tris(branched-alkyl) borate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on the absence of effects on F0 and F1 reproductive performance at any dosage level in a two-generation study, a dosage level of 400 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for parental reproductive toxicity of test item administered orally by gavage to Crl:CD(SD) rats. Based on the lower live litter size on PND 0 for F2 pups, decreased postnatal survival, and lower mean body weights and body weight gains during the pre-weaning period for F1 and F2 pups at 400 mg/kg/day, the NOAEL for neonatal toxicity was considered to be 100 mg/kg/day. Based on moribundity for F0 females and mortality for F1 males and females, as well as lower mean body weights, body weight gains, and/or food consumption for F0 males and F1 males and females at 400 mg/kg/day, the NOAEL for parental systemic toxicity was considered to be 100 mg/kg/day (OECD 415 and EPA OPPTS 870.3800).

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 October 2014 to 09 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

yes

Remarks:

various deviations with no negative impact on study (see Appendix A, attached)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

yes

Remarks:

various deviations with no negative impact on study (see Appendix A, attached)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION PERIOD
- Sexually mature male and virgin female Crl:CD(SD) rats were used as the test system on this study. This species and strain of animal is recognized as appropriate for reproduction studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, WIL Research has reproductive historical control data for the Crl:CD(SD) rat.
- The number of animals selected for the study (25 rats/sex/group) was based on the United States EPA Health Effects Test Guidelines: OPPTS 870.3800, Reproduction and Fertility Effects, Aug-1998 and the OECD Guidelines for the Testing of Chemicals Guideline 416, Two-Generation Reproduction Toxicity Study, 22-Jan-2001, which recommend evaluation of approximately, and not less than, 20 pregnant females.
- Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality in each generation of the study, this was an appropriate number of animals to achieve the desired number of animals in each generation.
- Crl:CD(SD) rats (110 males and 110 females) were received in good health from Charles River Laboratories, Inc., Stone Ridge, NY, on 23-Oct-2014.
- The animals were approximately 32 days old upon receipt.
- Each animal was examined by a qualified biologist on the day of receipt.
- The day following receipt, all animals were weighed and clinical observations were recorded.
- Each rat was uniquely identified by a Monel metal ear tag displaying the animal number and housed for 12 days for acclimation purposes.
- During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior.

F0, F1 AND F2 ANIMAL HOUSING
- Following receipt (F0) or selection (F1) and until cohabitation, all F0 and F1 parental animals were housed 2-3 per cage by sex in clean, solid-bottom cages with bedding material (Bed-O'Cobs; The Andersons, Cob Products Division, Maumee, OH). - The bedding material is periodically analyzed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research.
- The rats were paired for mating in a clean, solid-bottom cages with bedding material. Following the breeding period, the F0 and F1 males were returned to their original social group of 2-3 males until the scheduled necropsy of the parental generations.
- Following positive evidence of mating, the F0 and F1 females were individually housed in plastic maternity cages with bedding 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 returned to their original social group of 2-3 females until the scheduled necropsy of the parental generations, and the weaned F1 pups were housed together by litter for 1 week.
- Beginning on PND 28, the F1 offspring were housed 2-3 per cage by sex in clean, solid-bottom cages with bedding material until pairing.
- Females for which there was no evidence of mating were placed in solid-bottom cages with nesting material upon completion of a 14-day mating period.
- Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). The animal facilities at WIL Research are accredited by AAALAC International.
- Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER AND MAINTENANCE OF F0 AND F1 ANIMALS
- The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Feed lots used during the study were documented in the study records.
- Feeders were changed and sanitized once per week.
- Municipal water supplying the facility was sampled for contaminants according to WIL Research SOPs. Results of the diet and water analyses are maintained at WIL Research.
- No contaminants were present in animal feed or water at concentrations
sufficient to interfere with the objectives of this study.
- Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study.

ENVIRONMENTAL CONDITIONS
- All rats were housed throughout the acclimation period and during the study in an environmentally controlled room.
- The room temperature and relative humidity controls were set to maintain environmental conditions of 71°F ± 5°F (22°C ± 3°C) and 50% ± 20%, respectively.
- Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 68.2°F to 70.5°F (20.1°C to 21.4°C) and mean daily relative humidity ranged from 33.6% to 62.4% during the study.
- Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes.
- Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

VEHICLE
The vehicle used in preparation of the test substance formulations and for administration to the control group was arachis (peanut) oil, NF (lot nos. 2DK0142, 1DB0384, 2DF0016, 2EA0218, and 2EA0347; exp. dates: 31-Oct-2015, 31-Jan-2015, 30-Apr-2015, 31-Jan-2016, and 31-Jan-2016, respectively).

PREPARATION
- The vehicle suspension was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light.
- The vehicle was mixed throughout the preparation, sampling, and dose administration procedures. Dosing formulations were prepared at the test substance concentrations indicated in the table below.
- The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
- The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- The test substance and vehicle control formulations were administered to the F0 and F1 males and females orally by gavage, via an appropriately sized flexible, Teflon-shafted, stainless steel ball-tipped dosing cannula, 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.
- The F0 and F1 females continued to be dosed throughout mating, gestation, and lactation, through the day prior to euthanasia.
- The F0 males and females were dosed for 128-133 consecutive days, and F1 males and females were directly dosed for 136-147 consecutive days.
- A dosage volume of 5 mL/kg was used. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
- Dosage levels were selected by the Sponsor based on the results of previous studies with the test substance.
- In a previous developmental toxicity study (Herberth, 2015, WIL-168214), rats were administered the test substance from gestation days 6-19 at dosage levels of 250, 500, and 1000 mg/kg/day. All animals survived to the scheduled euthanasia on gestation day 20 and there were no significant clinical observations noted in any group during the study. Lower fetal body weights (male and female) were observed in all dose groups in the previous study; however, the reductions were most significant at 1000 mg/kg/day. As a result, dosage levels of 25, 100, and 400 mg/kg/day were chosen for use on the current study.
- 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).
- The selected route of administration for this study was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Details on mating procedure:

BREEDING PROCEDURES
- The animals were paired on a 1:1 basis within each treatment group after a minimum of 70 days of treatment.
- All animals were randomly selected for cohabitation, avoiding sibling matings.
- A breeding record containing the male and female identification
numbers and the start date of cohabitation was maintained.
- Each female was cohabitated with 1 male in a solid-bottom cage containing bedding material.
- Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist.
- Each mating pair was examined daily.
- The day when evidence of mating was identified was termed gestation day 0. The animals were separated, and the female was housed in an individual solid-bottom cage with bedding material.
- When evidence of mating was not apparent after 14 days, the female was placed in solid-bottom cages with bedding material, with no further opportunity for mating.
- Each generation was mated once to produce 1 litter per dam (the F1 and F2 litters). – Prior to the F0 cohabitation (study day 70), male body weights ranged from 374 g to 744 g and female body weights ranged from 211 g to 322 g. The animals were approximately 16 weeks old. All animals were randomly selected for pairing.
- Prior to the F1 cohabitation (PND 98), male body weights ranged from 407 g to 638 g and female body weights ranged from 222 g to 381 g. The animals were approximately 14-15 weeks old.
- For the purpose of calculating pre-coital intervals, rats cohabited over a 12-hour dark cycle were considered to have been paired for 1 day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLING AND ANALYSIS
- Homogeneity of the test substance in the vehicle for a minimum of 7 days was previously demonstrated at concentrations spanning the range used in the current study (Haas, 2015, WIL-168211). In addition, stability of the test substance in the vehicle was demonstrated in a previous validation study (Van Hoven et al., 2014).
- Samples for homogeneity and/or concentration determinations were collected from the top, middle, and bottom strata of each test substance dosing formulation and from the middle stratum of the control group formulations prepared during the first month of the study, and once a month thereafter for the remainder of the study.
- In addition, due to changes in batch size from previous studies, samples for resuspension homogeneity determination were collected from the top and bottom strata of an aliquot taken from the first test substance dosing formulations prepared on study following room temperature storage for 8 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection.
- All samples were stored at room temperature, protected from light until shipped to Wildlife International, Easton, MD, for analysis. All analyses were conducted using a validated method.

Duration of treatment / exposure:

- The F0 males and females were dosed for 128-133 consecutive days.
- The F1 males and females were directly dosed for 136-147 consecutive days.

Frequency of treatment:

Daily

Details on study schedule:

- See diagram summarising study design (attached).

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25 males and 25 females

Control animals:

yes, concurrent vehicle

Details on study design:

ASSIGNMENT OF F0 ANIMALS TO TREATMENT GROUPS
- At the conclusion of the acclimation period, all available F0 animals were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was selected for use in the computerized randomization procedure based on body weight stratification randomized in a block design.
- At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMS. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated and the animals were then arranged into groups according to the printout.
- Animals not assigned to study were transferred to the WIL Research colony.
- The experimental design for this study consisted of 3 test substance-treated groups and 1 control group, composed of 25 rats/sex each.
- The selected animals were approximately 6 weeks old at the initiation of test substance administration.
- Male body weights ranged from 174 g to 235 g and female body weights ranged from 126 g to 182 g on the initial day of test substance administration (study day 0).

Positive control:

Not applicable

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS AND SURVIVAL (F0 AND F1)
- All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity.
- Detailed physical examinations were recorded weekly for all parental animals throughout the study period.
- In addition, animals were observed for signs of toxicity approximately 1 hour following dose administration.
- The absence or presence of findings was recorded for all animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals.
- Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.
- Due to social housing, some observations could not be attributed to a single animal. In these instances, the observation was recorded in a separate computer protocol for the social group.

BODY WEIGHTS (F0 AND F1)
- 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.
- 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 treatment period (males and females) and for the entire F0 and F1 treatment period (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, 4, 7, 14, and 21. Body weight changes are presented for each of these intervals and for the entire gestation and lactation intervals (days 0-20 and 1-21, respectively).
- After weaning (lactation day 21), weekly body weights were recorded for these females until the scheduled necropsy.
- For F0 females with no evidence of mating, weekly body weights are presented on the individual report tables until necropsy. When body weights could not be determined for an animal during a given interval (due to an unscheduled death, as females enter gestation, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods when body weight values were unavailable for a given animal were left blank or designated as “NA” on the individual report tables.

FOOD CONSUMPTION (F0 AND F1)
- F0 and F1 male and female food consumption was measured weekly until cohabitation.
- Food consumption was measured on a per cage basis for the corresponding body weight
intervals.
- Food consumption was normalized to the number of animals/cage and was
reported as g/animal/day.
- Food intake was not recorded during the breeding period.
- Following the breeding period, food consumption for males and for females with no evidence of mating was measured on a weekly basis until the scheduled necropsy. – Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and lactation days 1, 4, 7, 14, and 21. - Food intake was reported as g/animal/day and g/kg/day for the corresponding intervals of gestation and lactation, and also for gestation days 0-20 and lactation days 1-21. Food efficiency (body weight gained as a percentage of food consumed) was also calculated and reported for these intervals.
- When food consumption could not be determined for an animal during a given interval
(due to an unscheduled death, weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable for a given animal were left blank or designated as“NA” on the individual report tables.

Oestrous cyclicity (parental animals):

ESTROUS CYCLES
- 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).
- Estrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. 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):

SPERMATOGENIC ENDPOINT EVALUATIONS
- Immediately upon euthanasia, the reproductive tract of each F0 and F1 male was exposed via a ventral mid-line incision.
- The right epididymis was excised and weighed.
- An incision was made in the distal region of the right cauda epididymis.
- The right cauda epididymis was then placed in Dulbecco's phosphate-buffered saline (maintained at approximately 37°C) with 10 mg/mL BSA. After a minimum 10-minute incubation period, a sample of sperm was loaded onto a slide with a 100-μm chamber depth for determination of sperm motility.
- Because sperm motility can be affected by temperature shock, all pipettes, slides, and diluents were warmed in an incubator, and motility determinations were performed under constant temperature (approximately 37°C).
- Analysis of a minimum of 200 motile and nonmotile spermatozoa per animal (if possible) in all groups was performed by the analyzer.
- The motility score (percent) for motile (showing motion only) and progressively motile (showing net forward motion) sperm was reported.
- Percent motile (or progressively motile) sperm = [Number of motile (or progressively motile) sperm / Total number of sperm counted] x 100]
- The right epididymis was then placed in modified Davidson’s solution for subsequent microscopic examination.
- Sperm morphology was evaluated by light microscopy via a modification of the wet mount evaluation technique (Linder et al., 1992).
- Abnormal forms of sperm (double heads, double tails, microcephalic, or megacephalic, etc.) from a differential count of 200 spermatozoa per animal, if possible, were recorded.
- The left testis and cauda epididymis from all F0 and F1 males from all test
substance-treated groups were weighed, stored frozen, homogenized, and analyzed for determination of homogenization resistant spermatid count and calculation of sperm production rate (Blazak et al., 1985).
- An aliquot of each sample was added to a solution containing a DNA-specific fluorescent dye (the dye stains DNA that is present in the head of the sperm).
- For analysis, each sample was mixed, and an aliquot was placed on a
slide with a 20-μm chamber depth. Illumination from a xenon lamp within the analyser allowed for the visualization and quantitation of the sperm. A minimum of 200 cells, if possible, or up to 20 fields were counted for each sample.
- The sperm production rate was calculated.
- Sperm production rate = Number of sperm per gram of tissue / 6.1 days where 6.1 days = the rate of turnover of the germinal epithelium.

Litter observations:

F0 AND F1 PARTURITION
- All females were allowed to deliver naturally and rear their young to weaning (PND 21).
- During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia.
- Beginning on the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery was first observed.

LITTER VIABILITY AND DEATHS
- Each litter was examined twice daily for survival, and all deaths were recorded.
- All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained.
- Intact offspring that were found dead or euthanized in extremis (by an intraperitoneal injection of sodium pentobarbital) from PND 0 to 4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984).
- Pups with external abnormalities that would warrant further skeletal examination were eviscerated and stained (Dawson, 1926) for subsequent skeletal evaluation. Findings were recorded as either developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter general body conformity, disrupt, or interfere with normal body function, or may be incompatible with life) as appropriate.
- A detailed gross necropsy was performed on any pup found dead after PND 4 and prior to weaning; tissues were preserved for possible future histopathological examination only as deemed necessary by the gross findings.

