/or walls (females only, beginning on study day 13), and/or head twitch were noted in the 1000 mg/kg/day group males and females at 1-2 and/or 3 hours following dose administration beginning on study day 12 and continuing sporadically throughout the remainder of treatment. Test substance-related, non-adverse, salivation or evidence thereof (clear material around the mouth) was noted in the 1000 mg/kg/day group males and females generally throughout the treatment period prior to dosing and 1 2 and/or 3 hours following dose administration. In addition, clinical findings of orange, orangish-red, and/or red material on various body surfaces were noted for males and females in all test substance-treated groups at all observation time points generally throughout the treatment period and were considered to be associated with the dark red color of the dosing formulations and were not considered adverse.

BODY WEIGHT AND WEIGHT GAIN
There were no test substance-related effects on mean body weights or body weight gains.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no test substance-related effects on food consumption for males throughout the treatment period or for females during the pre-mating, gestation, or lactation treatment periods at any dosage level.

ORGAN WEIGHTS
Females in the top dose group (1000 mg/kg/day) had a higher mean liver weight when compared with the control group.

GROSS AND MICROSCOPIC PATHOLOGY
One female from this group had yellow areas on the liver at the time of necropsy, which correlated histologically to areas of liquefactive necrosis. The affected rat also had the highest liver weight. The relationship between administration of the test substance and the hepatic necrosis is uncertain, as the liver was examined microscopically only for the animal with gross necropsy findings in the liver. There were no other test substance-related histologic changes.

In reproductive tissues, no test substance-related changes in organ weights, gross necropsy observations, or histologic changes were detected.

REPRODUCTIVE PERFORMANCE
The following indices were calculated: Male Mating Index (%), Female Mating Index (%), Male Fertility Index (%), Female Fertility Index (%), Male Copulation Index (%), Female Conception Index (%), Pre-Coital Interval (days). No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance treated groups. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value.

GESTATION LENGTH
Mean gestation lengths in the 100, 300, and 1000 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

Key result

Dose descriptor:

NOAEL

Remarks:

systemic effects

Effect level:

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

Sex:

male/female

Basis for effect level:

other: Based on behavior/CNS-related findings and increased mean liver weights at 1000 mg/kg/day.

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive effects

Effect level:

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

Sex:

male/female

Basis for effect level:

other: No effects seen at the highest dose level.

Target system / organ toxicity (P0)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

central nervous system

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The mean number of pups born, live litter size, and the percentage of males at birth in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values. Postnatal survival between birth and PND 0 (relative to number born), PND 0-1 and 1-4, and from birth to PND 4 in the 100, 300, and 1000 mg/kg/day groups was unaffected by parental test substance administration. No statistically significant differences from the control group were noted.
The numbers of pups (litters) found dead during PND 0-4 numbered 1(1), 2(2), 2(1), and 6(4) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Mean male and female pup body weights and body weight changes in the 100, 300, and 1000 mg/kg/day groups were unaffected by test substance administration during PND 1-4. No statistically significant differences from the control group were noted.

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

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)

PND 0 LITTER DATA AND POSTNATAL SURVIVAL
The mean number of pups born, live litter size, and the percentage of males at birth in the 100, 300, and 1000 mg/kg/day groups were similar to the control group values. Postnatal survival between birth and PND 0 (relative to number born), PND 0-1 and 1-4, and from birth to PND 4 in the 100, 300, and 1000 mg/kg/day groups was unaffected by parental test substance administration. No statistically significant differences from the control group were noted.

GENERAL PHYSICAL CONDITION
The general physical condition of all F1 pups in this study were unaffected by test substance administration.

OFFSPRING BODY WEIGHTS
Mean male and female pup body weights and body weight changes in the 100, 300, and 1000 mg/kg/day groups were unaffected by test substance administration during PND 1-4. No statistically significant differences from the control group were noted.

NECROPSIES OF PUPS FOUND DEAD
The numbers of pups (litters) found dead during PND 0-4 numbered 1(1), 2(2), 2(1), and 6(4) in the control, 100, 300, and 1000 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

neonatal toxicity

Generation:

F1

Effect level:

>= 1 000 other: mg/kg/bw (maternal)

Sex:

male/female

Basis for effect level:

other: No effects seen on litter data, postnatal survival or pup body weights through PND 4.

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on behavior/CNS-related findings and increased mean liver weights at 1000 mg/kg/day, the NOAEL for male and female systemic toxicity was considered to be 300 mg/kg/day. Based on the absence of effects on postnatal survival or pup body weights, the NOAEL for neonatal toxicity was determined to be 1000 mg/kg/day.

Executive summary:

EC# 457-320-2 has been tested in a guideline (OECD 421) reproductive/developmental toxicity GLP screening study. The test substance in mineral oil vehicle 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 100, 300, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 12 rats/sex received the vehicle (mineral oil, USP) on a comparable regimen. Males and females were approximately 12 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39–45 doses; females with no evidence of mating or those that failed to deliver were dosed through the day prior to euthanasia (post-cohabitation day 25 or post-mating day 25) for a total of 39–42 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. Necropsies were performed on pups that died. All other F₁ pups were euthanized on PND 4 and discarded without further examination. F₀ males were euthanized following completion of the mating period and F₀ females were euthanized on post-mating day 25 (females with evidence of mating), post-cohabitation day 25 (females with no evidence of mating), or on lactation day 4. Complete necropsies were conducted on all F₀ animals, and selected organs were weighed. Selected tissues of the reproductive system were examined microscopically from all F₀ animals in the control and high-dose groups.

All male and female rats survived to the scheduled necropsy. Test substance-related, adverse behaviour/CNS-related findings of intermittent tremors, repetitive movement of mouth and jaws, excessive pawing and/or licking of the cage floor and/or walls (females only, beginning on study day 13), and/or head twitch were noted in the 1000 mg/kg/day group males and females at 1–2 and/or 3 hours following dose administration beginning on study day 12 and continuing sporadically throughout the remainder of treatment. There were no test substance-related effects on mean body weights, body weight gains, or food consumption for males throughout the treatment period or for females during the pre-mating, gestation, or lactation treatment periods at any dosage level.

There were no test substance-related effects on male and female reproductive performance (mating, fertility, copulation, and conception indices and pre-coital intervals), mean gestation lengths, or the process of parturition at any dosage level.

Females in the top dose group (1000 mg/kg/day) had a higher mean liver weight when compared with the control group. In reproductive tissues, no test substance-related changes in organ weights, gross necropsy observations, or histologic changes were detected. In addition, the mean numbers of unaccounted-for sites, former implantation sites, and corpora lutea were similar between the control and test substance-treated groups.

There were no effects on the numbers of pups born, live litter size, percentage of males at birth, pup survival, or on mean male and female pup body weights and body weight gains during PND 1–4. No test substance-related clinical findings were noted for pups at any dosage level; no test substance-related macroscopic findings were observed in pups that were found dead.

In the absence of test substance-related effects on reproductive performance, gestation lengths, parturition, live litter size, numbers of pups born, and reproductive organs, a dosage level of 1000 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test substance when administered orally by gavage to Crl:CD(SD) rats. Based on behaviour/CNS-related findings and increased mean liver weights at 1000 mg/kg/day, the NOAEL for male and female systemic toxicity was considered to be 300 mg/kg/day. Based on the absence of effects on postnatal survival or pup body weights, the NOAEL for neonatal toxicity was determined to be 1000 mg/kg/day.