Phenol, paraalkylation products with C12-rich branched olefins derived from propene oligomerisation, ethane-1,2-diol, carbonate, sulfurized, calcium salts, overbased, reacted with alkylene carbonate, including distillates (petroleum), heavy paraffinic C10-C50

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fertility was measured with the registration substance up to a limit dose of 1000 mg/kg/day in both the OECD 421 and 416, and it was determined that there was no impact on fertility.

As per Annex IX and X, Section 8.7.3, Column 2 of Regulation (EC) No 1907/2006, a Two-generation reproductive toxicity study initiated before 13th March 2015 shall be considered appropriate to address the standard information requirement. The Two-generation reproductive toxicity study for the registration substance was initiated prior to the aforementioned data and is therefore appropriate to fulfil this data requirement.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2012 - May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study in accordance with OECD guidelines

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

EXP1200078
Lot no. E00048-256
Exp. date: 31 December 2014
Dark brown, opaque, viscous liquid

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: At the initiation of dose administration (study day 0 for females and study day 14 for males), the males and females were approximately 12.5 and 10.5 weeks old, respectively.
- Weight at study initiation: Male body weights 397-482 g on study day 14 and female body weights 199-281 g on study day 0
- Fasting period before study: no
- Housing: Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire mesh cages suspended above cage board. 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.
- Diet: ad libitum PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water: ad libitum reverse osmosis-purified (on site) drinking water
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21.4°C to 21.6°C
- Humidity: 32.5% to 46.2%
- Air changes: a minimum of 10 fresh air changes per hour
- Photoperiod: 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod

IN-LIFE DATES: From: 29 January 2013 To: 27 March 2013

Route of administration:

oral: gavage

Vehicle:

other: Mineral oil, USP

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18°C to 24°C). The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The dosage volume for all groups was 5 mL/kg.

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

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no.: lot nos. 1AL0557 and 2BC0539

Details on mating procedure:

- M/F ratio per cage: 1/1
- Proof of pregnancy: 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
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared during the in-life phase of the study. The analyzed dosing formulations were within WIL Research’s SOP range for suspensions (85% to 115%). The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).

Duration of treatment / exposure:

The males were dosed during study days 14-41 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females were dosed during study days 0 through the day prior to euthanasia (28 days prior to pairing through lactation day 3) for a total of 53-57 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 53-66 doses.

Frequency of treatment:

Daily.

Details on study schedule:

No further details.

Dose / conc.:

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

Remarks:

Vehicle control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Dosage levels were determined based on the results of previous studies, including a 14 day (Haas, 2013, WIL-537025) and a 28-day (Haas, 2013, WIL-537026) oral gavage toxicity study in male and female Sprague Dawley rats. In the 14-day study (Haas, 2013, WIL-537025), the test substance in the vehicle (mineral oil) was administered orally by gavage once daily for 14 consecutive days to 3 groups consisting of 5 rats/sex at dosage levels of 100, 300, and 1000 mg/kg/day. All animals survived to the scheduled necropsy. Test substance-related effects were limited to low incidence of clear material around the mouth noted in males at 1000 mg/kg/day. Based on these results, the maximum tolerated dose (MTD) was considered to be 1000 mg/kg/day. In the 28-day study (Haas, 2013, WIL-537026), the test substance in the vehicle (mineral oil) was administered orally by gavage once daily for 28 consecutive days to 3 groups consisting of 5 rats/sex at dosage levels of 100, 300, and 1000 mg/kg/day. There were no test substance-related deaths. Test substance-related effects were limited to clear material around the mouth and higher liver weights in the 1000 mg/kg/day group males and females and a lower seminal vesicles weight in the 1000 mg/kg/day group males. However, none of these effects were considered to be adverse. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day, the highest dosage level administered.
The selected route of administration for this study was oral (gavage) because this is a potential route of human exposure. Historically, this route has been used extensively for studies of this nature.

Positive control:

No, not required and not relevant.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily. Each male and female was also observed for signs of toxicity 1-2 hours following dose administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly. Individual male body weights were recorded weekly throughout the study, beginning on the first day of dose administration, 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 0 (when possible), 1, and 4.

FOOD CONSUMPTION: Yes
- Time schedule: Individual male and female food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the breeding 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 the breeding period, food consumption for females with no evidence of mating was measured on a weekly basis until the scheduled euthanasia.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
A complete necropsy was conducted on all F0 parental animals at the scheduled termination. All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period. 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) or post-cohabitation day 25 (females with no evidence of mating).

Necropsy 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. At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered
formalin (except as noted):
Brain
Pituitary gland
Coagulating glands
Prostate gland
Kidneys (2)
Seminal vesicles (2)
Liver (sections of 2 lobes)
Testes with epididymidesa (2)
Mammary glands and vas deferens
Ovaries and oviducts (2)
Uterus with cervix and vagina
All gross lesions

Microscopic examination was performed on the following tissues from all animals in the
control and 1000 mg/kg/day groups, and gross lesions from all groups, at the scheduled necropsies:
Cervix
Seminal vesicles
Coagulating glands
Testes
Epididymides
Uterus
Mammary glands (female only)
Vagina
Ovaries
Vas deferens
Pituitary gland
All gross lesions (internal)
Prostate gland

Oestrous cyclicity (parental animals):

Not examined.

Sperm parameters (parental animals):

Not examined.

Litter observations:

- Each litter was examined daily for survival, and all deaths were recorded.
- 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.
- Pups were individually weighed on PND 1 and 4.
- Mean pup weights were presented by sex for each litter and by dose group.
- Pups were individually sexed on PND 0 and 4.

Postmortem examinations (parental animals):

A complete necropsy was conducted on all F0 parental animals at the scheduled termination.
The following organs were weighed from all F0 animals at the scheduled necropsies: Brain, Ovaries with oviducts, Epididymides, Pituitary gland, Kidneys, Testes, Liver.
Microscopic examination was performed on the following tissues from all animals in the control and 1000 mg/kg/day groups, and gross lesions from all groups, at the scheduled necropsies:
Cervix, Seminal vesicles, Coagulating glands, Testes, Epididymides, Uterus, Mammary glands (female only), Vagina, Ovaries, Vas deferens, Pituitary gland, All gross lesions (internal), Prostate gland.
Tissues and organs from all animals in the 100 and 300 mg/kg/day groups (except for gross lesions) were maintained in the appropriate fixatives for possible future microscopic examination.

Postmortem examinations (offspring):

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

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). 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 former implantation sites, number of pups born, live litter size on PND 0, corpora lutea, unaccounted-for sites, absolute and relative organ weights, and pre-coital intervals were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences. If the 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. Histopathological findings of each treated group were compared to those of the control group by Fisher’s Exact test (Steel and Torrie, 1980).

Reproductive indices:

Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant) / Total No. of Males (Females) Used for Mating x 100

Male Fertility Index (%) = Total No. of Malese Siring a Litter / Total No. of Males Used for Mating x 100

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:

- Mean live litter size
- Postnatal survival between births and PND 0 and PND 4
- Postnatal survial for all other intervals

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical findings noted for males and females in the 300 and 1000 mg/kg/day groups (red material around the mouth) were considered non adverse.