LITTER REDUCTION
- To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4.
- Standardization of litter size was not performed on litters with fewer than 8 pups.
- All selections were performed by computerized randomization.
- The remaining offspring were weighed, euthanized by intraperitoneal injection of sodium pentobarbital, and discarded on PND 4.

CLINICAL OBSERVATIONS
- Litters were examined daily for survival and any adverse changes in appearance or behavior.
- A clinical observation was recorded for each pup on PND 1, 4, 7, 14, and 21.
- Any abnormalities in nesting and nursing behavior were recorded.

BODY WEIGHTS
- Pups were individually weighed on PND 1, 4, 7, 14, and 21. Mean pup weights were
presented by sex for each litter and by group.
- When body weight values were unavailable for a given animal (due to unscheduled death, weighing error, etc.) group mean values were calculated for that interval using the available data.
- The time periods when body weight values were unavailable for a given animal were designated as “NA”on the individual report tables.

SEX DETERMINATION
- Pups were individually sexed on PND 0, 4, 14, and 21.

WEANING AND SELECTION
- Each dam and litter remained housed together until weaning on lactation day 21. - Twenty-five male and 25 female F1 pups/litter from each group (control, 25, 100, and 400 mg/kg/day) were randomly selected prior to weaning (PND 21) to comprise the F1 generation. These pups (a minimum of 1 male and 1 female per litter, when available) were administered the test substance daily beginning on PND 21.
- Pups were selected for the F1 generation as follows. A computerized randomization
procedure was used to select a minimum of 1 male and 1 female per litter, when
available. An additional male and/or female were selected from a litter, if necessary, to obtain 25 males and 25 females for each group. Pups selected for the F1 generation retained the dam number, followed by a hyphen "-" and the digit tattoo marking (i.e., 99999-01). F2 pups retained the dam number. However, due to software spacing constraints, the -01, -02, etc. designation in the F1 dam number was replaced with the corresponding letter of the alphabet (i.e., -01 = A, -02 = B, etc.) on some report tables.
- Following weaning, F1 and F2 animals were uniquely identified by a Monel metal ear tag displaying the animal number.

DEVELOPMENT LANDMARKS – BALANOPREPUTIAL SEPARATION
- Each male pup was observed for balanopreputial separation beginning on PND 35
(Korenbrot et al., 1977).
- The age at which balanopreputial separation was first observed was recorded for each pup.
- Examination of the pups continued daily until balanopreputial separation was present.
- Body weights were recorded at the age of attainment of this landmark.

DEVELOPMENT LANDMARKS – VAGINAL PATENCY
- Each female pup was observed for vaginal perforation beginning on PND 25 (Adams
et al., 1985).
- The age at which the vaginal lumen was first observed to open was recorded for each pup.
- Examination of the females was continued daily until vaginal patency was present or an animal was paired for breeding.
- Body weights were recorded at the age of attainment of this landmark.

Postmortem examinations (parental animals):

SCHEDULED EUTHANASIA – F0 AND F1 GENERATIONS (ADULTS)
- All surviving F0 adults were euthanized by carbon dioxide inhalation following the selection of the F1 generation and completion of a detailed physical examination.
- All surviving F1 adults were euthanized by carbon dioxide inhalation following weaning of the F2 pups.
- A complete necropsy and selective histopathologic examination were conducted.

MACROSCOPIC EXAMINATION – F0 AND F1 ANIMALS
- A complete necropsy was conducted on all parental animals (F0 and F1) found dead,
euthanized in extremis, or at termination.
- 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.
- Clinical findings that were verified at necropsy were designated CEO. For females that delivered or had macroscopic evidence of implantation, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded.
- The number of unaccounted-for sites was calculated for each female by subtracting the number of pups born from the number of former implantation sites observed.
- Numbers of corpora lutea were also recorded for females with macroscopic evidence of implantation and for females necropsied during gestation through lactation day 4.
- For females that failed to deliver, a pregnancy status was determined, and specific emphasis was placed on anatomic or pathologic findings that may have interfered with pregnancy.
- The F0 and F1 parental tissues and organs shown in the table below were collected and were placed in 10% neutral-buffered formalin.

ORGAN WEIGHTS
- Except as noted in the table below, paired organs were weighed together. Absolute weights and organ to final body weight and brain weight ratios were reported.
- When organ weights could not be determined for an animal (due to weighing error, lost or damaged organ, etc.), group mean values were calculated using the available data.
- The organs for which weights could not be determined were designated as “NA”on the individual report tables.

HISTOPATHOLOGY AND MICROSCOPIC EXAMINATION
- Microscopic evaluations were performed on the tissues shown in the table below for all F0 and F1 parental animals from the control and high-dose groups and for all adult animals found dead and euthanized in extremis.
- After fixation, protocol-specified tissues were trimmed according to WIL Research SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research’s SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin.
- After fixation, the ovaries were shipped to WIL Research Hillsborough for processing. The tissues were trimmed according to WIL Research SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to WIL Research SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin.
- Microscopic examination was performed on all tissues listed above from all animals in the control and 400 mg/kg/day groups at the scheduled necropsies and all parental animals that were found dead or euthanized in extremis. In addition, reproductive organs of all animals suspected of reduced fertility, e.g., those that failed to mate, conceive, sire, or deliver healthy offspring, or for which estrous cyclicity or sperm number, motility, or morphology were affected, were subjected to a histopathologic evaluation.
- Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, or other designations as appropriate. Tissues may appear on the report tables as not examined due to the tissue not being in the plane of section, not present at trimming, etc.

Postmortem examinations (offspring):

SCHEDULED EUTHANASIA – F1 AND F2 WEANLINGS
- All remaining nonselected F1 and F2 weanlings were euthanized by carbon dioxide
inhalation and necropsied on PND 21 (see Deviations from the Protocol).
- Necropsies were performed and/or organ weights were recorded for these animals.

MACROSCOPIC EXAMINATION – F1 AND F2 WEANLINGS
- 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 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.

ORGAN WEIGHTS – F1 AND F2 WEANLINGS
- The brain, spleen, and thymus were weighed from 1 pup/sex/litter at the scheduled necropsies on PND 21.

Statistics:

- Details of statistical analysis are given in the section titled 'any other information on materials and methods incl. tables'.

Reproductive indices:

- Mating, fertility, copulation and conception indices were calculated.
- Male (female) mating index (%) = [Number of males (females) with evidence of mating (or females confirmed pregnant) / Total number of males (females) used for mating] x 100
- Male fertility index (%) = [Number of males siring a litter / Total number of males used for mating] x 100
- Male copulation index (%) = [Number of males siring a litter / Number of males with evidence of mating (or females confirmed pregnant)] x 100
- Female fertility index (%) = [Number of females with confirmed pregnancy / Total number of females used for mating] x 100
- Female conception index (%) = [Number of females with confirmed pregnancy / Number of females with evidence of mating (or females confirmed pregnant)] x 100

Offspring viability indices:

- Litter parameters were calculated.
- Mean live litter size = Total viable pups on PND 0 / Number litters with vaiable 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) / Number of pups born per litter] 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) / Number of litters per group] x 100 where N = PND 0 to 1, 1 to 4 (pre-selection), 4 (post-selection) to 7, 7 to 14, 14 to 21, birth to PND 4 (pre-selection) or 4 (post-selection) to 21,
- Total litter loss was determined when the last pup in the litter was found dead prior to the scheduled euthanasia.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- No test substance-related clinical findings were noted at the detailed physical examinations.
- Findings noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
- Approximately 1 hour following test substance administration, increased incidences of clear material on various body surfaces, including the mouth, nose, forelimb(s), and/or ventral neck, were noted for F0 males in the 100 mg/kg/day group and F0 males and females in the 400 mg/kg/day group compared to the control group. The clear material findings were primarily observed around the mouth, which were noted for all 25 F0 males and females in the 400 mg/kg/day group, as well as 17 of 25 F0 males in the 100 mg/kg/day group throughout the respective treatment periods. The other clear material findings (nose, forelimb[s], and ventral neck) were noted predominantly in the 400 mg/kg/day group (7 to 16 F0 males and 3 to 13 F0 females each), and occurred intermittently during the respective treatment periods.
- In addition, test substance-related increased incidences of red material around the mouth were noted for F0 males and females in the 400 mg/kg/day group compared to the control group. Red material around the mouth was noted for 17 males and 19 females in the 400 mg/kg/day group as early as study day 7 and continued throughout the remainder of the respective treatment periods.
- The aforementioned clear and red material findings occurred at a similar frequency in F0 males and females. No other test substance-related clinical findings were noted approximately 1 hour following dose administration at any dosage level.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- Two F0 females in the 400 mg/kg/day group were euthanized in extremis during the study.
- Female no. 30740 was euthanized in extremis on lactation day 0. On the day of euthanasia, this female was noted with the clinical findings piloerection, unkempt appearance, red material around the nose, mouth, and ventral trunk, and red vaginal discharge; however, no noteworthy changes in body weight or food consumption were noted for this female prior to euthanasia. Based upon macroscopic and microscopic findings the cause of death was determined to be ulceration of the ileum, characterized by ulceration of the ileal mucosa with associated subacute inflammation. The relationship of this death to administration of the test substance was unclear.
- Female no. 30721 was euthanized in extremis on gestation day 22 following clinical findings of red material on various body surfaces, pale and cool extremities, a cool body, yellow material around the urogenital area, and decreased defecation at the detailed physical examinations. With the exception of 16 live fetuses retained in utero, no noteworthy macroscopic or microscopic findings were noted at necropsy, and therefore a cause of death was unable to be determined; however, the moribund condition of this female was likely related to a difficult delivery, although the relationship to administration of the test substance was unclear.
- No other possible treatment-related effects on survival were noted for F0 males and females at any dosage level.
- In the 100 mg/kg/day group, F0 male no. 30826 was found dead on study day 44 (prior to mating); no remarkable clinical findings or noteworthy changes in body weight or food consumption were noted for this male prior to death. However, based upon macroscopic and microscopic findings, the cause of death was determined to be an intubation error, and unrelated to administration of the test substance.
- All other F0 animals in the control, 25, 100, and 400 mg/kg/day groups survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

WEEKLY BODY WEIGHTS
- Test substance-related lower mean body weight gains were noted for the 400 mg/kg/day group F0 males compared to the control group generally throughout the entire generation; differences achieved significance (p<0.05 or p<0.01) during study day intervals 0-7, 28-35, 35-42, 42-49, 63-70, and 112-119. As a result, mean cumulative body weight gains for F0 males in the 400 mg/kg/day group were significantly (p<0.05) lower than the control group when the pre-mating period (study days 0-70) and the entire generation (study days 0-126) were evaluated. Consequently, mean F0 male body weights in this group were 4.7% to 10.7% lower than the control group during study days 21-126, with the differences achieving significance (p<0.05) during study days 49-126.
- No test substance-related effects on mean body weights and body weight gains were noted for F0 males in the 25 and 100 mg/kg/day groups and for F0 females at all dosage levels. The values in the test substance-treated groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males), with the following exceptions. Significantly (p<0.05 or p<0.01) higher mean body weight gains were noted for F0 males during study days 49-56 (25 mg/kg/day group) and 91-98 (100 mg/kg/day group), and for F0 females in the 400 mg/kg/day group during study days 42-49 compared to the control group. However, the differences in mean body weight gain noted for F0 males and females in these groups were transient, did not occur in a dose-related manner, and/or were not of sufficient magnitude to affect mean body weights and overall mean body weight gains; therefore, they were not attributed to test substance administration.
GESTATION
- Mean F0 maternal body weights and body weight gains were unaffected by test substance administration during gestation. Differences between the control, 25, 100, and 400 mg/kg/day groups were slight and not statistically significant.
LACTATION
- Mean F0 maternal body weights and body weight gains were unaffected by test substance administration during lactation. Differences between the control, 25, 100, and 400 mg/kg/day groups were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- See discussion of food consumption and food efficiency (below).