Mortality:

no mortality observed

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:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

ORGAN WEIGHTS (PARENTAL ANIMALS):
Slightly higher mean liver weights (absolute, relative to final body weight, and relative to brain weight) were noted in the 1000 mg/kg/day F0 group males [5.9%, 5.9%, and 3.8% (values not statistically significant)] and females [14.2% (p<0.05), 13.8% (p<0.01), and 13.5% (p<0.05)]. These changes were of a minimal magnitude. Slightly higher mean liver weights were also noted at 1000 mg/kg/day in a previous 28-day study (Haas, 2013, WIL-537026) (approximately 12% higher in males and females) and in a concurrent 90-day study (Haas, Draft, WIL-537030) (9.5% and 8.1% higher in males and females, respectively) of the same test substance with no correlating adverse clinical pathology or histologic changes. Based on the lack of any correlating adverse clinical pathology or histologic changes in two additional studies on the same test substance with similar minimal liver weight elevations, microscopic examination of the liver was not performed in the current study and the slightly higher mean liver weights were considered and toxicologically irrelevant.

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse systemic or local toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day)

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

body weight and weight gain

Remarks on result:

not determinable due to adverse toxic effects at highest dose / concentration tested

Remarks:

Exposure via F0 female

Reproductive effects observed:

not specified

Conclusions:

Under the conditions of this screening study, no test substance-related effects were noted on F0 reproductive endpoints, including male and female mating and fertility, male copulation, and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition at any dosage level. Therefore, the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive toxicity was considered to be 1000 mg/kg/day (the highest dosage level evaluated) when the test substance was administered orally by gavage to Crl:CD(SD) rats. There were no adverse test substance-related clinical findings and no effects on mean body weights, body weight changes, and food consumption noted at any dosage level. Furthermore, there were no adverse effects on organ weights or macroscopic/microscopic alterations in F0 males and females at any dosage level. Therefore, the NOAEL for F0 male and female systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for neonatal toxicity was considered to be 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.

Executive summary:

The test substance was administered by daily oral gavage to male and female Sprague Dawley rats at dose levels of 0, 100, 300 or 1000 mg/kg bw/day according to the OECD 421 guideline. Males were exposed for 2 weeks prior to mating, during mating and up to termination (total of 29 doses). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and during 3 days of lactation (53-57 doses).

All F0 males and females in the control, 100, 300, and 1000 mg/kg/day groups survived to the scheduled necropsies. Test substance-related clinical findings were noted for males and females in the 300 and 1000 mg/kg/day groups. Clear material around the mouth was noted for 11 of 12 males and all females at 1000 mg/kg/day and to a lesser extent (5 males and 6 females) at 300 mg/kg/day at 1-2 hours following dose administration generally throughout the respective treatment periods. The clinical findings noted for males and females in the 300 and 1000 mg/kg/day groups were considered non-adverse. Body weight and food consumption were unaffected by test substance administration.

Slightly higher mean liver weights (absolute, relative to final body weight, and relative to brain weight) were noted in the 1000 mg/kg/day F0 group males [5.9%, 5.9%, and 3.8% (values not statistically significant)] and females [14.2% (p<0.05), 13.8% (p<0.01), and 13.5% (p<0.05)]. These changes were of a minimal magnitude. Slightly higher mean liver weights were also noted at 1000 mg/kg/day in a previous 28-day study (Haas, 2013, WIL-537026) (approximately 12% higher in males and females) and in a concurrent 90-day study (Haas, Draft, WIL-537030) (9.5% and 8.1% higher in males and females, respectively) of the same test substance with no correlating adverse clinical pathology or histologic changes. Based on the lack of any correlating adverse clinical pathology or histologic changes in two additional studies on the same test substance with similar minimal liver weight elevations, microscopic examination of the liver was not performed in the current study and the slightly higher mean liver weights were considered toxicologically irrelevant. Therefore, the NOAEL for F0 male and female systemic toxicity was considered to be 1000 mg/kg/day.

No test substance-related effects were noted on F0 reproductive endpoints, including male and female mating and fertility, male copulation, and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition at any dosage level. Therefore, the no-observed-adverse-effect level (NOAEL) for F0 male and female reproductive toxicity was considered to be 1000 mg/kg/day (the highest dosage level evaluated) when the test substance was administered orally by gavage to Crl:CD(SD) rats.

Reference 2

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2013 - November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and EPA guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Name of test material (as cited in study report): EXP1200078
- Physical state: dark brown viscous liquid
- Storage condition of test material: at room temperature in the dark
- Analytical purity: 100%
- Lot/batch No.: E00007-285
- Expiration date of the lot/batch: end-2015

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: (P) 6 wks
- Weight at study initiation: (P) Males: 174 g to 234 g ; Females: 139 g to 183 g. Prior to the F0 cohabitation (study day 69), male body weights ranged from 437 g to 624 g and female body weights ranged from 254 g to 413 g. The animals were approximately 16 weeks old. All animals were randomly selected for pairing. Prior to the F1 cohabitation (PND 91), male body weights ranged from 441 g to 646 g and female body weights ranged from 228 g to 364 g. The animals were 97-105 days old at pairing.
- Fasting period before study: no
- Housing: all F0 and F1 parental test animals were housed individually, except during the mating period, in stainless steel wire-mesh cages suspended above cage-board. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the males remained housed in suspended wire mesh cages until the scheduled necropsy of the parental generations, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding. The dams were housed in these cages until weaning on lactation day 21. Following weaning of the litters in each generation (F1 and F2), the dams were individually housed in suspended wire-mesh cages until the scheduled necropsy, and the weaned F1 pups were housed together by litter for 1 week. Beginning on PND 28, the F1 offspring were individually housed in suspended wire-mesh cages until pairing. Following pairing, F1 females were housed in litter boxes as described for the F0 females. F1 males remained in the suspended wire-mesh cages until necropsy. Females for which there was no evidence of mating were placed in plastic maternity cages with nesting material upon completion of a 14 day mating period. If these animals did not deliver after 25 days, they were returned to individual suspended wire-mesh cages.
- Diet: ad libitum PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water: ad libitum reverse osmosis-purified (on site) drinking water
- Acclimation period: 16 days (and 7 days to allow for adaptation to the automatic watering system)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 to 23.2
- Humidity (%): 34.2 to 51.9
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 08 January 2014 To: 25 September 2014

Route of administration:

oral: gavage

Vehicle:

other: mineral oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The vehicle was dispensed approximately weekly for administration to the control group and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light. The vehicle was mixed throughout the sampling and dose administration procedures. Test substance concentrations of 0, 20, 60 and 200 mg/mL were prepared.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- When evidence of mating was not apparent after 14 days, the female was placed in a plastic maternity cage with nesting material, with no further opportunity for mating.
- After successful mating each pregnant female was housed in an individual plastic cage with nesting material.
All females were allowed to deliver naturally and rear their young to weaning (PND 21).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. Test substance formulations at concentrations of 0.1 and 400 mg/mL were previously assessed for homogeneity and stability following 12 days of room temperature storage (Coffee, 2013, WIL-537029). Therefore, stability was not assessed in the current study. samples for homogeneity determination were collected from the top, middle, and bottom strata of the first test substance dosing formulations and on the largest-volume formulations prepared for use on the study. In addition, samples for resuspension homogeneity determinations were collected from the top and bottom strata of aliquots taken from the first 20 and 200 mg/mL dosing formulations following room temperature storage for 9 days; the aliquots were stirred for at least 30 minutes prior to sample collection. Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared during the first month of dose administration, then monthly thereafter.