Food efficiency:

effects observed, non-treatment-related

Description (incidence and severity):

WEEKLY FOOD CONSUMPTION AND FOOD EFFICIENCY
- Mean F0 food consumption, evaluated as g/animal/day, and food efficiency in the 25, 100, and 400 mg/kg/day groups was unaffected by test substance administration. The values in the test substance-treated groups were generally similar to the control group values for the pre-mating period (females) or the entire generation (males).
- Differences were slight and not statistically significant, with the following exceptions. Significantly (p<0.01) lower mean food efficiency was noted for F0 males in the 400 mg/kg/day group during study days 14-21, 28-35, 63-70, and 112-119, and for F0 females in this group during study days 42-49 compared to the control group. In addition, significantly (p<0.05) higher mean food efficiency was noted for F0 males in the 25 mg/kg/day group during study days 49-56. These differences were not considered test substance-related.
GESTATION
- Test substance-related higher mean food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 400 mg/kg/day group F0 females sporadically throughout gestation; differences were significant (p<0.05 or p<0.01) during gestation days 4-7 and 17-20. As a result, significantly (p<0.05 or p<0.01) higher mean food consumption was noted in this group when the overall gestation treatment period (gestation days 0-20) was evaluated. However, no test substance-related effects on mean gestational body weights were noted for F0 females in the 400 mg/kg/day group; therefore, the increased mean food consumption noted in this group was not considered adverse.
- Mean food efficiency for F0 females in the 400 mg/kg/day group was similar to the control group during gestation days 0-4, 4-7, and 7-11, but generally significantly (p<0.05 or p<0.01) lower during the remainder of gestation (days 11-14, 14-17, and 17-20), and when the overall gestation treatment period (gestation days 0-20) was evaluated. The differences in mean food efficiency at 400 mg/kg/day were not considered test substance-related due to the absence of corresponding effects on mean gestational body weights.
- Mean F0 maternal food consumption and food efficiency in the 25 and 100 mg/kg/day groups were unaffected by test substance administration during gestation. Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p<0.05) lower mean g/animal/day food consumption during gestation days 0-4 and higher mean food efficiency during gestation days 4-7 were noted in the 100 mg/kg/day group compared to the control group. However, these transient differences were not of sufficient magnitude to affect mean food consumption or food efficiency when the overall gestation treatment period (gestation day 0-20) was evaluated, nor mean body weight gains during these intervals. Therefore, the differences were not attributed to test substance administration.
LACTATION
- Mean F0 maternal food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency were unaffected by test substance administration during lactation. - Differences between the control, 25, 100, and 400 mg/kg/day groups were slight and not statistically significant.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- Female nos. 30721 and 30740 in the 400 mg/kg/day group were euthanized in extremis on gestation day 22 (prior to parturition) and lactation day 0, respectively.
- The only significant histologic findings for female no. 30721 were severe necrosis and severe lymphoid depletion of the thymus. Because both of these findings were non-specific, a cause of death could not be determined; however, the moribund condition of this female was likely related to a difficult delivery. For female no. 30740, the cause of death was ulceration of the ileum, characterized by ulceration of the ileal mucosa with associated subacute inflammation. This animal also had non-inflammatory mucosal erosions of the glandular stomach consistent with stress. The relationship of this death to administration of the test substance was unclear.
- In the 100 mg/kg/day group, F0 male no. 30826 was found dead on study day 44. Microscopically, acute hemorrhage of the esophageal submucosa, consistent with acute trauma, was noted for this male. The cause of death was an intubation error and unrelated to test substance administration. All other F0 males and females survived to the scheduled necropsies.
- There were no test substance-related microscopic findings in males or females. All histologic changes were considered to be incidental findings unrelated to test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
- In one control group female (no. 30707), two 100 mg/kg/day group females (nos. 30761 and 30800), and one 400 mg/kg/day group female (no. 30783), there was an adenoma reported as a mass in the skin (no. 30761) or subcutaneous tissue (nos. 30707, 30800, and 30783). These adenomas were benign, well delineated, well differentiated, and consistent with mammary gland adenomas.
- In one 400 mg/kg/day group female (no. 30720), there was a gross finding of pale thyroid glands and a microscopic finding of mild hypertrophy of the follicular cells of the thyroid gland. This change in thyroid gland epithelium is commonly associated with induction of metabolic enzymes in the liver. The higher liver weights in the 400 mg/kg/day group females would be compatible with this microscopic, non-adverse change in the thyroid gland. Since the finding was in an isolated animal, and there were no group mean changes in thyroid weights or higher thyroid weight in this particular animal, the follicular epithelial hypertrophy was not considered test substance-related.
- One control group female (no. 30786) had a follicular cyst; one 400 mg/kg/day group female (no. 30776) had a luteal cyst; and one 400 mg/kg/day group female (no. 30805) had a paraovarian cyst in the ovary. Any variation in number or types of cysts were considered to represent normal biological variability unrelated to test substance.
- In one 100 mg/kg/day group male with reduced fertility (no. 30890), there were changes in the testis that would explain the clinical findings. There was Sertoli cell vacuolation, a focal infiltrate of lymphocytes, syncytial giant cells, and hypospermia in the epididymis. Since there was no dose-response profile, this isolated finding was considered spurious and unrelated to the test substance.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

- See discussion of reproductive performance (below) and Table 2 (attached).

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

SPERMATOGENIC ENDPOINT EVALUATIONS
- No test substance-related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage concentration.
- Differences from the control group were slight and were not statistically significant.

Reproductive performance:

no effects observed

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE
- F0 male and female reproductive parameters are presented in Table 2 (attached).
- No test substance-related effects on F0 reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. Two, 1, 2, and 2 male and female pairs in the control, 25, 100, and 400 mg/kg/day groups, respectively, had evidence of mating but did not produce a litter.
- There was reduced fertility in 8 female rats: 2 control group females (nos. 30697 and 30749), one 25 mg/kg/day group female (no. 30750), two 100 mg/kg/day group females (nos. 30702 and 30765), and three 400 mg/kg/day group females (nos. 30717, 30721, and 30793). The ovaries in these animals contained both current and old corpora lutea, suggesting these animals had been cycling. The uteri of these animals had relatively small circumferences, with condensed and eosinophilic endometrial stroma having little glandular development, when compared to animals without reduced fertility. At the time of necropsy, the vagina was microscopically consistent with diestrus in 6 of these animals (nos. 30697, 30750, 30702, 30765, 30717, and 30793). The microscopic appearance of the reproductive tissues in these 6 animals was compatible with a normal nulliparous female adult rat. There was no microscopic cause for reduced fertility noted clinically.
- One control group female (no. 30749) with reduced fertility had a retained antral follicles and old corpora lutea, a normal dilated uterus compatible with estrus in a nulliparous female, and a vagina in estrus. The ovary had a follicle indicative of proestrus, indicative of pending ovulation. There was no primary or apparent cause for the reduced fertility noted clinically or the absence of current corpora lutea.
- One 400 mg/kg/day group female (no. 30721) with reported reduced fertility was pregnant and was euthanized in extremis. This females was euthanized prior to parturition on gestation day 22, and had retained 16 live fetuses in utero.
- There were 2 control group males (nos. 30806 and 30852), one 25 mg/kg/day group male (no. 30856), two 100 mg/kg/day group males (nos. 30815 and 30890), and three 400 mg/kg/day group males (nos. 30840, 30849, and 30901) with reduced fertility. There were no microscopic correlates to the reduced fertility, except for male no. 30890 in the 100 mg/kg/day group. This animal had hypospermia and degenerative changes in the testis consisting of vacuolated Sertoli cells and syncytial giant cells. These changes in 1 mid-dose group animal were considered spurious and unrelated to the test substance. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.
GESTATION LENGTH AND PARTURITION
- No test substance-related effects were noted on mean gestation lengths or the process of parturition at any dosage level. The mean gestation lengths in the 25, 100, and 400 mg/kg/day groups were 21.7, 21.7, and 22.1 days, respectively, compared to a mean gestation length of 21.7 days in the concurrent control group.
- The slight difference in the 400 mg/kg/day group was significant (p<0.05) compared to the concurrent control group; however, the value was within the WIL Research historical control data range (21.5 to 22.3 days). Therefore, the slightly longer mean gestation length in this group was not considered test substance-related. No signs of dystocia were noted at any dosage level.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: no effects on F0 and F1 reproductive performance at any dosage level

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: parental systemic toxicity

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- No test substance-related clinical findings were noted at the detailed physical examinations. Findings noted in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
- Approximately 1 hour following test substance administration, increased incidences of clear material on various body surfaces, including the mouth, nose, forelimb(s), and/or ventral neck, were noted for F1 males in the 100 mg/kg/day group and F1 males and females in the 400 mg/kg/day group compared to the control group.
- The clear material findings were primarily observed around the mouth, and were noted for 24 of 25 F1 males and 23 of 25 F1 females in the 400 mg/kg/day group, as well as 15 of 25 F1 males in the 100 mg/kg/day group throughout the respective treatment periods.
- The other clear material findings (nose, forelimb[s], and/or ventral neck) were noted exclusively in the 400 mg/kg/day group (2 to 4 F1 males and 7 F1 females), and occurred intermittently during the respective treatment periods.
- In addition, test substance-related increased incidences of red material around the mouth were noted for F1 males and females in the 400 mg/kg/day group compared to the control group. Red material around the mouth was noted for 17 males and 18 females in the 400 mg/kg/day group as early as PND 35 and continued sporadically throughout the remainder of the respective treatment periods.
- No other test substance-related clinical findings were noted approximately 1 hour following dose administration at any dosage level.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- Male no. 30712-09 in the 25 mg/kg/day group was found dead on PND 27. Based on macroscopic and microscopic findings, the cause of mortality for this male was dilatation of ventricles of the brain, which was considered a congenital developmental defect unrelated to the test substance.
- In the control group, 1 male (no. 30734-06) and 1 female (no. 30734-16) were euthanized in extremis on PND 27 and gestation day 10, respectively. The moribund condition of male no. 30734-06 was determined to be dilatation of ventricles of the brain (congenital defect), while female no. 30734-16 was had a grossly visible nodule that correlated to an exostoses adjacent to the pituitary; this exostoses was considered the cause of morbidity and a spontaneous occurrence.
- All other F1 animals in the control, 25, 100, and 400 mg/kg/day groups survived to the scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

WEEKLY BODY WEIGHTS
- The effects on mean body weight and body weight gain for F1 males in the 400 mg/kg/day group during the pre-weaning period continued into the post-weaning period. Test substance-related lower weekly mean body weight gains were noted for F1 males in the 400 mg/kg/day group generally throughout the entire generation; the differences were significant (p<0.05 or p<0.01) during PND 21-42, 49-56, 70-77, 84-91, and 119-126. As a result, mean cumulative body weight gains for F1 males in this group were significantly (p<0.01) lower than the control group when the pre-mating period (PND 21-98) and the entire generation (PND 21-154) were evaluated. Consequently, mean F1 male body weights in this group were 10.6% to 16.1% lower than the control group during PND 21-154; the differences were significant (p<0.01).
- No test substance-related effects on mean body weight and body weight gain were noted for F1 males in the 25 and 100 mg/kg/day groups. The values in these test substance-treated groups were generally similar to the control group values throughout the entire generation (PND 21-154), with the exception of a significantly (p<0.05) higher mean body weight gain in the 100 mg/kg/day group during PND 126-133. This difference in mean body weight gain noted for F1 males in this group was transient, did not occur in a dose-related manner, and was not of sufficient magnitude to affect mean body weight and overall mean body weight gain; therefore, it was not attributed to test substance administration.
- Similarly to the F1 males, the effects on mean body weight and body weight gain for F1 females in the 400 mg/kg/day group during the pre-weaning period continued into the post-weaning period. Test substance-related, significantly (p<0.05 or p<0.01) lower mean body weight gains were noted for F1 females in the 400 mg/kg/day group during PND 21-28 and 28-35 compared to the control group. Thereafter, mean body weight gains in this group were similar to the control group throughout the remainder of the pre-mating period (PND 35-98) and when the overall pre-mating period (PND 21-98) was evaluated, with the exception of a transient, significantly (p<0.05) higher mean body weight gain during PND 56-63. However, mean body weights in the 400 mg/kg/day group F1 females were 5.4% and 16.3% lower than the control group during PND 21-98; the differences were generally significant (p<0.05 or p<0.01).
- No test substance-related effects on mean body weight and body weight gain were noted for F1 females in the 25 and 100 mg/kg/day groups. The values in these test substance-treated groups were generally similar to the control group values throughout the pre-mating period (PND 21-98), with the exception of significantly (p<0.05 or p<0.01) higher mean body weight gains in these groups during PND 56-63. These differences in mean body weight gain noted for F1 females in the 25 and 100 mg/kg/day groups were transient, did not occur in a dose-related manner, and was not of sufficient magnitude to affect mean body weight and overall mean body weight gain; therefore, they were not attributed to test substance administration.
GESTATION
- Mean F1 maternal body weight gains were unaffected by test substance administration during gestation at all dosage levels. Any statistically significant differences between the control, 25, 100, and 400 mg/kg/day groups were slight, transient, and/or occurred in a manner that was not dose-related. However, mean body weight in the 400 mg/kg/day group was significantly (p<0.05) lower (6.8%) than the control group on gestation day 0, the result of test substance-related effects on mean body weight noted in this group during the pre-mating period. Mean body weights in the 400 mg/kg/day group were generally similar to the control group during the remainder of gestation (days 4-20); differences were slight and not statistically significant.
LACTATION
- Mean F1 maternal body weights and body weight gains were unaffected by test substance administration during lactation. Differences between the control, 25, 100, and 400 mg/kg/day groups were slight and not statistically significant, with the following exceptions. Significantly (p<0.05 or p<0.01) higher mean body weight gains were noted in the 400 mg/kg/day group compared to the control group during lactation days 4-7 and 14-21. The statistical significance noted in this group during these intervals was attributed to the low values noted for control group animals, rather than the test substance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- See discussion of food consumption and food efficiency (below).