Duration of treatment / exposure:

The F0 and F1 males and females were administered the test substance once daily for a minimum of 70 consecutive days prior to mating. Dose administration for the F0 and F1 males continued throughout mating and through the day prior to euthanasia, for a total of 127 132 doses and 138-148 doses, respectively. The F0 and F1 females continued to be dosed throughout mating, gestation, and lactation, through the day prior to euthanasia, for a total of 127-132 doses and 138-148 doses, respectively. A dosage volume of 5 mL/kg bw was used. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose.
The offspring of the F0 and F1 generations (F1 and F2 litters, respectively) in the present study were potentially exposed to the test substance in utero, as well as via the milk while nursing. The F1 pups selected for mating (25 sex/group) were directly administered the test substance following weaning (beginning on PND 21).

Frequency of treatment:

once daily

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on a previous 28-day toxicity study in rats (WIL-537026), in which male and female rats were dosed for 28 consecutive days at dosage levels of 100, 300 and 1000 mg/kg bw/day. No significant adverse effects were noted in that study, and therefore the same dosage levels were assessed in the current reproduction toxicity study.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: 1 hour following dose administration and weekly for all parental animals throughout the study period. Females expected to deliver were observed twice daily for dystocia or other difficulties.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual F0 and F1 male body weights were recorded weekly throughout the study and prior to the scheduled necropsy. Individual F0 and F1 female body weights were 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 lactation days 0 (when possible), 1, 4, 7, 14, and 21. After weaning (lactation day 21), weekly body weights were recorded for these females until the scheduled necropsy. For F0 and F1 females with no evidence of mating, weekly body weights are presented on the individual report tables until necropsy.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION : No

Oestrous cyclicity (parental animals):

Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the estrous cycle of each F0 and F1 female for 21 days prior to cohabitation and continuing until evidence of mating was observed or until the end of the mating period. The average cycle length was calculated for complete estrous cycles (i.e., the total number of returns to metestrus [M] or diestrus [D] from estrus [E] or proestrus [P], beginning 21 days prior to initiation of the mating period and continuing until the detection of evidence of mating). The cycle during which evidence of mating was observed for a given animal was not included in the mean individual estrous cycle length calculation. Vaginal lavages were also performed on the day of necropsy to determine the stage of the estrous cycle.

Sperm parameters (parental animals):

Parameters examined in P/F1 male parental generations:
testis weight, epididymis weight, prostate weight, sperm production rate, sperm motility, sperm morphology

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, age of balanopreputial separation/vaginal patancy (perforation)

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

The brain, spleen, and thymus were weighed from all F1 and F2 weanlings at the scheduled necropsies.

Postmortem examinations (parental animals):

SACRIFICE
All surviving F0 adults were euthanized by carbon dioxide inhalation following the selection of the F1 generation and completion of a clinical observation. All surviving F1 adults were euthanized by carbon dioxide inhalation following weaning of the F2 pups.

GROSS NECROPSY
- The necropsy included examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, and the thoracic, abdominal, and pelvic cavities, including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
according to guideline

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Gross necropsies with emphasis on developmental morphology and organs of the reproductive system were performed on nonselected F1 and F2 weanlings euthanized by carbon dioxide inhalation on PND 21. F1 and F2 organs (brain, spleen, and thymus) were collected from 1 randomly selected pup/sex/litter that survived to the scheduled termination on PND 21. These tissues and all gross lesions from F1 and F2 weanlings were preserved in 10% neutral buffered formalin for possible future histopathologic examination; all other tissues and the carcasses were discarded.

HISTOPATHOLOGY / ORGAN WEIGTHS
according to guideline

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.
Parental mating, fertility, copulation, and conception indices were analyzed using the Chi square test with Yates’ correction factor. Parental (weekly, gestation, and lactation) and offspring body weights and body weight changes, parental food consumption, food efficiency, estrous cycle lengths, pre-coital intervals, gestation lengths, former implantation sites, live litter sizes, unaccounted-for sites, numbers of pups born, balanopreputial separation data (day of attainment and body weight), vaginal patency data (day of attainment and body weight), anogenital distance data, absolute and relative organ weights, sperm production rates, epididymal and testicular sperm numbers, and ovarian primordial follicle counts were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of postnatal pup survival and pup sexes at birth (percentage of males per litter), percentages of motile and progressively motile sperm, and percentages of sperm with normal morphology were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance treated groups to the control group. Histopathological findings in the test substance-treated groups were compared to the control group using a two-tailed Fisher’s Exact test.

Reproductive indices:

Male (Female) Mating Index (%) = (No. of Males (Females) with Evidence of Mating, or Females Confirmed Pregnant) / Total No. of Males (Females) Used for Mating) x100

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 Confirmed Pregnant) x100

Female Fertility Index (%) = (No. of Females with Confirmed Pregnancy / Total No. of Females Used for Mating) x100

Female Conception Index (%) = (No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating, or Females Confirmed Pregnant)) x100

Offspring viability indices:

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

Postnatal Survival Between Birth and PND 0 or PND 4 (Pre Selection) (% Per Litter) = ((Sum of (Viable Pups Per Litter on PND 0 or PND 4 [Pre-Selection]/No. of Pups Born Per Litter)* )/ No. of Litters Per Group) x 100

Postnatal Survival for All Other Intervals (% Per Litter) = ((Sum of (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, 1 -4 (Pre Selection), 4 (Post Selection)-7, 7-14,14-21, birth -PND 4 (Pre-Selection), or 4 (Post Selection)-21


* = Pups that were found dead due to mechanical injury were excluded from pup viability calculations.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

clear material around the mouth

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:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): F0: Increased incidences of clear material around the mouth were noted at approximately 1 hour following dose administration in the 300 mg/kg bw/day group males beginning as early as study day 24 and in the 1000 mg/kg bw/day group males and females beginning as early as study day 4 and 22, respectively, and continuing sporadically throughout the study. This finding was attributed to the test substance but was not considered to be adverse due to the lack of signs of toxicity.
F1: Increased incidences of clear and/or red material around the mouth were noted at approximately 1 hour following dose administration in the 300 and 1000 mg/kg bw/day group F1 males and females generally beginning after the fourth week of dose administration and continuing sporadically throughout the study in a dose-related manner, with a higher incidence in the males. This finding was attributed to the test substance but was not considered to be adverse due to the lack of signs of toxicity.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): F0: Occasional differences from the control group (increases or decreases) were transient and/or did not demonstrate a dose-response profile; the changes in food consumption and mean body weights and body weight gains were not attributed to the test substance.
F1: The observed significant (p<0.05) difference from the control group consisted of lower mean body weight gain in the 1000 mg/kg bw/day group males during PND 112-119. Because the decrease was transient, was not of sufficient magnitude to result in statistically significantly lower mean body weights, and/or did not demonstrate a dose-response profile, the lower mean body weight gain in the 1000 mg/kg bw/day group males was not attributed to the test substance. The lower mean body weight gain in the 100 mg/kg bw/day group during gestation days 14-17 and subsequently during the overall gestation period (days 0-20) was without a dose response. Therefore, the lower mean body weight gain observed in the 100 mg/kg bw/day group was not attributed to the test substance.
Occasional significant (p<0.01) differences in lower mean food consumption in the 300 and 1000 mg/kg bw/day group males during PND 119-126 were transient and/or did not demonstrate a dose response profile; the changes in mean food consumption were not attributed to test substance administration. The lower mean food efficiency in the 100 mg/kg bw/day group during gestation day 14-17 and subsequently during the overall gestation period (days 0-20) was in the absence of a dose response. Therefore, the lower mean food efficiency noted in the 100 mg/kg bw/day group was not attributed to the test substance. During lactation, the only significant differences from the control group were lower mean food consumption (g/animal/day and/or g/kg/day) during lactation days 1-4 in the 100 mg/kg bw/day group and higher mean food consumption during lactation days 4-7 in the 1000 mg/kg bw/day group. Mean food consumption and food efficiency in these groups were similar to the control group when the entire lactation period (days 1-21) was evaluated, and the changes were transient and not indicative of a dose-response. Therefore, the lower mean food consumption noted in the 100 mg/kg bw/day group and the higher mean food consumption noted in the 1000 mg/kg bw/day group were not attributed to the test substance.

REPRODUCTIVE FUNCTION: No effect on male/female reproduction function, gestation length and the process of parturition was noted (F0/F1), as indicated by the reported indices.
ESTROUS CYCLE (PARENTAL ANIMALS) F0: The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. Mean estrous cycle lengths in the control, 300, and 1000 mg/kg bw/day group were higher than the maximum mean value in the WIL historical control data. The higher mean values were due to 8, 7, and 6 females with mean estrous cycle lengths of 7-19 days; 1 female in the 100 mg/kg bw/day group also had an extended estrous cycle length (16 days). Because longer estrous cycle lengths were observed similarly in the concurrent control group and the females in the 300 and 1000 mg/kg bw/day group mated and were pregnant, no relationship to the test substance was evident; no statistically significant differences were noted.
F1: Although mean estrous cycle lengths in the 300 and 1000 mg/kg bw/day groups were slightly longer than the control group, the values were within the WIL reproductive historical control range. Two females each in the 300 and 1000 mg/kg bw/day group were outliers, with mean estrous cycle lengths of 9.5 18 days. These females mated within the first estrous cycle during the mating period and were pregnant. Therefore, there were no effects on reproductive function in these females. In addition, none of these differences from the concurrent control group in mean estrous cycle length in the test substance-treated groups were statistically significant.

ORGAN WEIGHTS (PARENTAL ANIMALS) F0: There were test substance-related significantly (p<0.05 or p<0.01) higher liver weights relative to final body weight in 300 and 1000 mg/kg bw/day group males. Although these liver weights relative to final body weight in the 300 and 1000 mg/kg bw/day groups slightly exceeded the WIL Research historical control reference range, the relative liver weights for the control and 100 mg/kg bw/day groups were also out of the WIL Research reference range. Mean absolute liver weights in the 300 and 1000 mg/kg bw/day group males were 6.3% and 5.0% higher, respectively, but the changes were not statistically significant, and the absolute weights were within the WIL Research historical control range. There were no microscopic correlations to higher liver weights. The slightly higher weights were considered nonadverse.
Other significant (p<0.05) differences included lower absolute right epididymis weight in the 1000 mg/kg bw/day group males and higher uterus/cervix/oviduct weights (absolute and relative to final body weight and brain weight) in the 300 mg/kg bw/day group females. The right epididymis weight was within the WIL Research historical control range.
The higher uterus/cervix/oviduct weights in the 300 mg/kg bw/day group were primarily due to one female for which the weight of these organs was more than double the next highest weight in that group. In the absence of a dose-response profile (uterus/cervix/oviduct weights) and the presence of a single outlier value for the 300 mg/kg bw/day group females or the absence of the weight change affecting both absolute and relative weights (right epididymis weight), and the lack of a similar effect on the left epididymis in the 1000 mg/kg bw/day group males, these variations were considered to represent biological variability.
F1: There was a significantly (p<0.05) higher liver weight relative to final body weight in the 1000 mg/kg bw/day group F1 males. The change was slightly out of the WIL Research historical control range. However, there was no histologic correlation to this higher relative liver weight and no change in group mean absolute liver weight or in group mean liver weight relative to brain weight. Also, the change was in part a consequence of the slightly lower group mean final body weight in the 1000 mg/kg bw/day group F1 males. Therefore, any variation in group mean liver weights in the F1 males was considered unrelated to the test substance. There was a slightly lower (significant, p<0.05) group mean absolute spleen weight in the 1000 mg/kg bw/day group F1 females. Since there were no changes in the group mean relative spleen weights (relative to final body weight or relative to brain weight), and since the change in spleen weight was within the WIL Research historical control range, the change in absolute spleen weight was considered unrelated to the test substance.

GROSS PATHOLOGY (PARENTAL ANIMALS) F0: The mean number of implantation sites in the 1000 mg/kg bw/day group (13.4 per dam) was significantly (p<0.05) lower than the concurrent control group (15.9 per dam). A similar effect was not observed in the F1 generation. Therefore, this finding was not attributed to the test substance. No test substance-related effects were observed on the number of former implantation sites and the number of unaccounted-for sites in the 100 and 300 mg/kg bw/day groups. The differences between the control and test substance-treated groups were slight and not statistically significant.
F1: No test substance related effects were observed on the number of pups born, the number of former implantation sites, and the number of unaccounted-for sites.

HISTOPATHOLOGY (PARENTAL ANIMALS) F0: All microscopic findings were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance-related alteration in the prevalence, severity or histologic character of those incidental tissue alterations.
F1: There were no test substance-related histologic changes in F1 males or females. All histologic changes were considered to be incidental findings. There was no test substance-related alteration in the prevalence, severity or histologic character of those incidental tissue alterations.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of test substance related adverse effects

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING) F1: The mean number of F1 pups born (11.9 per dam) and mean live litter size on PND 0 (11.7 pups per dam) were lower in the 1000 mg/kg bw/day group compared to the concurrent control group (14.5 and 14.4 per dam, respectively). The differences were significant (p<0.01). The lower mean number of pups born in this group was attributed to the lower mean number of implantation sites and the slightly higher mean number of unaccounted for sites. A similar effect was not observed in the F1 generation, and the values were within the ranges of the WIL historical control data. Therefore, the lower mean number of F1 pups born and live litter size on PND 0 was not considered to be test substance-related.
F2: The only significant (p<0.05) difference from the control group was a lower mean live litter size on PND 0 in the 100 mg/kg bw/day group. No dose-response relationship was evident; therefore, this finding was not attributed to the test substance.