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

WEEKLY FOOD CONSUMPTION AND FOOD EFFICIENCY
- Mean weekly food consumption, evaluated as g/animal/day, in the 400 mg/kg/day group was significantly (p<0.05 or p<0.01) lower during PND 28-35, 35-42, 84-91, and 119-126 for F1 males and during PND 28-35 for F1 females. The lower food consumption was attributed to test substance administration and corresponded to lower mean body weight gains noted in this group during these intervals.
- Mean food consumption in the 400 mg/kg/day group F1 females was comparable to the control group during the remainder of the pre-mating period.
- Mean food efficiency was unaffected by test substance administration for F1 males and females.
- Significantly (p<0.05) higher mean food efficiency was noted for F1 males in the 100 mg/kg/day group during PND 126-133 compared to the control group. This difference was considered transient and not test substance-related, as there was no dose response or corresponding effect on mean body weight.
- Mean food consumption and food efficiency in the 25 and 100 mg/kg/day group F1 males and females were unaffected by test substance administration. The values in the test substance-treated groups were similar to the control group during the pre-mating period (females) and entire generation (males), with the following exception. Significantly (p<0.05) lower mean food consumption was noted for F1 males in the 25 mg/kg/day group compared to the control group during PND 35-42. This difference was considered transient and not test substance-related, as there was no corresponding effect on mean body weight or body weight gain.
GESTATION
- Test substance-related higher mean food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 400 mg/kg/day group F1 females sporadically throughout gestation; differences were generally significant (p<0.05 or p<0.01). As a result, significantly (p<0.05 or p<0.01) higher mean food consumption was noted in this group when the overall gestation treatment period (gestation days 0-20) was evaluated. However, mean body weights in the 400 mg/kg/day group were similar to or lower than the control group throughout gestation; therefore, the increased mean food consumption noted in this group was not considered adverse.
- Significantly (p<0.05 or p<0.01) higher mean food efficiency was noted in the 400 mg/kg/day group during gestation days 0-4 and 7-11 compared to the control group; however, these differences were not of sufficient magnitude to affect the overall gestation period (days 0-20).
- Mean maternal food consumption and food efficiency were unaffected by test substance administration in the 25 and 100 mg/kg/day groups during gestation.
- Differences were slight and not statistically significant, with the following exceptions. Significantly (p<0.01) lower mean food consumption was noted in the 100 mg/kg/day group during gestation days 14-17, and significantly (p<0.01) higher mean food efficiency was noted in the 25 and 100 mg/kg/day groups during gestation days 7-11 compared to the control group. These transient differences were not considered test substance-related.
LACTATION
- Mean F1 maternal food consumption, evaluated as g/animal/day and g/kg/day, and food efficiency were unaffected by test substance administration during lactation. - Differences between the control, 25, 100, and 400 mg/kg/day groups were slight, not statistically significant, and/or did not occur in a dose-related manner.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

ORGAN WEIGHTS
- There was a test substance-related lower mean final body weight (15.2%, p<0.01) in the 400 mg/kg/day group males.
- This change in group mean final body weight was the reason for many additional statistically significant changes in group mean organ weights in the 400 mg/kg/day males, including lower adrenal gland weights (absolute and relative to brain weight); lower brain weights (absolute and relative to final body weight); lower left cauda epididymis weight (absolute); higher right cauda epididymis weight (relative to final body weight); lower left epididymis weight (absolute); higher right epididymis weights (relative to final body weight); higher kidney weight (relative to final body weight); lower pituitary weights (absolute and relative to final body and brain weights); lower spleen weight (absolute); higher seminal vesicle/coagulating gland/accessory gland fluid (relative to final body weight); higher left and right testes weights (relative to final body weight); higher liver weight (relative to final body weight); higher thyroid gland weight (relative to final body weight). These changes in group mean organ weight parameters were within the WIL Research historical control data ranges.
- Several statistically significant changes in group mean organ weight parameters were considered a result of slightly lower final body weights in the 25 and 100 mg/kg/day groups that were not considered test substance-related. These changes included lower left cauda epididymis weights (absolute and relative to brain weight) in the 100 mg/kg/day group males; lower left epididymis weights (absolute and relative to brain weight) in the 100 mg/kg/day group males; lower right epididymis weights (absolute and relative to brain weight) in the 100 mg/kg/day group males; lower left testis weight (absolute and relative to brain weight) in the 25 and 100 mg/kg/day group males; and lower pituitary weights (absolute and relative to brain weight) in the 25 and 100 mg/kg/day group males. The values were within the WIL Research historical control data ranges and were considered unrelated to test substance administration.
- There was a lower group mean right epididymis weight (absolute) in the 25 mg/kg/day group males. This lower weight was due to an outlier (male no. 30730-05) that had severe atrophy of the right testis and severe hypospermia of the right epididymis. The lesion was unilateral and considered spurious and unrelated to test substance administration.
- There was no test substance-related effect on group mean final body weights in F1 females. There were test substance-related higher liver weights (absolute, relative to final body weight, and relative to brain weight) in the 400 mg/kg/day group females. These values exceeded the values in the WIL Research historical control data ranges.
- There were higher group mean thymus weights (absolute and relative to body weight and brain weight) in the 400 mg/kg/day group females, but the values were within the WIL Research historical control data ranges, and therefore were not considered a test substance-related effect.
- Several statistically significant changes in group mean organ weight parameters were present in the 400 mg/kg/day group females, but these changes were considered a result of the slightly lower (2.1%) group mean final body weight in this group. These changes included: lower brain weight (absolute) and lower pituitary weights (absolute and relative to brain weight). These changes in organ weights were within the WIL Research historical control data ranges.
- There was a lower kidney weight relative to brain weight in the 25 mg/kg/day group females; however, this change did not show a dose-response relationship, did not affect absolute kidney weight or kidney weight relative to brain weight, was not associated with histologic findings, and was therefore considered spurious and unrelated to test substance administration.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

MACROSCOPIC EXAMINATION
- Three F1 animals in the 400 mg/kg/day group were found dead prior to the scheduled necropsy. Male no. 30751-01 was found dead on PND 34; at necropsy, gross findings for this male included pale lung lobes that were not fully collapsed and dark red discoloration of the thyroid glands.
- Female nos. 30755-08 and 30782-14 were found dead on PND 32; at necropsy, pale lung lobes that were not fully collapsed were noted for each of these females. Based on the macroscopic findings of lungs not fully collapsed, a gavage error during dosing was considered a possible cause of death for these animals; however, a relationship to treatment could not be dismissed. No other possible treatment-related effects on survival were noted in the test substance-treated groups.
- In the 25 mg/kg/day group, male no. 30712-09 was found dead on PND 27; at necropsy, gross findings for this male included dilatation of the ventricles of the brain, white areas on the liver, and dark red contents in the cranial cavity.
- In the control group, male no. 30734-06 was euthanized in extremis on PND 27 and female no. 30734-16 was euthanized in extremis on gestation day 10. At necropsy, male no. 30734-06 had dilatation of the ventricles of the brain, gas filled cecum, ileum, and jejunum, and a pale liver, and female no. 30734-16 had gas filled cecum, colon, duodenum, ileum, jejunum, and stomach, a small thymus, and a dark red nodule on the bone adjacent to the pituitary; female no. 30734-16 was also gravid with 17 normally developing implantations in utero.
- All other F1 animals survived until scheduled necropsy.
- At the scheduled necropsies, no test substance-related internal findings were observed for F1 males and females at any dosage level. Macroscopic findings observed in the test substance-treated groups occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.
- 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. The differences between the control and test substance-treated groups were slight and not statistically significant.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MICROSCOPIC EXAMINATIONS
- Three F1 animals in the 400 mg/kg/day group were found dead prior to the scheduled necropsy; all 3 deaths were attributed to test substance administration. Male no. 30751-01 in this group was found dead on PND 34. The cause of death for this male was undetermined microscopically, as findings were limited to congestion of the thyroid glands.
- Female nos. 30755-08 and 30782-14 in this group were found dead on PND 32. Microscopically, findings were limited to mineralization in the kidneys for female no. 30755-08, and therefore the cause of death for these females was undetermined.
– No other test substance-related effects on survival were noted for F1 animals in the test substance-treated groups.
- In the 25 mg/kg/day group, male no. 30712-09 was found dead on PND 27; microscopically, findings for this male included dilatation of the ventricles of the brain and cystic kidneys. Dilatation of the ventricles was considered the cause of death, and is a congenital developmental defect unrelated to the test substance.
- In the control group, F1 male no. 30734-06 was euthanized in extremis on PND 27 and F1 female no. 30734-16 was euthanized in extremis on gestation day 10. Microscopically, male no. 30734-06 had dilatation of the ventricles of the brain, which was considered the cause of death, while female no. 30734-16 had an exostoses adjacent to the pituitary, which was considered the cause of morbidity and a spontaneous occurrence.
- All other F1 males and females survived to the scheduled necropsies.
- There were no test substance related histologic changes noted for F1 males at any dosage level.
- There was test substance-related hepatocellular hypertrophy in the 400 mg/kg/day group females. The change was minimal to mild, correlated to higher liver weights in this group, and was considered an adaptive, non-adverse change.
- Incidence of selected histopathologic findings at scheduled necropsy of F1 parental animals is presented in Table 4 (attached).
- There were no other test substance-related histologic changes. Remaining histologic changes 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.
- There were no test substance-related alterations in ovarian primordial follicle counts among the ovaries examined.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

- See discussion of reproductive performance.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

SPERMATOGENIC ENDPOINT EVALUATIONS
- No test substance-related effects were observed on F1 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, progressive motility, and morphology) in males at any dosage level.
- Significantly (p<0.05) lower mean left cauda epididymis weights in the 100 and 400 mg/kg/day groups and significantly (p<0.05) lower left testis weights in the 25 and 100 mg/kg/day groups were noted compared to the control group. These findings were not considered test substance-related.
- No other statistically significant differences were noted at any dosage level.

Reproductive performance:

no effects observed

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE
- F1 male and female reproductive parameters are presented in Table 3 (attached).
- No test substance-related effects on F1 reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. Males that did not sire a litter numbered 2, 0, 2, and 3 in the control, 25, 100, and 400 mg/kg/day groups, respectively. Females that had evidence of mating but were nongravid numbered 3, 0, 2, and 3 in the same respective groups.
- The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean lengths of estrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.
GESTATION LENGTH AND PARTURITION
- No test substance-related effects were noted on mean gestation lengths or the process of parturition at any dosage level.
- Mean F1 gestation lengths in the test substance-treated groups were similar to the control group value. Differences were slight and were not statistically significant.
- The mean gestation lengths in the 25, 100, and 400 mg/kg/day groups were 21.9, 21.7, and 22.0 days, respectively, compared to mean gestation lengths of 21.8 days in the concurrent control group and 21.9 days in the WIL Research historical control data.
- No signs of dystocia were noted at any dosage concentration.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: no effects on F0 and F1 reproductive performance at any dosage level

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: parental systemic toxicity

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

GENERAL PHYSICAL CONDITION
- The general physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study were unaffected by test substance administration.
- Pups (litters) that were found dead numbered 18(9), 7(7), 6(5), and 56(12) in the control, 25, 100, and 400 mg/kg/day groups, respectively. Sixteen (7), 5(5), 9(4), and 34(12) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.
- A higher number of pups were found dead in the 400 mg/kg/day group compared to the control group. The increased mortality in this group was primarily attributed to 3 females (nos. 30757, 30740, and 30720) that combined for 41 of the 56 found dead pups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

PND 0 LITTER DATA AND POSTNATAL SURVIVAL
- The mean number of pups born, live litter size, and percentage of males per litter at birth were unaffected by administration of the test substance to the F0 parental animals at all dosage levels.
- Test substance-related effects on F1 postnatal survival were noted in the 400 mg/kg/day group. Lower mean postnatal survival was noted in this group from PND 0-1, 1-4, and 4-7, which resulted in lower postnatal survival from birth to PND 4 and from PND 4-21. Although none of the differences were statistically significant when compared to the control group, the values from the aforementioned intervals were lower than the respective minimum mean values in the WIL Research historical control database.
- The lower values in the 400 mg/kg/day group were primarily attributed to female nos. 30720 and 30740 (total litter losses on PND 1 and 0, respectively) and no. 30757 (10 dead pups during PND 1-7), but viability was also reduced (≤80.0%) for 8 females during birth to PND 4 and for 4 females during PND 4-21.
- Mean postnatal survival between birth and PND 0 (relative to number born), PND 0-1, 1-4 (pre-selection), 4 (post-selection)-7, 7-14, 14-21, and from birth to PND 4 (pre-selection) and PND 4 (post-selection) to PND 21 were unaffected by administration of the test substance to the F0 parental animals at 25 and 100 mg/kg/day. Differences from the control group were slight, were not statistically significant, and/or did not occur in a dose-related manner.
CLINICAL OBSERVATIONS AND SURVIVAL
- Likely test substance-related effects on survival were noted for F1 animals in the 400 mg/kg/day group. One male (no. 30751-01) was found dead on PND 34 and 2 females (nos. 30755-08 and 30782-14) were found dead on PND 32. No clinical findings during the detailed physical examinations and post-dosing observations, and no noteworthy changes in mean body weight were noted for these animals prior to death. The cause of mortality was undetermined for these animals based on the gross and microscopic findings however, macroscopic findings of lungs not fully collapsed, indicative of a dosing error, was considered a possible cause of death for these animals; however, a relationship to treatment could not be dismissed.
- There were no other possible test substance-related effects on survivability in the F1 generation.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

OFFSPRING BODYWEIGHTS
- Test substance-related effects on pup body weights were noted in the 400 mg/kg/day group during the pre-weaning period (PND 1-21).
- Mean F1 male and female pup birth weights (PND 1) in the 400 mg/kg/day group were generally similar to the control group. However, lower mean male and female pup body weight gains were noted in this group during the entire pre-weaning period (PND 1-21) compared to the control group (significant [p<0.05 or p<0.01] during PND 4-21). As a result, mean male and female pup body weights in this group were up to 12.9% and 15.0%, respectively, lower than the control group during PND 7-21; differences from the control group were significant (p<0.01) on PND 14 and 21.
- Mean male and female pup body weights and body weight changes in the 25 and 100 mg/kg/day groups were unaffected by test substance administration throughout the postnatal period. No statistically significant differences from the control group were noted.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- See discussion for second parental generation.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

- See discussion for second parental generation.