SEXUAL MATURATION (OFFSPRING) F1: Test substance administration had no effect on mean ages of attainment of F1 balanopreputial separation. The mean age of attainment of F1 vaginal patency in the 1000 mg/kg bw/day group (31.5 days) was significantly earlier than the concurrent control group (32.6 days). Mean body weight at the age of attainment of vaginal patency in this group (111.8 g) was slightly lower than the control group (119.1 g); the difference was not statistically significant. To further evaluate if the test substance affected pup development, anogenital distance was measured in the F2 pups. No test substance related effects were noted in the F2 pups. Therefore, the early age and body weight at attainment of vaginal patency in the F1 females in the 1000 mg/kg bw/day group was not attributed to the test substance.
F2: Mean F2 anogenital distances and/or anogenital distances relative to the cube root of the body weight in the 100 and 300 mg/kg bw/day group males were longer than the concurrent control group; the differences were generally significant (p<0.05 or p<0.01). However, the values for the 1000 mg/kg bw/day group F2 males were similar to the concurrent control group. Therefore, no dose-response relationship was evident and the differences for the males in the 100 and 300 mg/kg bw/day groups were not attributed to the test substance.
In the 300 and 1000 mg/kg bw/day group F2 females, the mean anogenital distances and/or anogenital distances relative to the cube root of the body weight were significantly (p<0.05 or p<0.01) longer than the concurrent control group. However, the values were similar to the mean in the WIL reproductive historical control data; the concurrent control group value was near the minimum mean value of the WIL reproductive historical control data. In addition, a test substance-related effect on vaginal patency would result in a smaller anogenital distance for females, the opposite of that observed in the F2 females in the 300 and 1000 mg/kg bw/day groups. Therefore, the longer anogenital distance in these females was not considered to be test substance-related.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of test substance related adverse effects

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of test substance related adverse effects

Reproductive effects observed:

not specified

The analyzed dosing formulations (between 95.3% and 108%) were within the acceptable range of 85% to 115% for suspensions and were homogenous.

The test substance was not detected in the vehicle formulation that was administered to the control group

Conclusions:

Based on the absence of substance related toxicological relevant adverse effects in a two-generation toxicity study in rats, the oral NOAELs for EXP1200078 for parental systemic toxicity, reproductive toxicity and neonatal toxicity are 1000 mg/kg bw/day (highest dose tested).

Executive summary:

EXP1200078 was tested for potential adverse effects in a 2-generation reproduction toxicity study in rats according to OECD 416 guideline.

Male and female rats (25/sex) were administered the test substance in mineral oil (0, 10, 300 or 1000 mg/kg bw/day) daily by oral gavage for 70 days prior to mating. The test substance was administered to offspring selected to become the F₁parental generation following weaning (beginning on PND 21). The F0 and F1 males continued to receive the test substance throughout mating and through the day prior to euthanasia. The F0 and F1 females continued to receive the test substance throughout mating, gestation, and lactation, and through the day prior to euthanasia. Offspring (25/sex/group) from the pairing of the F0 animals were selected prior to PND 21 to constitute the F1 generation. F0 males and females were exposed for 127 -132 days, and F1 males and females were exposed for 138 -148 days.

There were no test substance-related deaths in the F0 and F1 generations at any dosage level. Test substance-related clinical findings (red and/or clear material around the mouth) were noted in the 300 and 1000 mg/kg bw/day group males and females at approximately 1 hour following dose administration in both parental generations. Because there were no other indications of systemic toxicity at any dosage level, these findings were not considered adverse.

Higher mean liver weights relative to final body weight were noted in the F0 males in the 300 and 1000 mg/kg bw/day groups and the F1 males in the 1000 mg/kg bw/day group. The differences from the respective control groups were statistically significant, but the slight increase in weight was considered not adverse.

The mean number of former implantation sites in the 1000 mg/kg b/day group F0 females was statistically significantly lower than the control group, which resulted in a lower mean number of F1 pups born and lower mean live litter size on PND 0. However, this effect was not observed in the F1 females. Therefore, the finding observed in the F0 females was not attributed to the test substance.

The F1 females in the 1000 mg/kg bw/day group attained vaginal patency earlier than the control group. Therefore, anogenital distance was measured in the F2 pups on PND 1. No test substance-related effects on F2 anogenital distance were noted at any dosage level. Therefore, the early attainment of vaginal patency in the F1 females in the 1000 mg/kg bw/day group was not considered to be test substance-related.

Based on the absence of substance related toxicological relevant adverse effects the oral NOAELs in this study with EXP1200078 for parental systemic toxicity, reproductive toxicity and neonatal toxicity are 1000 mg/kg bw/day (highest dose tested).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

Studies were conducted according to GLP methodology with no adverse effects recorded at any dose up to the limit of 1000 mg/kg/day.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Quality of whole database:

Not a relevant route of exposure; material is non-volatile at ambient temperatures.

Effects on developmental toxicity

Description of key information

Two GLP studies were conducted up to the limit dose of 1000 mg/kg/day using the registration substance, and no adverse effects were observed in the shorter term study (OECD 414) or the two longer term studies (OECD 421 and 416).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2013 - Mar 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

EXP1200078
Lot no. E00048-256
Exp. date: 31 December 2014
Dark brown, opaque, viscous liquid

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, MI, USA
- Age at study initiation: females were 13 weeks old when paired for breeding
- Weight at study initiation: 205-296 g on gestation day 0
- Housing: Upon arrival and until pairing, all rats were individually housed in clean, stainless steel wire-mesh cages. The rats were paired for mating in the home cage of the male. Following positive evidence of mating, the females were returned to individual suspended wire-mesh cages.
- Diet (ad libitum): basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (ad libitum): municipal water
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 to 23.2
- Humidity (%): 39.2 to 54.7
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 03 December 2013 To: 10 January 2014

Route of administration:

oral: gavage

Vehicle:

other: mineral oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Lot/batch no.: 2BK0397 and 1CK0210

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the test substance formulations in the vehicle at concentrations of 0.1 and 400 mg/mL following 12 days of room temperature storage were previously established in a separate study (Coffee, 2013, WIL-537029). Therefore, homogeneity and stability assessments were not conducted in the current study.
Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared during the first and last weeks of the in-life phase of the study. One set of samples from each collection was subjected to the appropriate analyses. The remaining set of samples was stored at room temperature as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a validated high performance liquid chromatography method with fluorescence detection.

Details on mating procedure:

- Impregnation procedure: cohoused 1:1 with each animal judged to be in good health and meeting acceptable body weight requirements was placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding. Resident males were untreated, sexually mature rats. These rats were maintained under similar laboratory conditions as the females.
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug or sperm in vaginal lavage referred to as day 0 of pregnancy

Duration of treatment / exposure:

Gestation days 6-19

Frequency of treatment:

Once daily

Duration of test:

Gestation day 0 through 20

Dose / conc.:

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

Remarks:

Vehicle control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dosage levels were selected based on the results of a previous 28-day toxicity study in rats (Haas, 2013, WIL-537026). In that study, the test substance was administered orally by gavage once daily to male and female rats for 28 consecutive days at dosage levels of 100, 300, and 1000 mg/kg/day. All animals survived to the scheduled euthanasia and there were no treatment-related effects on body weights or food consumption at any dosage level tested. Test substance-related higher mean liver weights were noted in the 1000 mg/kg/day group males and females at the scheduled necropsy, but were not considered adverse. As a result, dosage levels of 100, 300, and 1000 mg/kg/day (the same dosage levels used in the previous study) were assessed in the current study.
- Rationale for animal assignment: The bred females were assigned to groups using a WTDMS™ computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design.

Maternal examinations:

CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual clinical observations were recorded daily from gestation days 0 through 20. Animals were also observed for signs of toxicity approximately 1 hour following dose administration.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual maternal body weights were recorded on gestation days 0 and 6-20 (daily). Group mean body weights were calculated for each of these days. Mean body weight changes were calculated for each corresponding interval and also for gestation days 6-9, 9-12, 12-15, 15-20, and 6-20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: The thoracic, abdominal, and pelvic cavities were opened by a ventral mid line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: placentae examined, uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10% ammonium sulfide solution for detection of early implantation loss.

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [all per litter]
Half of the littr by Wilson's sectioning technique and hlaf by midcoronal slice.

Statistics:

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group. Data obtained from nongravid animals were excluded from statistical analyses.
Maternal body weights (absolute and net), body weight changes (absolute and net), and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites, and viable fetuses, and fetal body weights (separately by sex and combined) were subjected to a parametric one-way ANOVA to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and fetal sex distribution), total fetal malformations and developmental variations (external, visceral, skeletal, and combined) and each particular external, visceral, and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the nonparametric ANOVA revealed significant (p<0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.

Indices:

Postimplantation Loss/Litter = (No. Dead Fetuses, Resorptions (Early/Late)/Group) / (No. Gravid Females/Group)
Summation Per Group (%) = (Sum of Postimplantation Loss/Litter (%)) / (No. Litters/Group)
Postimplantation Loss/Litter (%) = (No. Dead Fetuses, Resorptions (Early/Late)/Litter) / (No. Implantation Sites/Litter) x 100
Summation per Group (%) = (Sum of Viable Fetuses Affected/Litter (%)) / (No. Litters/Group)
Viable Fetuses Affected/Litter (%) = (No. Viable Fetuses Affected/Litter) / (No. Viable Fetuses/Litter) x 100

Historical control data:

These are available.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test substance-related absence of body weight gain (0 g) was noted in the 1000 mg/kg/day group following the onset of dose administration (gestation day 6-7) and resulted in a lower mean body weight gain in this group during the gestation day 6-9 cumulative interval; differences from the control group were significant (p<0.01). Although mean body weight gains in the 1000 mg/kg/day group were similar to the control group throughout the remainder of the treatment period (gestation days 9-12, 12 15, and 15-20), the initial decrement was of sufficient magnitude to result in a significantly (p<0.01) lower mean body weight gain when the entire treatment period (gestation days 6-20) was evaluated. In addition, mean body weights in this group were up to 5.6% lower than the control group during the treatment period; the differences were significant (p<0.05) during gestation days 9-11 and 14-20. Therefore, the effect on mean body weight gain noted at the initiation of dose administration was considered to be adverse. Mean net body weight and net body weight gain in the 1000 mg/kg/day group were significantly (p<0.05 or p<0.01) lower than the control group, while mean gravid uterine weight in this group was similar to the control group.

In the 300 mg/kg/day group, sporadically lower mean body weight gains, primarily during gestation days 6-9 and 15-20, were noted during the treatment period compared to the control group; the differences were significant (p<0.05) on gestation day 17-18 only. Although these sporadic lower mean body weight gains resulted in a significantly (p<0.05) lower mean body weight gain when the entire treatment period (gestation days 6-20) was evaluated, these differences were not of sufficient magnitude to affect mean absolute body weights. Therefore, the lower mean body weight gains noted in the 300 mg/kg/day group were considered to be test substance-related but not adverse.

Mean maternal body weight gains in the 100 mg/kg/day group were unaffected by test substance administration during the treatment period; differences from the control group were generally slight and not statistically significant. Despite the absence of effects on mean body weight gain, significantly (p<0.05) lower (4.7% to 5.2%) mean body weights were noted in this group during gestation days 9-11 and 14-16 compared to the control group.

Significantly (p<0.05 or p<0.01) lower mean net body weight gains were noted in the 100 and 300 mg/kg/day groups compared to the control group, resulting in a significantly (p<0.05) lower mean net body weight at 100 mg/kg/day and a slightly lower (not statistically significant) mean net body weight at 300 mg/kg/day. In the absence of a dose response, the effects on mean net body weight and net body weight gain were not considered test substance-related. Mean gravid uterine weights in these groups were comparable to the control group.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Lower (generally significant, p<0.05 or p<0.01) mean food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 1000 mg/kg/day group during the study (gestation days 6-9, 12-15, and/or 15-20) and resulted in significantly (p<0.01) lower mean food consumption when the entire treatment period (gestation days 6-20) was evaluated. The lower mean food consumption noted during gestation days 6-9 and 6-20 corresponded to the lower mean body weight gains noted during these intervals, and therefore was considered to be test substance-related and adverse.

Mean food consumption in the 100 and 300 mg/kg/day groups were unaffected by test substance administration. In the 300 mg/kg/day group, mean food consumption was significantly (p<0.05) lower than the control group on gestation day 17-18 (evaluated as g/animal/day only), which corresponded to a lower mean body weight gain on this day.

However, the lower mean food consumption on this day was not considered to be test substance-related, due to the transient nature. Furthermore, mean food consumption in the 300 mg/kg/day group was similar to the control group during the cumulative intervals (gestation days 6-9, 9-12, 12-15, and 15-20) and when the entire treatment period (gestation days 6-20) was evaluated. In the 100 mg/kg/day group, significantly (p<0.05) lower mean food consumption (evaluated as g/animal/day only) was noted during gestation days 6-9 and subsequently when the overall treatment period (gestation
days 6-20) was evaluated. However, due to the slight nature of the differences (1 g to 2 g) and in the absence of effects on body weight gain in the group, the lower mean food consumption noted in the 100 mg/kg/day group was not considered to be test substance related. No other statistically significant differences were noted in the 100 and 300 mg/kg/day groups.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Intrauterine growth and survival were unaffected by test substance administration at dosage levels of 100, 300, and 1000 mg/kg/day. Parameters evaluated included postimplantation loss, live litter size, mean fetal body weights, and fetal sex ratios. Mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre implantation loss were similar across all groups. Differences from the control group were slight and not statistically significant.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Differences from the control group were slight and not statistically significant.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Differences from the control group were slight and not statistically significant.

Dead fetuses:

no effects observed

Description (incidence and severity):

None found in controls or any dose group.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

With the exception of 1 and 2 females in the control and 300 mg/kg/day groups, respectively, all females were determined to be gravid.