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Description (incidence and severity):

BALANOPREPUTIAL SEPARATION
- Mean ages of attainment of balanopreputial separation were unaffected by test substance administration.
- The mean ages of attainment of balanopreputial separation were 46.1, 46.4, and 46.5 days in the 25, 100, and 400 mg/kg/day groups, respectively, when compared to 45.7 days in the control group and 45.6 days in the WIL Research historical control data.
- Mean body weights at the age of attainment were 234.5 g, 236.3 g, and 209.5 g in the same respective groups compared to 233.1 g in the control group and 231.7 g in the WIL Research historical control data; the difference in mean body weight in the 400 mg/kg/day group was significant (p<0.01) compared to the control group. This body weight deficit (10.1%) corresponded to the lower mean body weights and body weight gains in this group during the weekly body weight evaluations
VAGINAL PATENCY
- Mean ages of attainment of vaginal patency were unaffected by test substance administration.
- The mean ages of attainment of vaginal patency were 35.2, 34.7, and 35.1 days in the 25, 100, and 400 mg/kg/day groups, respectively, when compared to 34.3 days in the control group and 33.6 days in the WIL Research historical control data.
- Mean body weights at the age of attainment were 118.1 g, 118.4 g, and 106.6 g in the same respective groups when compared to 119.7 g in the control group and 111.0 g in the WIL Research historical control data; the difference in mean body weight in the 400 mg/kg/day group was significant (p<0.01) compared to the control group. This body weight deficit (10.9%) corresponded to the lower mean body weights and body weight gains in this group during the weekly body weight evaluations.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

ORGAN WEIGHTS
- During necropsies of weanlings selected for organ weights (PND 21), test substance-related, significantly (p<0.05 or p<0.01) lower mean final body weights were noted for the 400 mg/kg/day group F1 males (11.8%) and females (10.6%) compared to the control group.
- Lower mean absolute brain and absolute spleen weights were also noted for F1 males and females in the 400 mg/kg/day group compared to the control group; the differences were generally significant (p<0.05 or p<0.01). Although the mean absolute brain and spleen weights in this group were within the respective WIL Research historical control data ranges, the differences generally occurred in a dose related manner, and were considered test substance-related.
- No test substance-related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed at 25 and 100 mg/kg/day. None of the differences from the control group were statistically significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

NECROPSIES OF PUPS FOUND DEAD
- The numbers of pups (litters) found dead from PND 0 through the selection of the F1 generation were 18(9), 7(7), 6(5), and 56(12) in the control, 25, 100, and 400 mg/kg/day groups, respectively. No internal findings that could be attributed to parental administration to the test substance were noted at the necropsies of pups that were found dead.
- Aside from the presence or absence of milk in the stomach, internal findings were limited to the 400 mg/kg/day group, and consisted of dilated renal pelves for pup no. 30735-01 and dark red tracheal contents and dark red discolored lungs (all lobes) for pup nos. 30757-04 and 30799-05. Because these findings were noted infrequently in single pups, they were not attributed to the test substance. No other internal findings were noted.
NECROPSIES OF WEANLINGS NOT SELECTED FOR ORGAN WEIGHTS (PND 21)
- No internal findings that could be attributed to parental administration with the test substance were noted at the necropsy of weanlings euthanized on PND 21. – Internal findings were limited to 2 pups in the 25 mg/kg/day group, and consisted of a missing tail (entire length) and cystic kidneys for pup no. 30804-01 and cystic kidneys for pup no. 30804-07. These findings were noted infrequently, in single pups, and in a manner that was not dose related, and therefore were not attributed to the test substance. No other internal findings were noted.
NECROPSIES OF WEANLINGS SELECTED FOR ORGAN WEIGHTS (PND 21)
- At the PND 21 necropsy of F1 weanlings selected for organ weights, no internal findings that could be attributed to parental administration with the test substance were noted.
- Internal findings were limited to dark red areas on the thymus for 1 pup (no. 30762-09) in the 100 mg/kg/day group. Because this finding was noted in a single pup and in a manner that was not dose related, it was not attributed to the test substance. No other internal findings were noted.

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

- See discussion for second parental generation.

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Remarks on result:

other:

Remarks:

neonatal toxicity

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

GENERAL PHYSICAL CONDITION
- The general physical condition (defined as the occurrence and severity of clinical findings) of all F2 pups in this study were unaffected by F1 parental test substance administration.
- Pups that were found dead or euthanized in extremis numbered 9(8), 12(6), 30(9), and 40(13) in the control, 25, 100, and 400 mg/kg/day groups, respectively. Eight (5), 2(2), 6(3), and 16(9) pups (litters) in the same respective groups were missing and presumed to have been cannibalized.
- A higher number of pups were found dead in the 400 mg/kg/day group compared to the control group. The increased mortality in this group was primarily attributed to 2 female (nos. 30748-12 and 30748-09) that combined for 21 of the 40 found dead or euthanized in extremis pups.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

PND 0 LITTER DATA AND POSTNATAL SURVIVAL
- A lower live litter size on PND 0 was noted in the 400 mg/kg/day group (11.7 per dam) compared to the control group (13.5 per dam). The difference was not statistically significant, but the value was comparable to the minimum mean value in the WIL Research historical control data (11.6 per dam). As a result, a lower (not statistically significant) mean litter proportion (89.7% per litter) of postnatal survival was noted at birth (PND 0) in the 400 mg/kg/day group compared to the control group (98.4% per litter). A lower mean litter proportion of postnatal survival continued to be noted in the 400 mg/kg/day group during PND 0-1 (89.9% per litter), and consequently from birth to PND 4 (79.1% per litter) compared to the control group (95.7% and 90.9% per litter, respectively). The differences from the control group were not statistically significant, but the values in the 400 mg/kg/day group were below the minimum mean litter proportion values in the WIL Research historical control data (93.3% [PND 0], 94.6% [PND 0-1], and 83.8% [birth to PND 4]). The lower postnatal survival in the 400 mg/kg/day group was partially attributed to 2 females from a single litter (nos. 30748-09 and 30748-12), with total litter loss on PND 0 and a litter survival value of 21.4% from birth to PND 4, respectively. The mean litter proportions of postnatal survival in the 400 mg/kg/day group were similar to the control group during PND 4-21. No test substance-related effects on the percentage of males at birth and the mean number of pups born were noted at 400 mg/kg/day.
- There were no test substance-related effects on the mean number of pups born, live litter size, percentage of males per litter at birth, and postnatal survival in the 25 and 100 mg/kg/day groups. Differences from the control group were slight, not statistically significant, and/or did not occur in an dose-related manner.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

OFFSPRING BODY WEIGHTS
- Mean F2 male and female pup birth weights on PND 1 were similar between the control and 400 mg/kg/day group. Additionally, mean F2 male and female pup body weights and body weight gains in the 400 mg/kg/day group were generally similar to the control group during PND 1-7.
- However, significantly (p<0.01) lower mean F2 male and female pup body weight gains were noted in this group during the remainder of the pre-weaning period (PND 7-21) compared to the control group. As a result, mean F2 male and female pup body weights in this group were up to 15.4% and 14.6%, respectively, lower than the control group from PND 14-21; differences from the control group were significant (p<0.01).
- Mean F2 male and female pup body weights and body weight changes in the 25 and 100 mg/kg/day groups were unaffected by test substance administration throughout the postnatal period. No statistically significant differences from the control group were noted.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

ORGAN WEIGHTS
- Test substance-related, significantly (p<0.01) lower mean final body weights were noted for the 400 mg/kg/day group F2 males (14.3%) and females (15.4%) compared to the control group.
- Lower mean absolute and relative (to final body and/or brain weights) brain and spleen weights were also noted for F2 males and females in the 400 mg/kg/day group compared to the control group; the differences were generally significant (p<0.05 or p<0.01). Although the mean absolute brain and spleen weights in this group were within the respective WIL Research historical control data ranges, the differences occurred in a dose-related manner, and were considered test substance-related.
- No test substance-related effects on organ weights (absolute, relative to final body weight, and relative to brain weight) were observed at 25 and 100 mg/kg/day.
- None of the differences from the control group were statistically significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

NECROPSIES OF PUPS FOUND DEAD
- The numbers of pups (litters) found dead or euthanized in extremis from PND 0 through the selection of the F2 generation were 9(8), 12(6), 30(9), and 40(13) in the control, 25, 100, and 400 mg/kg/day groups, respectively.
- No internal findings that could be attributed to parental administration of the test substance were noted at the necropsies of pups that were found dead or euthanized in extremis.
- Aside from the presence or absence of milk in the stomach, internal findings consisted of the following. In the 400 mg/kg/day group, pup no. 30751-04-01 had the variation small lungs (all lobes) and pup nos. 30748-09-05 and 30748-09-10 had the variation hemorrhagic ring around the iris (unilateral or bilateral). In the 100 mg/kg/day group, pup no. 30722-14-11 had the variation renal papilla(e) not developed and distended ureters and pup no. 30762-16-02 had a small thymus and a dark red discolored right eye.
- In the 25 mg/kg/day group, pup no. 30766-07-01 had the malformation cleft palate (palatine plates not joined; entire length) and the variations sternebrae malaligned (slight or moderate; nos. 2-5), thoracic vertebral centra not fully ossified (nos. 5 and 10-12), and 14th rudimentary ribs. These findings were not attributed to the test substance as they occurred in single pups (litters) and/or did not occur in a dose-related manner.
- No other internal findings were noted.
NECROPSIES OF WEANLINGS NOT SELECTED FOR ORGAN WEIGHTS (PND 21)
- No internal findings that could be attributed to parental administration of the test substance were noted at the necropsy of weanlings euthanized on PND 21.
- Internal findings, including distended ureter(s) and dilated renal pelves, were noted in single pups, similarly in the control group, and in a manner that was not dose-related.
NECROPSIES OF WEANLINGS SELECTED FOR ORGAN WEIGHTS (PND 21)
- At the PND 21 necropsy of F2 weanlings selected for organ weights, no internal findings were observed at any dosage level.

Histopathological findings:

not examined

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Remarks on result:

other: neonatal toxicity

Critical effects observed:

no

Reproductive effects observed:

no

ANALYSIS OF DOSING FORMULATIONS

- Results of homogeneity and concentration analysis are summarised in Table 1 (attached).

- The mean concentrations of the 5 and 80 mg/mL formulations prepared on 03-Nov-2014 were 78.4% and 116% of the target concentration, and the mean concentrations of the 5 and 20 mg/mL formulations prepared on 16-Jun-2015 were 63.5% and 83.0% of the target concentration; all other samples were within WIL Research SOP range for suspensions (85% to 115%).

- The out-of-specification results noted in the 5 mg/mL group had no impact on the study because the NOAEL for parental systemic toxicity was determined to be a higher dosage level (20 mg/mL).

- The out-of-specification results noted in the 20 and 80 mg/mL groups had no impact on the study because the difference from the WIL Research SOP range was minimal (≤2%), and the formulations were prepared the same way on each day.

- Additionally, the results of the analyses on 16-Jun-2015 were consistent with the results obtained during the method validation 70-day storage assessment;i.e., formulations at lower prepared concentrations have lower mean percent recovery following prolonged storage (Haas, 2015, WIL-168211).

- For logistical reasons (i.e., sample shipment and method development times) sample analyses on 16-Jun-2015 in this study were conducted approximately 27 days after formulation and dosing. However, because all formulations were used for dosing within 8 days of preparation, it is believed that the animals on this study received the intended dose concentration/dosage levels.

- All analyzed dosing formulations were homogeneous and the test substance was not detected in the vehicle formulation that was administered to the control group (Group 1).

Conclusions:

Based on the absence of effects on F0 and F1 reproductive performance at any dosage level, a dosage level of 400 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for parental reproductive toxicity of test item administered orally by gavage to Crl:CD(SD) rats. Based on the lower live litter size on PND 0 for F2 pups, decreased postnatal survival, and lower mean body weights and body weight gains during the pre-weaning period for F1 and F2 pups at 400 mg/kg/day, the NOAEL for neonatal toxicity was considered to be 100 mg/kg/day. Based on moribundity for F0 females and mortality for F1 males and females, as well as lower mean body weights, body weight gains, and/or food consumption for F0 males and F1 males and females at 400 mg/kg/day, the NOAEL for parental systemic toxicity was considered to be 100 mg/kg/day.

Executive summary:

GUIDELINE

The study was conducted to evaluate the potential adverse effects of the test item on reproduction in a two generation study. This included determining the effects of the test substance on male and female reproductive processes, including gonadal function, estrous cyclicity, mating behavior, conception, gestation, parturition, lactation, weaning, and on growth and development of the offspring. A minimum of 1 litter per dam was produced in each generation. The protocol was designed in general accordance with the United States EPA Health Effects Test Guidelines: OPPTS 870.3800, Reproduction and Fertility Effects, Aug-1998, and the OECD Guidelines for the Testing of Chemicals, Guideline 416, Two-Generation Reproduction Toxicity Study, 22-Jan-2001.

 

METHODS

Four groups of male and female Crl:CD(SD) rats (25/sex/group) were administered the test substance, daily by oral gavage, for at least 70 consecutive days prior to mating. Dosage levels were 25, 100, and 400 mg/kg/day for the F0 and F1 generations. A concurrent control group of 25 rats/sex/group received the vehicle (arachis [peanut] oil) on a comparable regimen. F0 animals were approximately 6 weeks of age at the initiation of test substance administration. The test substance was administered to offspring selected to become the F1 parental 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. For both generations (F1 and F2), 8 pups per litter (4 per sex, when possible) were selected on PND 4 to reduce the variability among the litters. Offspring (25/sex/group) from the pairing of the F0 animals were selected on PND 21 to constitute the F1 generation. F0 males and females were dosed for 128-133 consecutive days and F1 males and females were exposed for 136-147 consecutive days, respectively.

 

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals for males and females throughout the study (including during gestation and lactation for females). Vaginal lavages were performed daily for determination of estrous cycles beginning 21 days prior to cohabitation. All F0 and F1 females were allowed to deliver and rear their pups until weaning on lactation day 21. Detailed physical examinations, body weights, and sexes for F1 and F2 pups were recorded at appropriate intervals. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the F1 rats selected to constitute the F1 generation. Nonselected F1 pups were necropsied on PND 21, and F2 pups were necropsied on PND 21. Selected organs were weighed for 1 pup/sex/litter from both F1 and F2 pups that were necropsied on PND 21. Each surviving F0 and F1 parental animal received a complete detailed gross necropsy following the completion of weaning of the F1 and F2 pups, respectively; selected organs were weighed. Spermatogenic endpoints (sperm motility [including progressive motility], morphology, and numbers) were recorded for all F0 and F1 males, and ovarian primordial follicle counts were recorded for all F1 females in the control and high-dose groups and for all F1 females suspected of reduced fertility. Designated tissues from F0 and F1 parental animals were examined microscopically.

 

RESULTS

Test substance-related effects on F0 and F1 parental survival occurred at 400 mg/kg/day. In the F0 generation, 1 female was euthanizedin extremison lactation day 0 following noteworthy clinical findings of piloerection, unkempt appearance, red material around the nose, mouth, and ventral trunk, and red vaginal discharge. Ulceration of the ileum, characterized by ulceration of the ileal mucosa with associated subacute inflammation,

was determined as the cause of the moribund condition for this female; the relationship of this death to administration of the test substance was unclear. An additional F0 female in this group was euthanizedin extremison gestation day 22 following noteworthy clinical findings of pale and cool extremities, a cool body, and red material on various body surfaces. Although a cause of death was undetermined microscopically, the aforementioned clinical signs, in conjunction with the retention of 16 live fetusesinutero, pointed to a difficult delivery as the likely cause of death; however, a relationship to administration of the test substance was possible. In the F1 generation, 1 male was found dead on PND 34 and 2 females were found dead on PND 32. The cause of death for these animals was undetermined based on macroscopic and microscopic findings. However, based on macroscopic lung findings, gavage error was considered a likely cause of death, although a relationship to treatment with the test substance could not be completely dismissed. No other possible test substance-related effects on survival were noted for F0 and F1 parental animals at any dosage level.

 

One F0 male in the 100 mg/kg/day group was found dead on study day 44 due to an intubation error and 1 F1 male in the 25 mg/kg/day group was found dead on PND 27; dilatation of the ventricles of the brain was determined to be the cause of moribundity for the F1 male in the 25 mg/kg/day group. In the control group, 1 F1 male was euthanizedinextremison PND 27 and 1 F1 female was euthanizedin extremisgestation day 10 due to clinical signs and effects on body weight; dilatation of the ventricles of the brain and an exostoses adjacent to the pituitary, respectively, were determined to be the causes for the moribund condition of these animals. All other F0 and F1 parental animals survived to the scheduled necropsies.

 

Test substance-related clinical findings, including clear material on various body surfaces and red material around the mouth, were noted for F0 and F1 parental animals in the 100 and/or 400 mg/kg/day groups approximately 1 hour following dose administration. Clear material findings, primarily observed around the mouth, were noted for the majority of F0 and F1 males and females in the 400 mg/kg/day group, as well as to a slightly lesser extent for F0 and F1 males in the 100 mg/kg/day group, throughout the respective treatment periods. Red material around the mouth was noted predominantly for F0 and F1 males and females in the 400 mg/kg/day group throughout the respective treatment periods. No test substance-related clinical findings were noted at the detailed physical examinations for F0 and F1 parental animals at any dosage level. Therefore, because these clear and red material findings noted during the post-dosing observations did not generally persist to the detailed physical examinations the following day, they were not considered adverse.

 

Test substance-related lower mean body weight gains, in the absence of corresponding reduced mean food consumption, were noted for F0 males in the 400 mg/kg/day group throughout the entire generation, resulting in a mean body weight on study day 126 that was 10.4% lower than the control group. Mean body weights, body weight gains, and food consumption for F0 males in the 25 and 100 mg/kg/day groups were similar to the control group throughout the entire generation. For F0 females, mean body weights and body weights gains were similar to the control group throughout the pre-mating period, as well as during gestation and lactation at all dosage levels. Test substance-related higher mean food consumption and food efficiency were noted for F0 females in the 400 mg/kg/day group during gestation; however, mean body weights and body weight gains were similar to the control group throughout gestation; therefore, the increases were not considered adverse. No test substance-related effects on mean food consumption and/or food efficiency were noted for F0 females in the 400 mg/kg/day group during the pre-mating period and lactation, as well as in the 25 and 100 mg/kg/day groups throughout pre-mating, gestation, and lactation.

 

In the F1 generation, lower mean body weights and body weight gains were noted for males and females in the 400 mg/kg/day group, which were continuations of effects that originated during the pre-weaning period. Lower mean body weight gains, with generally reduced mean food consumption, were noted for F1 males throughout the entire generation, resulting in a mean body weight on PND 154 that was 15.5% lower than the control group. Mean body weights, body weight gains, and food consumption for F1 males in the 25 and 100 mg/kg/day groups were similar to the control group throughout the entire generation. For the F1 females, lower mean body weight gains and/or reduced mean food consumption were noted in the 400 mg/kg/day group for the first 2 weeks following weaning (PND 21-35), but similar to the control group thereafter. However, mean body weights in this group were 5.4% to 16.3% lower than the control group throughout the pre-mating period (PND 21-98). Mean body weights and body weight changes for F1 females were similar to the control group at 25 and 100 mg/kg/day during the pre-mating period, and at all dosage levels during gestation and lactation. Test substance-related higher mean food consumption was noted for F1 females in the 400 mg/kg/day group during gestation; however, mean body weights and body weight gains were similar to the control group throughout gestation; therefore, the increases were not considered adverse. No test substance-related effects on mean food consumption and/or food efficiency were noted for F1 females in the 400 mg/kg/day group during lactation, as well as in the 25 and 100 mg/kg/day groups throughout pre-mating, gestation, and lactation.

 

No test substance-related effects were observed on F0 and F1 reproductive performance (estrous cycles, mating, fertility, copulation or conception indices, the mean number of days between pairing and coitus, and the mean length of gestation), parturition, or the mean numbers of implantation sites and unaccounted-for sites. Spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate, motility, progressive motility, and the percentage of morphologically normal sperm) were also unaffected by test substance administration.

 

There were no test substance-related macroscopic findings noted for F0 and F1 males or females at any dosage level. Test substance-related higher mean liver weights (absolute and relative to final body weight and/or brain weight) were noted in the 400 mg/kg/day group F0 males and females and F1 females. With the exception of the F1 females, there were no microscopic correlates; therefore, these organ weight changes were not considered adverse. Test substance-related hepatocellular hypertrophy was noted for F1 females in the 400 mg/kg/day group; the change was minimal to mild, correlated to higher liver weights in this group, and was considered an adaptive, non-adverse change. No test substance-related organ weight changes or microscopic findings were noted for F0 and F1 males and females at 25 and 100 mg/kg/day, organ weight changes for F1 males at 400 mg/kg/day, and microscopic findings for F0 males and females and F1 males at 400 mg/kg/day.

 

Total litter losses and increased numbers of pups found dead between PND 1-7 were noted for F0 females in the 400 mg/kg/day group. As a result, test substance-related lower mean postnatal survival was noted from birth to PND 4 and PND 4-21 for F1 pups in the 400 mg/kg/day group. The mean numbers of F1 pups born, percentage of males at birth, live litter size on PND 0, and general physical condition of pups at all dosage levels and postnatal survival at 25 and 100 mg/kg/day were unaffected by F0 parental test substance administration. A test substance-related lower mean F2 live litter size on PND 0 was noted in the 400 mg/kg/day group. Additionally, test substance-related lower postnatal survival was observed for F2 pups in the 400 mg/kg/day group during birth to PND 4, primarily due to a total litter loss for 1 F1 female on PND 0 and an increased numbers of pups found dead between PND 0-4 for an additional F1 female in this group. Postnatal survival for F2 pups at 400 mg/kg/day was similar to the control group during the remainder of the postnatal period (PND 4-21). The mean number of F2 pups born, live litter size on PND 0, and postnatal survival at 25 and 100 mg/kg/day, and the percentages of males at birth and general physical condition of pups at all dosage levels were unaffected by F1 parental test substance administration.

 

Test substance-related effects on mean F1 and F2 pup body weight were noted during the pre-weaning period. Lower mean pup body weight gains were noted for F1 males and females during PND 4-21 and for F2 males and females during PND 7-21. As a result, mean F1 and F2 pup body weights were up to 15.0% and 15.4% lower for males and females, respectively, during the pre-weaning period. Mean F1 and F2 male and female pup body weights and body weight gains in the 25 and 100 mg/kg/day groups were unaffected by parental test substance administration.

 

No test substance-related effects on the mean ages of attainment of balanopreputial separation or vaginal patency were noted in the F1 weanlings. However, mean body weights at the age of attainment of these developmental landmarks were lower in the 400 mg/kg/day group males and females due to the aforementioned decrements in mean body weights and body weight gains.

 

No macroscopic findings noted at any dosage level in the F1 and F2 pups that were found dead, euthanizedin extremis, or euthanized at the scheduled necropsy on PND 21 that was attributed to F0 or F1 parental test substance administration.

 

Test substance-related lower mean final body weights were noted for F1 and F2 males and females in the 400 mg/kg/day group on PND 21. In addition, test substance-related lower mean absolute brain and spleen weights for F1 male and female pups and lower mean absolute and relative (to final body weight and brain weight) brain and spleen weights for F2 males and females were noted on PND 21. No test substance-related effects on organ weights were observed for F1 and F2 male and female pups in the 25 and 100 mg/kg/day groups.

 

CONCLUSIONS

Based on the absence of effects on F0 and F1 reproductive performance at any dosage level, a dosage level of 400 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for parental reproductive toxicity of test item administered orally by gavage to Crl:CD(SD) rats. Based on the lower live litter size on PND 0 for F2 pups, decreased postnatal survival, and lower mean body weights and body weight gains during the pre-weaning period for F1 and F2 pups at 400 mg/kg/day, the NOAEL for neonatal toxicity was considered to be 100 mg/kg/day. Based on moribundity for F0 females and mortality for F1 males and females, as well as lower mean body weights, body weight gains, and/or food consumption for F0 males and F1 males and females at 400 mg/kg/day, the NOAEL for parental systemic toxicity was considered to be 100 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study

GUIDELINE

The study was conducted to evaluate the potential adverse effects of the test item on reproduction in a two generation study. This included determining the effects of the test substance on male and female reproductive processes, including gonadal function, estrous cyclicity, mating behavior, conception, gestation, parturition, lactation, weaning, and on growth and development of the offspring. A minimum of 1 litter per dam was produced in each generation. The protocol was designed in general accordance with the United States EPA Health Effects Test Guidelines: OPPTS 870.3800, Reproduction and Fertility Effects, Aug-1998, and the OECD Guidelines for the Testing of Chemicals, Guideline 416, Two-Generation Reproduction Toxicity Study, 22-Jan-2001.

 

METHODS

Four groups of male and female Crl:CD(SD) rats (25/sex/group) were administered the test substance, daily by oral gavage, for at least 70 consecutive days prior to mating. Dosage levels were 25, 100, and 400 mg/kg/day for the F0 and F1 generations. A concurrent control group of 25 rats/sex/group received the vehicle (arachis [peanut] oil) on a comparable regimen. F0 animals were approximately 6 weeks of age at the initiation of test substance administration. The test substance was administered to offspring selected to become the F1 parental 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. For both generations (F1 and F2), 8 pups per litter (4 per sex, when possible) were selected on PND 4 to reduce the variability among the litters. Offspring (25/sex/group) from the pairing of the F0 animals were selected on PND 21 to constitute the F1 generation. F0 males and females were dosed for 128-133 consecutive days and F1 males and females were exposed for 136-147 consecutive days, respectively.

 

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals for males and females throughout the study (including during gestation and lactation for females). Vaginal lavages were performed daily for determination of estrous cycles beginning 21 days prior to cohabitation. All F0 and F1 females were allowed to deliver and rear their pups until weaning on lactation day 21. Detailed physical examinations, body weights, and sexes for F1 and F2 pups were recorded at appropriate intervals. Developmental landmarks (balanopreputial separation and vaginal patency) were evaluated for the F1 rats selected to constitute the F1 generation. Nonselected F1 pups were necropsied on PND 21, and F2 pups were necropsied on PND 21. Selected organs were weighed for 1 pup/sex/litter from both F1 and F2 pups that were necropsied on PND 21. Each surviving F0 and F1 parental animal received a complete detailed gross necropsy following the completion of weaning of the F1 and F2 pups, respectively; selected organs were weighed. Spermatogenic endpoints (sperm motility [including progressive motility], morphology, and numbers) were recorded for all F0 and F1 males, and ovarian primordial follicle counts were recorded for all F1 females in the control and high-dose groups and for all F1 females suspected of reduced fertility. Designated tissues from F0 and F1 parental animals were examined microscopically.

 

RESULTS

Test substance-related effects on F0 and F1 parental survival occurred at 400 mg/kg/day. In the F0 generation, 1 female was euthanizedin extremison lactation day 0 following noteworthy clinical findings of piloerection, unkempt appearance, red material around the nose, mouth, and ventral trunk, and red vaginal discharge. Ulceration of the ileum, characterized by ulceration of the ileal mucosa with associated subacute inflammation, was determined as the cause of the moribund condition for this female; the relationship of this death to administration of the test substance was unclear. An additional F0 female in this group was euthanizedin extremison gestation day 22 following noteworthy clinical findings of pale and cool extremities, a cool body, and red material on various body surfaces. Although a cause of death was undetermined microscopically, the aforementioned clinical signs, in conjunction with the retention of 16 live fetusesinutero, pointed to a difficult delivery as the likely cause of death; however, a relationship to administration of the test substance was possible. In the F1 generation, 1 male was found dead on PND 34 and 2 females were found dead on PND 32. The cause of death for these animals was undetermined based on macroscopic and microscopic findings. However, based on macroscopic lung findings, gavage error was considered a likely cause of death, although a relationship to treatment with the test substance could not be completely dismissed. No other possible test substance-related effects on survival were noted for F0 and F1 parental animals at any dosage level.

 

One F0 male in the 100 mg/kg/day group was found dead on study day 44 due to an intubation error and 1 F1 male in the 25 mg/kg/day group was found dead on PND 27; dilatation of the ventricles of the brain was determined to be the cause of moribundity for the F1 male in the 25 mg/kg/day group. In the control group, 1 F1 male was euthanized in extremis on PND 27 and 1 F1 female was euthanized in extremis on gestation day 10 due to clinical signs and effects on body weight; dilatation of the ventricles of the brain and an exostoses adjacent to the pituitary, respectively, were determined to be the causes for the moribund condition of these animals. All other F0 and F1 parental animals survived to the scheduled necropsies.