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: lower mean body weight gain

Details on maternal toxic effects:
No mortality nor adverse clinical signs were observed: increased incidences of clear and red material around the mouth were noted for females in the 1000 mg/kg/day group, starting at gestation day 12 and continued sporadically through gestation day 19. These findings did not persist to the daily examinations the following day and therefore were not considered adverse.
Body weight: A statistically significant lower mean body weight gain in the 1000 mg/kg/day group was noted during gestation days 6-9.This resulted in a significant lower mean body weight gain for the entire treatment period, although mean body weight gains in the 1000 mg/kg/day group were similar the the control group from gestation days 9 onwards. Mean body weights in this group were statistically significantly decreased during the treatment period: 5.6% lower compared to controls. In addition, some effects on mean body weight and/or body weight gain were observed in the 100 and 300 mg/kg/day dose groups. However, no dose response was observed, and the actual mean body weights were only approx 4% lower compared to controls. Therefore, these effects are not considered substance related.
A significantly lower mean food consumption was observed in the 1000 mg/kg/day dose goup.
At necropsy no test substance related findings were observed in any dose group.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food efficiency

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Key result

Abnormalities:

no effects observed

Description (incidence and severity):

No abnormalities / malformations detected in any soft tissue, skeletal exam, external or any other parameter.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Changes in sex ratio:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No significant changes in the laparohysterectomy data were observed. Malformations were observed in all treatment (and control) groups, and were considered spontaneous in origin. No statistically significant difference from the control group was noted, no dose related manner was observed, and/or effects were within the historical control data ranges.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks:

Test material given to maternal animals.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

changes in postnatal survival

external malformations

skeletal malformations

visceral malformations

other:

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

The analyzed dosing formulations were within the range 85% to 115%. The test substance was not detected in the analyzed vehicle formulation (Group 1).

Results of the analyses of dosing formulations are summarized below.

Results of Concentration Analyses

	Mean Concentration, mg/mL (% of Target)
Date of Preparation	Group 2 (20 mg/mL)	Group 3 (60 mg/mL)	Group 4 (200 mg/mL)
19 December 2013	20.2 (101)	60.6 (101)	207 (104)
02 January 2014	20.7 (104)	61.7 (103)	207 (103)

 

Conclusions:

In an OECD414 test gudeline study, female rats orally dosed with EXP1200078 during gestation days 6 through 19, did show adverse effects at the highest dose tested and no effects were observed on the fetuses. Therefore, the NOAEL for maternal toxicity was determined to be 300 mg/kg bw/day and for developmental toxicity the NOAEL is >=1000 mg/kg bw/day.

Executive summary:

EXP1200078 was administered by daily oral gavage to female Sprague Dawley rats during gestation days 6 through 19 at dose levels of 0, 100, 300 or 1000 mg/kg bw/day according to the OECD 414 guideline. On gestation day 20 a laparohysterectomy was performed on each female. All females in the 0, 100, 300 and 1000 mg/kg/day groups survived to the scheduled necropsy. A lower mean body weight gain was noted in the 1000 mg/kg/day group during gestation days 6 -9 resulting in a lower mean body weight gain and body weight mean over the entire treatment period. Mean food consumption in this group was lower than the control group throughout the treatment period. Mean gravid uterine weight in the 1000 mg/kg/day group was similar to the control group. No test substance related macroscopic findings were observed at any dose level. Therefore, the NOAEL for maternal toxicity is determined to be 300 mg/kg/day.

Intrauterine growth, survival and fetal morphology were unaffected by maternal test substance administration at all dose levels. Therefore, the NOAEL for embryo/fetal development was considered to be >=1000 mg/kg bw/day based on the absence of adverse effects.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2012 - May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study in accordance with OECD guidelines

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD 421 Reproduction/Developmental Toxicity Screening Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

EXP1200078
Lot no. E00048-256
Exp. date: 31 December 2014
Dark brown, opaque, viscous liquid

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: At the initiation of dose administration (study day 0 for females and study day 14 for males), the males and females were approximately 12.5 and 10.5 weeks old, respectively.
- Weight at study initiation: Male body weights 397-482 g on study day 14 and female body weights 199-281 g on study day 0
- Fasting period before study: no
- Housing: Following receipt and until pairing, all F0 animals were housed individually in clean, stainless steel wire mesh cages suspended above cage board. 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.
- Diet: ad libitum PMI Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water: ad libitum reverse osmosis-purified (on site) drinking water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21.4°C to 21.6°C
- Humidity: 32.5% to 46.2%
- Air changes: a minimum of 10 fresh air changes per hour
- Photoperiod: 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod

IN-LIFE DATES: From: 29 January 2013 To: 27 March 2013

Route of administration:

oral: gavage

Vehicle:

other: Mineral oil, USP

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18°C to 24°C). The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The dosage volume for all groups was 5 mL/kg.

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

VEHICLE
- Concentration in vehicle: 0, 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg
- Lot/batch no.: lot nos. 1AL0557 and 2BC0539

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared during the in-life phase of the study. The analyzed dosing formulations were within WIL Research’s SOP range for suspensions (85% to 115%). The test substance was not detected in the analyzed vehicle formulation that was administered to the control group (Group 1).

Details on mating procedure:

- M/F ratio per cage: 1
- Proof of pregnancy: vaginal plug or the presence of sperm following vaginal lavage referred to as day 0 of pregnancy.
- After successfull mating each pregnant female was caged: individually into plastic maternity cages with nesting material, ground concob bedding.

Duration of treatment / exposure:

The males were dosed during study days 14-41 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 doses. The females were dosed during study days 0 through the day prior to euthanasia (28 days prior to pairing through lactation day 3) for a total of 53-57 doses. Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 25) for a total of 53-66 doses.

Frequency of treatment:

Daily.

Duration of test:

Until lactation day 4.

Dose / conc.:

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

Remarks:

Vehicle control

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

Dosage levels were determined based on the results of previous studies, including a 14 day (Haas, 2013, WIL-537025) and a 28-day (Haas, 2013, WIL-537026) oral gavage toxicity study in male and female Sprague Dawley rats. In the 14-day study (Haas, 2013, WIL-537025), the test substance in the vehicle (mineral oil) was administered orally by gavage once daily for 14 consecutive days to 3 groups consisting of 5 rats/sex at dosage levels of 100, 300, and 1000 mg/kg/day. All animals survived to the scheduled necropsy. Test substance-related effects were limited to low incidence of clear material around the mouth noted in males at 1000 mg/kg/day. Based on these results, the maximum tolerated dose (MTD) was considered to be 1000 mg/kg/day. In the 28-day study (Haas, 2013, WIL-537026), the test substance in the vehicle (mineral oil) was administered orally by gavage once daily for 28 consecutive days to 3 groups consisting of 5 rats/sex at dosage levels of 100, 300, and 1000 mg/kg/day. There were no test substance-related deaths. Test substance-related effects were limited to clear material around the mouth and higher liver weights in the 1000 mg/kg/day group males and females and a lower seminal vesicles weight in the 1000 mg/kg/day group males. However, none of these effects were considered to be adverse. Therefore, the no-observed-adverse-effect level (NOAEL) was considered to be 1000 mg/kg/day, the highest dosage level administered.
The selected route of administration for this study was oral (gavage) because this is a potential route of human exposure. Historically, this route has been used extensively for studies of this nature.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily. Each male and female was also observed for signs of toxicity 1-2 hours following dose administration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly. Individual male body weights were recorded weekly throughout the study, beginning on the first day of dose administration, 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 0 (when possible), 1, and 4.

FOOD CONSUMPTION: Yes
- Time schedule: Individual male and female food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the breeding 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 the breeding period, food consumption for females with no evidence of mating was measured on a weekly basis until the scheduled euthanasia.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
A complete necropsy was conducted on all F0 parental animals at the scheduled termination. All surviving F0 adults were euthanized by carbon dioxide inhalation. Males were euthanized following completion of the mating period. 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) or post-cohabitation day 25 (females with no evidence of mating).

Necropsy 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. At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered
formalin (except as noted):
Brain
Pituitary gland
Coagulating glands
Prostate gland
Kidneys (2)
Seminal vesicles (2)
Liver (sections of 2 lobes)
Testes with epididymidesa (2)
Mammary glands and vas deferens
Ovaries and oviducts (2)
Uterus with cervix and vagina
All gross lesions

Microscopic examination was performed on the following tissues from all animals in the
control and 1000 mg/kg/day groups, and gross lesions from all groups, at the scheduled necropsies:
Cervix
Seminal vesicles
Coagulating glands
Testes
Epididymides
Uterus
Mammary glands (female only)
Vagina
Ovaries
Vas deferens
Pituitary gland
All gross lesions (internal)
Prostate gland

Ovaries and uterine content:

Examination included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes (dams delivered)
- Number of late resorptions: Yes (dams delevered)

Fetal examinations:

- External examinations: Yes [all per litter)
- Each litter was examined daily for survival, and all deaths were recorded.
- Litters were examined daily for survival and any adverse changes in appearance or behavior.
- Pups were individually weighed on PND 1 and 4. Mean pup weights were presented by sex for each litter and by dose group.
- Pups were individually sexed on PND 0 and 4.

Statistics:

Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Parental mating, fertility, conception, and copulation indices were analyzed using the Chi-square test with Yates’ correction factor (Hollander and Wolfe, 1999). 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 former implantation sites, number of pups born, live litter size on PND 0, corpora lutea, unaccounted-for sites, absolute and relative organ weights, and pre-coital intervals were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA (Kruskal and Wallis, 1952) to determine intergroup differences. If the 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. Histopathological findings of each treated group were compared to those of the control group by Fisher’s Exact test (Steel and Torrie, 1980).

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) = (Sum of (Viable Pups Per Litter on PND 0 or PND 4/No. of Pups Born Per Litter)/ No. of Litters Per Group) x 100

Other Intervals (% Per Litter) = (Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)/ No. of Litters Per Group) x 100

Where N = PND 0-1 and 1-4

Historical control data:

Data from this stuey were compared to historic control data from similar studies. All results from this current study were in bounds of the historic controls.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Slight higher mean liger weights (absolute, relative to final body weight, and relative to brain weight) were noted in the 1000 mg/kg bw/day F0 group males [5.9%, 5.9% and 3.8% (values not statistically significant)] and females [14.2% (p<0.05), 13.8% (p<0.01), and 13.5% (p<0.05)].
Based on lack of any correlating adverse clinical pathology or histologic changes in two additional studies on the same test substance with similar minimal liver weight elevations, these findings were not considered adverse and toxicologically relevant.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

mortality

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The mean number of pups born, live litter size and the percentage of males at birth in the 100, 300, and 1000 mg/kg bw/day groups were comparable to the control group values.
Postnatal survival in the 100, 300, and 1000 mg/kg bw/day groups were unaffected by parental test substance administration.
Mean male and female pup birth weights (PND 1) and body weights and body weight changes in the 100, 300, and 1000 mg/kg bw/day groups were unaffected by parental administration of the test substance during PND 1-4.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Remarks:

This refers to maternal dose within the context of this study

Sex:

male/female

Basis for effect level:

other: No effects seen on any up parameter inculding survival to PND 4 and subsequent examinations.

Remarks on result:

other:

Remarks:

The NOAEL for neonatal toxicity was considered to be 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.

Key result

Abnormalities:

no effects observed

Developmental effects observed:

not specified

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Conclusions:

There were no adverse test substance-related clinical findings and no effects on mean body weights, body weight changes, and food consumption noted at any dosage level. Furthermore, there were no adverse effects on organ weights or macroscopic/microscopic alterations in F0 males and females at any dosage level. Therefore, the NOAEL for F0 male and female systemic toxicity was considered to be 1000 mg/kg bw/day.
The NOAEL for neonatal toxicity was considered to be 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.

Executive summary:

EXP1200078 was administered by daily oral gavage to male and female Sprague Dawley rats at dose levels of 0, 100, 300 or 1000 mg/kg bw/day according to the OECD 421 guideline. Males were exposed for 2 weeks prior to mating, during mating and up to termination (total of 29 doses). The females were exposed for 2 weeks prior to mating, during mating, during post-coitum, and during 3 days of lactation (53-57 doses).

All F0 males and females in the control, 100, 300, and 1000 mg/kg bw/day groups survived to the scheduled necropsies. Test substance-related clinical findings were noted for males and females in the 300 and 1000 mg/kg bw/day groups. Clear material around the mouth was noted for 11 of 12 males and all females at 1000 mg/kg bw/day and to a lesser extent (5 males and 6 females) at 300 mg/kg bw/day at 1-2 hours following dose administration generally throughout the respective treatment periods. The clinical findings noted for males and females in the 300 and 1000 mg/kg bw/day groups were considered non-adverse. Body weight and food consumption were unaffected by test substance administration.

Slightly higher mean liver weights (absolute, relative to final body weight, and relative to brain weight) were noted in the 1000 mg/kg bw/day F0 group males [5.9%, 5.9%, and 3.8% (values not statistically significant)] and females [14.2% (p<0.05), 13.8% (p<0.01), and 13.5% (p<0.05)]. These changes were of a minimal magnitude. Slightly higher mean liver weights were also noted at 1000 mg/kg/day in a previous 28-day study (Haas, 2013, WIL-537026) (approximately 12% higher in males and females) and in a concurrent 90-day study (Haas, Draft, WIL-537030) (9.5% and 8.1% higher in males and females, respectively) of the same test substance with no correlating adverse clinical pathology or histologic changes. Based on the lack of any correlating adverse clinical pathology or histologic changes in two additional studies on the same test substance with similar minimal liver weight elevations, microscopic examination of the liver was not performed in the current study and the slightly higher mean liver weights were considered toxicologically irrelevant. Therefore, the NOAEL for F0 male and female systemic toxicity was considered to be 1000 mg/kg bw/day.

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 comparable to the control group values. Postnatal survival in the 100, 300, and 1000 mg/kg/day groups were unaffected by parental test substance administration. Mean male and female pup birth weights (PND 1) and body weights and body weight changes in the 100, 300, and 1000 mg/kg/day groups were unaffected by parental administration of the test substance during PND 1-4.

Therefore, the NOAEL for neonatal toxicity was considered to be 1000 mg/kg bw/day based on the absence of effects on F1 offspring at all dose levels.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

All the studies were GLP, and no adverse effects were noted at any dose up to the limit of 1000 mg/kg/day.

Effect on developmental toxicity: via inhalation route

Quality of whole database:

Not a relevant route of exposure; material is non-volatile at ambient temperatures.

Justification for classification or non-classification

The registration substance had a NOAEL of 1000 mg/kg/day in rodents in a OECD 414, 421 and 416, as such, in accordance to Regulation (EC) 1272/2008 the substnace does not meet the classification criteria for reproductive toxicity.