 

Test substance-related clinical findings, including clear material on various body surfaces and red material around the mouth, were noted for F0 and F1 parental animals in the 100 and/or 400 mg/kg/day groups approximately 1 hour following dose administration. Clear material findings, primarily observed around the mouth, were noted for the majority of F0 and F1 males and females in the 400 mg/kg/day group, as well as to a slightly lesser extent for F0 and F1 males in the 100 mg/kg/day group, throughout the respective treatment periods. Red material around the mouth was noted predominantly for F0 and F1 males and females in the 400 mg/kg/day group throughout the respective treatment periods. No test substance-related clinical findings were noted at the detailed physical examinations for F0 and F1 parental animals at any dosage level. Therefore, because these clear and red material findings noted during the post-dosing observations did not generally persist to the detailed physical examinations the following day, they were not considered adverse.

 

Test substance-related lower mean body weight gains, in the absence of corresponding reduced mean food consumption, were noted for F0 males in the 400 mg/kg/day group throughout the entire generation, resulting in a mean body weight on study day 126 that was 10.4% lower than the control group. Mean body weights, body weight gains, and food consumption for F0 males in the 25 and 100 mg/kg/day groups were similar to the control group throughout the entire generation. For F0 females, mean body weights and body weights gains were similar to the control group throughout the pre-mating period, as well as during gestation and lactation at all dosage levels. Test substance-related higher mean food consumption and food efficiency were noted for F0 females in the 400 mg/kg/day group during gestation; however, mean body weights and body weight gains were similar to the control group throughout gestation; therefore, the increases were not considered adverse. No test substance-related effects on mean food consumption and/or food efficiency were noted for F0 females in the 400 mg/kg/day group during the pre-mating period and lactation, as well as in the 25 and 100 mg/kg/day groups throughout pre-mating, gestation, and lactation.

 

In the F1 generation, lower mean body weights and body weight gains were noted for males and females in the 400 mg/kg/day group, which were continuations of effects that originated during the pre-weaning period. Lower mean body weight gains, with generally reduced mean food consumption, were noted for F1 males throughout the entire generation, resulting in a mean body weight on PND 154 that was 15.5% lower than the control group. Mean body weights, body weight gains, and food consumption for F1 males in the 25 and 100 mg/kg/day groups were similar to the control group throughout the entire generation. For the F1 females, lower mean body weight gains and/or reduced mean food consumption were noted in the 400 mg/kg/day group for the first 2 weeks following weaning (PND 21-35), but similar to the control group thereafter. However, mean body weights in this group were 5.4% to 16.3% lower than the control group throughout the pre-mating period (PND 21-98). Mean body weights and body weight changes for F1 females were similar to the control group at 25 and 100 mg/kg/day during the pre-mating period, and at all dosage levels during gestation and lactation. Test substance-related higher mean food consumption was noted for F1 females in the 400 mg/kg/day group during gestation; however, mean body weights and body weight gains were similar to the control group throughout gestation; therefore, the increases were not considered adverse. No test substance-related effects on mean food consumption and/or food efficiency were noted for F1 females in the 400 mg/kg/day group during lactation, as well as in the 25 and 100 mg/kg/day groups throughout pre-mating, gestation, and lactation.

 

No test substance-related effects were observed on F0 and F1 reproductive performance (estrous cycles, mating, fertility, copulation or conception indices, the mean number of days between pairing and coitus, and the mean length of gestation), parturition, or the mean numbers of implantation sites and unaccounted-for sites. Spermatogenic endpoints (mean testicular and epididymal sperm numbers, sperm production rate, motility, progressive motility, and the percentage of morphologically normal sperm) were also unaffected by test substance administration.

 

There were no test substance-related macroscopic findings noted for F0 and F1 males or females at any dosage level. Test substance-related higher mean liver weights (absolute and relative to final body weight and/or brain weight) were noted in the 400 mg/kg/day group F0 males and females and F1 females. With the exception of the F1 females, there were no microscopic correlates; therefore, these organ weight changes were not considered adverse. Test substance-related hepatocellular hypertrophy was noted for F1 females in the 400 mg/kg/day group; the change was minimal to mild, correlated to higher liver weights in this group, and was considered an adaptive, non-adverse change. No test substance-related organ weight changes or microscopic findings were noted for F0 and F1 males and females at 25 and 100 mg/kg/day, organ weight changes for F1 males at 400 mg/kg/day, and microscopic findings for F0 males and females and F1 males at 400 mg/kg/day.

 

Total litter losses and increased numbers of pups found dead between PND 1-7 were noted for F0 females in the 400 mg/kg/day group. As a result, test substance-related lower mean postnatal survival was noted from birth to PND 4 and PND 4-21 for F1 pups in the 400 mg/kg/day group. The mean numbers of F1 pups born, percentage of males at birth, live litter size on PND 0, and general physical condition of pups at all dosage levels and postnatal survival at 25 and 100 mg/kg/day were unaffected by F0 parental test substance administration. A test substance-related lower mean F2 live litter size on PND 0 was noted in the 400 mg/kg/day group. Additionally, test substance-related lower postnatal survival was observed for F2 pups in the 400 mg/kg/day group during birth to PND 4, primarily due to a total litter loss for 1 F1 female on PND 0 and an increased numbers of pups found dead between PND 0-4 for an additional F1 female in this group. Postnatal survival for F2 pups at 400 mg/kg/day was similar to the control group during the remainder of the postnatal period (PND 4-21). The mean number of F2 pups born, live litter size on PND 0, and postnatal survival at 25 and 100 mg/kg/day, and the percentages of males at birth and general physical condition of pups at all dosage levels were unaffected by F1 parental test substance administration.

 

Test substance-related effects on mean F1 and F2 pup body weight were noted during the pre-weaning period. Lower mean pup body weight gains were noted for F1 males and females during PND 4-21 and for F2 males and females during PND 7-21. As a result, mean F1 and F2 pup body weights were up to 15.0% and 15.4% lower for males and females, respectively, during the pre-weaning period. Mean F1 and F2 male and female pup body weights and body weight gains in the 25 and 100 mg/kg/day groups were unaffected by parental test substance administration.

 

No test substance-related effects on the mean ages of attainment of balanopreputial separation or vaginal patency were noted in the F1 weanlings. However, mean body weights at the age of attainment of these developmental landmarks were lower in the 400 mg/kg/day group males and females due to the aforementioned decrements in mean body weights and body weight gains.

 

No macroscopic findings noted at any dosage level in the F1 and F2 pups that were found dead, euthanized in extremis, or euthanized at the scheduled necropsy on PND 21 that was attributed to F0 or F1 parental test substance administration.

 

Test substance-related lower mean final body weights were noted for F1 and F2 males and females in the 400 mg/kg/day group on PND 21. In addition, test substance-related lower mean absolute brain and spleen weights for F1 male and female pups and lower mean absolute and relative (to final body weight and brain weight) brain and spleen weights for F2 males and females were noted on PND 21. No test substance-related effects on organ weights were observed for F1 and F2 male and female pups in the 25 and 100 mg/kg/day groups.

 

CONCLUSIONS

Based on the absence of effects on F0 and F1 reproductive performance at any dosage level, a dosage level of 400 mg/kg/day (the highest dosage level tested) was considered to be the no-observed-adverse-effect level (NOAEL) for parental reproductive toxicity of test item administered orally by gavage to Crl:CD(SD) rats. Based on the lower live litter size on PND 0 for F2 pups, decreased postnatal survival, and lower mean body weights and body weight gains during the pre-weaning period for F1 and F2 pups at 400 mg/kg/day, the NOAEL for neonatal toxicity was considered to be 100 mg/kg/day. Based on moribundity for F0 females and mortality for F1 males and females, as well as lower mean body weights, body weight gains, and/or food consumption for F0 males and F1 males and females at 400 mg/kg/day, the NOAEL for parental systemic toxicity was considered to be 100 mg/kg/day.

Effects on developmental toxicity

Description of key information

Key study

Maternal toxicity was evidenced at 1000 mg/kg/day by lower mean body weight gains, body weights, and food consumption. No evidence of maternal toxicity was noted at 250 or 500 mg/kg/day. Based on these results, a dosage level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Developmental toxicity was noted at 500 and 1000 mg/kg/day as evidenced by lower mean fetal body weights, which, in conjunction with a lower number of viable fetuses, correlated with a lower mean gravid uterine weight in the 1000 mg/kg/day group. Higher mean litter proportion of postimplantation loss with a corresponding lower mean number and litter proportion of viable fetuses were also noted at 1000 mg/kg/day. In addition,test substance-related fetal malformations were noted at 1000 mg/kg/day and test substance-related developmental variations were noted at 500 and 1000 mg/kg/day. Intrauterine growth in the 250 mg/kg/day group and survival in the 250 and 500 mg/kg/day groups were unaffected by test substance administration. Based on these results, a dosage level of 250 mg/kg/day was considered to be the NOAEL for embryo/fetal development when test item was administered orally by gavage to bred Crl:CD(SD) rats (OECD 414 and OPPTS 870.3700).

Expert opinion

Based on detailed review of the study data, fetal skeletal variations noted at 500 and 1000 mg/kg/day occurred in the presence of maternal toxicity and fetal development delay and should be considered secondary. Given the excessive reductions in maternal body weight at 1000 mg/kg/day and consequent drastic reductions in fetal weight (up to 35.9 % versus controls), the dose was excessively high. Thus, any development effects noted in these fetuses should be interpreted with caution as they were noted in the presence of, and are likely secondary to, frank maternal toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July 2014 to 19 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

various with no impact on the result of the study (see Appendix A, Attached)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

various with no impact on the result of the study (see Appendix A, Attached)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rat

Strain:

other: Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION
- Sexually mature, virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. This species and strain of animal is recognized as appropriate for developmental toxicity studies. WIL Research has historical control data on the background incidence of fetal malformations and developmental variations in the
Crl:CD(SD) rat. This animal model has been proven to be susceptible to the effects of developmental toxicants.
- The number of animals selected for the study was based on the United States EPA Health Effects Test Guidelines: OPPTS 870.3700, Prenatal Development Toxicity Study, Aug-1998 and the OECD Guidelines for the Testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, 22-Jan-2001, which recommends evaluation of approximately 20 females with implantation sites at necropsy. Given the possibility of nongravid animals, unexpected deaths, or treatment-related moribundity and/or mortality, 25 females/group was an appropriate number of animals to obtain a sample size of 20 at termination.
- Crl:CD(SD) rats (125 females) were received in good health from Charles River Laboratories, Inc., Stone Ridge, NY, on 24-Jul-2014. The animals were approximately 80 days old upon receipt. Each female was examined by a qualified biologist on the day of receipt. The day following receipt, all animals were weighed and clinical observations were recorded. Each rat was uniquely identified by a Monel metal ear tag displaying the animal number and housed for a minimum of 12 days for acclimation purposes. During the acclimation period, the rats were observed twice daily for mortality and changes in general appearance and behavior.

ANIMAL HOUSING
- Upon arrival and until pairing, all rats were individually housed in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week.
- The rats were paired for mating in the home cage of the male.
- Following positive evidence of mating, the females were returned to individual suspended wire-mesh cages; nesting material was not required as the females were euthanized prior to the date of expected parturition.
- Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National
Research Council, 2011). The animal facilities at WIL Research are fully accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitized weekly.

DIET, DRINKING WATER AND MAINTENANCE
- The basal diet used in this study, PMI Nutrition International, LLC Certified Rodent LabDiet 5002, was a certified feed with appropriate analyses performed by the manufacturer and provided to WIL Research. Feed lots used during the study were documented in the study records. The feeders were changed and sanitized once per week.
- Municipal water supplying the facility was sampled for contaminants according to WIL Research SOPs. The results of the diet and water analyses are maintained at WIL Research. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.
- Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet
were provided ad libitum throughout the acclimation period and during the study.


ENVIRONMENTAL CONDITIONS
- All rats were housed throughout the acclimation period and during the study in an environmentally controlled room. The room temperature and relative humidity controls were set to maintain environmental conditions of 71 °F ± 5 °F (22 °C ± 3 °C) and 50 % ± 20 %, respectively.
- Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis. Actual mean daily temperature ranged from 69.8 °F to 70.7°F (21.0 °C to 21.5 °C) and mean daily relative humidity ranged from 47.2 % to 58.6 % during the study.
- Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes.
- Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Route of administration:

oral: gavage

Vehicle:

peanut oil

Remarks:

Lot number 1DB0384; expiry date 31 January 2015

Details on exposure:

PREPARATION
- The vehicle suspension was prepared approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature, protected from light.
- The vehicle was mixed throughout the preparation, sampling, and dose administration procedures.
- Dosing formulations were prepared at the test substance concentrations indicated in the table below.
- The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature , protected from light. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
- The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

ORGANIZATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- The vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, Teflon-shafted, stainless steel ball-tipped dosing cannula once daily during gestation days 6-19.
- The dosage volume for all groups was 5 mL/kg.
- Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose.
- All animals were dosed at approximately the same time each day.
- Study group assignment is shown in the table below.
- Dosage levels were selected based on results of a previous 28-day toxicity study in rats (Haas, 2015, WIL-168211) in which dosage levels of 250, 500, and 1000 mg/kg/day were used. There were no apparent effects on body weight gains or food consumption in the previous study and no significant clinical signs noted in either sex. A single female in the 1000 mg/kg/day group was found dead on study day 18; however, no cause of death could be determined for this animal. Based on the limited toxicity noted in the previous study, dosage levels of 250, 500, and 1000 (limit dose for OECD 414 studies) mg/kg/day were chosen for evaluation on the current study.
- The selected route of administration for this study was oral (gavage) because this is a potential route of exposure for humans. Historically, this route has been used extensively for studies of this nature.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLING AND ANALYSES
- Homogeneity and stability of the test substance in the vehicle for up to 7 days were previously demonstrated at concentrations spanning the range used in the current study (Haas, 2015, WIL-168211).
- Samples for concentration and/or homogeneity determinations were collected from the middle stratum of the first and last control group dosing formulations and from the top, middle, and bottom strata of the first and last test substance dosing formulations.
- Samples were shipped at room temperature, protected from light, to Wildlife International for analysis. All analyses were conducted by Wildlife International using a validated method (Wildlife International Project No. 796C-102).

Details on mating procedure:

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS AND BREEDING PROCEDURES
- At the conclusion of the acclimation period, all available females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was placed in a suspended wire-mesh cage with a resident male from the same strain and source for breeding.
- Resident males were untreated, sexually mature rats utilized exclusively for breeding. These rats were maintained under similar laboratory conditions as the females.
- A breeding record containing the male and female identification numbers and the dates of cohabitation was maintained.
- The selected females were approximately 13 weeks old when paired for breeding.
- Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm in a vaginal lavage and verified by a second biologist. Each mating pair was examined daily.
- The day on which evidence of mating was identified was termed gestation day 0 and the animals were separated.
- The experimental design consisted of 3 test substance-treated groups and 1 control group, composed of 25 rats per group.
- The bred females were assigned to groups using a WTDMS computer program which randomized the animals based on stratification of the gestation day 0 body weights in a block design.
- Animals not assigned to the study were transferred to the WIL Research stock colony or euthanized by carbon dioxide inhalation and discarded.
- Body weight values ranged from 216 g to 302 g on gestation day 0.

Duration of treatment / exposure:

Gestation days 6 to 19

Frequency of treatment:

Daily

Duration of test:

20 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25 females

Control animals:

yes, concurrent vehicle

Details on study design:

- See diagram summarising study design (attached).

Maternal examinations:

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

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

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

Ovaries and uterine content:

GESTATION DAY 20 LAPAROHYSTERECTOMY
- Laparohysterectomies and macroscopic examinations were performed blind to treatment group.
- All females were euthanized on gestation day 20 by carbon dioxide inhalation.
- The thoracic, abdominal, and pelvic cavities were opened by a ventral mid-line incision, and the contents were examined. In all instances, the postmortem findings were correlated with the antemortem observations, and any abnormalities were recorded. The uterus and ovaries were then exposed and excised.
- The number of corpora lutea on each ovary was recorded. The trimmed uterus was weighed and opened, and the number and location of all fetuses, early and late resorptions, and the total number of implantation sites were recorded. The placentae were also examined.
- The individual uterine distribution of implantation sites was documented using the following procedure. All implantation sites, including resorptions, were numbered in consecutive order beginning with the left distal to the left proximal uterine horn, noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn.
- Maternal tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as indicated by the gross findings. Representative sections of corresponding organs from a sufficient number of control animals were retained for comparison. The carcass of each female was then discarded.

Fetal examinations:

FETAL MORPHOLOGICAL EXAMINATION
- Fetal examinations were performed blind to treatment group.
- Each viable fetus was examined externally, individually sexed, weighed, euthanized by a subcutaneous injection of sodium pentobarbital in the scapular region, and tagged for identification. Fetal tags contained the WIL Research study number, the female number, and the fetus number.
- The detailed external examination of each fetus included, but was not limited to, an examination of the eyes, palate, and external orifices, and each finding was recorded.
- Nonviable fetuses (if the degree of autolysis was minimal or absent) were examined, the crown-rump length measured, weighed, sexed, and tagged individually. Crown-rump measurements and degrees of autolysis were recorded for late resorptions, a gross external examination was performed (if possible), and the tissues were discarded.
- Each viable fetus was subjected to a visceral examination using a modification of the Stuckhardt and Poppe fresh dissection technique to include the heart and major blood vessels (Stuckhardt and Poppe, 1984). The sex of each fetus was confirmed by internal examination. Fetal kidneys were examined and graded for renal papillae development (Woo and Hoar, 1972).
- Heads from approximately one-half of the fetuses in each litter were placed in Harrison’s fixative for subsequent soft-tissue examination by the Wilson sectioning technique (Wilson, 1965). The heads from the remaining one-half of the fetuses were examined by a midcoronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol.
- Following fixation in alcohol, each fetus was stained with Alizarin Red S (Dawson, 1926) and Alcian Blue (Inouye, 1976). Fetuses were then examined for skeletal malformations and developmental variations. External, visceral, and skeletal findings were recorded as developmental variations (alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity and/or occur at high incidence, representing slight deviations from normal) or malformations (those structural anomalies that alter
general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).

Statistics:

- Statistical analyses are described below.

Indices:

- Intrauterine data were summarized using two methods of calculation (group mean litter basis calculated as postimplantation loss and proportional litter basis calculated as summation per group (%).
- 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 where Postimplantation Loss/Litter (%) = [No Dead fetuses, Resorptions (Early/Late)/Litter / No Implantation Sites/Litter] x 100
- The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis using the equation Summation per Group (%) = [(Sum of Viable Fetuses Affected / Litter (%)) / No Litters/Group] where Viable Fetuses Affected / Litter (%) = [(No Viable Fetuses Affected/Litter) / No Viable Fetuses/Litter] x 100.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Test substance-related increased incidences of yellow material around the ventral trunk and urogenital areas were noted in the 1000 mg/kg/day group generally throughout the treatment period and were not considered to be adverse.
- Increased incidences of clear and red material around the mouth were noted in all test substance-treated groups at approximately 1 hour following dose administration when compared to the control group. These findings were generally noted throughout the treatment period. Because clear and red material around the mouth did not persist to the daily examinations on the following day they were not considered adverse.
- No other test substance-related clinical findings were noted approximately 1 hour following dose administration or at the daily examinations at any dosage level.
- Findings noted in the treated groups, including hair loss on the forelimbs and ventral trunk, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

- All females in the control, 250, 500, and 1000 mg/kg/day groups survived to the scheduled necropsy on gestation day 20.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

MATERNAL BODY WEIGHT CHANGES AND GRAVID UTERINE WEIGHTS
- Test substance-related, significantly (p<0.01) lower mean body weight gains were noted in the 1000 mg/kg/day group compared to the control group generally throughout the treatment period (gestation days 6-20). As a result, mean body weights in the 1000 mg/kg/day group were 5.4% to 11.8% lower than the control group during gestation days 17-20; the differences were significant (p<0.05 or p<0.01). Mean gravid uterine weight, net body weight, and net body weight gain in this group were also significantly (p<0.05 or p<0.01) lower than the control group.
- In the 500 mg/kg/day group, mean body weight gains were similar to the control group during gestation days 6-9. During gestation days 9-12 a significantly (p<0.01) higher mean body weight gain was noted in the 500 mg/kg/day group due to a significantly (p<0.01) higher mean body weight gain during gestation days 11-12 when compared to the control group. Test substance-related, significantly (p<0.05 or p<0.01) lower mean body weight gains were noted in this group compared to the control group during the remainder of the study (gestation days 12-15 and 15-20). However, the lower mean body weight gains noted in the 500 mg/kg/day group were not of sufficient magnitude to affect the overall treatment period (gestation days 6-20) and mean body weights in this group were similar to the control group throughout the study; therefore, these differences were not considered to be adverse. Mean net body weight, net body weight gain, and gravid uterine weight in the 500 mg/kg/day group were unaffected by test substance administration.
- Mean maternal body weights, body weight gains, net body weight, net body weight gain, and gravid uterine weight in the 250 mg/kg/day group were unaffected by test substance administration. Differences from the control group were slight and not statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Test substance-related lower mean food consumption, evaluated as g/animal/day and g/kg/day, was noted in the 1000 mg/kg/day group throughout the treatment period (gestation days 6-20). The differences from the control group were generally significant (p<0.05 or p<0.01) and corresponded to the lower mean body weight gains noted for these females.
- Mean maternal food consumption in the 250 and 500 mg/kg/day groups was unaffected by test substance administration. Differences from the control group were slight and not statistically significant, with the following exceptions. Significantly (p<0.05) lower mean food consumption was noted in the 500 mg/kg/day group during gestation days 14-15 and 17-18. Because these differences were transient and did not affect the overall treatment interval, they were not considered to be test substance-related.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

- At the scheduled necropsy on gestation day 20, no test substance-related internal findings were observed at dosage levels of 250, 500, and 1000 mg/kg/day.
- Macroscopic findings observed in the test substance-treated groups occurred infrequently, at similar frequencies
in the control group, and/or in a manner that was not dose-related.
- All females were determined to be gravid.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

GESTATION DAY 20 LAPAROHYSTERECTOMY DATA
- Test substance-related effects on intrauterine survival were noted in the 1000 mg/kg/day group. The mean litter proportion of postimplantation loss (early and late resorptions) in the 1000 mg/kg/day group (18.5% per litter) was significantly (p<0.01) higher than the control group (3.1% per litter) and the value exceeded the maximum mean value in the WIL Research historical control data (11.3% per litter). As a result, a significantly (p<0.05 or p<0.01) lower mean number (12.7 per dam) and litter proportion (81.5% per litter) of viable fetuses was noted in this group when compared to the concurrent control group (15.1 per dam and 96.9% per litter, respectively) and the minimum mean value in the WIL Research historical control data (12.2 fetuses per dam and 88.7% per litter, respectively).
- Test substance-related effects on intrauterine growth were noted at dosage levels of 500 and 1000 mg/kg/day. In the 500 and 1000 mg/kg/day groups, test substance-related lower mean male (3.4 g and 2.6 g, respectively), female (3.2 g and 2.5 g, respectively), and combined-sex (3.3 g and 2.5 g, respectively) fetal body weights were noted compared to the concurrent control group values (4.0 g, 3.8 g, and 3.9 g, respectively); the differences were significant (p<0.01). The mean fetal body weights at 500 and 1000 mg/kg/day were also below the minimum mean male, female, and combined-sex fetal body weight values in the WIL Research historical control data (3.5 g, 3.4 g, and 3.4 g, respectively). Significantly (p<0.01) lower mean fetal body weights (male, female, and combined) were noted in the 250 mg/kg/day group compared to the control group. However, the magnitude (5.0% to 7.9% lower than the concurrent control group) of the differences was small and the values were within the WIL Research historical control ranges. Therefore, the differences in fetal body weights observed in the 250 mg/kg/day group were not considered to be test substance-related.
- Intrauterine survival at 250 and 500 mg/kg/day was unaffected by maternal test substance administration. Mean numbers of corpora lutea and implantation sites, mean fetal sex ratios, 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.

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: lower mean bodyweight gains, bodyweights and food consumption at 1000 mg/kg bw/day

External malformations:

effects observed, treatment-related

Description (incidence and severity):

EXTERNAL MALFORMATIONS AND VARIATIONS
- External malformations were noted in 0(0), 2(2), 1(1), and 52(12) fetuses (litters) in the control, 250, 500, and 1000 mg/kg/day groups, respectively. Test substance-related external malformations were noted in the 1000 mg/kg/day group and consisted of increased mean litter proportions of fetal anasarca, cleft palate (confirmed skeletally for 1 of 2 fetuses as posterior portion of palatine plates not joined), and localized fetal edema (neck and/or thorax) compared to the concurrent control group.
- The differences from the concurrent control group were not statistically significant, but the mean litter proportions of these external malformations exceeded the maximum mean values in the WIL Research historical control data. Table 2 (attached) summarizes the absolute number of fetuses and mean litter proportions of the test substance-related external malformations noted in the 1000 mg/kg/day group.
- In the 1000 mg/kg/day group, fetus no. 24035-07 had a filamentous tail (skeletally all vertebrae posterior to sacral vertebra no. 4 were absent) and fetus no. 24048-13 had anal atresia and vertebral agenesis (anury; skeletally, all vertebrae posterior to sacral vertebra no. 2 were absent). These findings were noted in single fetuses and the mean litter proportions were not statistically significantly different from the concurrent control group and were within the WIL Research historical control data ranges, therefore, they were not considered test substance-related.
- Fetus no. 24014-14 in the 500 mg/kg/day group had craniorachischisis (cranial vault and vertebral column were open through the sacral region) which consisted skeletally of dorsal aspects of cervical arch no. 1 through sacral arch no. 4 that were reflected more laterally than normal. In the 250 mg/kg/day group, fetus no. 24000-08 had a facial cleft (upper left rostral region) which skeletally consisted of absent nasal and premaxilla bones and misshapen maxilla bones and exencephaly with an open eyelid; skeletally this finding consisted of and absent frontal, parietal, interparietal, and supraoccipital bones. Fetus no. 24019-09 in the 250 mg/kg/day group had a cleft palate (entire length; not confirmed skeletally).
- The external malformations observed in the 250 and 500 mg/kg/day groups were noted for single fetuses, did not occur in a dose-related manner, and the mean litter proportions of these findings were not statistically significantly different from the concurrent control group and therefore they were not attributed to the test substance.
- There were no external developmental variations noted for fetuses in this study.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

SKELETAL MALFORMATIONS AND VARIATIONS
- Skeletal malformations were noted in 6(3), 3(3), 2(2), and 20(12) fetuses (litters) in the control, 250, 500, and 1000 mg/kg/day groups, respectively.
- Test substance-related skeletal malformations were noted in the 1000 mg/kg/day group and consisted of increased mean litter proportions of rib anomaly (small, fused, and/or reduced ossification of ribs), interrupted ossification of the rib(s), and bent limb bone(s) (radius, ulna, humerus, tibia, and/or fibula) compared to the concurrent control group. Although the mean litter proportions of these skeletal malformations were not statistically significantly different from the concurrent control group, the values exceeded the maximum mean values in the WIL Research historical control data. Table 4 (attached) summarizes the absolute number of fetuses and mean litter proportions of the test substance-related skeletal malformations noted at 1000 mg/kg/day. No other test substance-related skeletal malformations were noted at any dosage level.
- One fetus each in the 250 and 1000 mg/kg/day groups had a skull anomaly (basispheniod misshapen and supraoccipital bone absent, respectively). Two fetuses each in the 500 and 1000 mg/kg/day group had vertebral anomalies with or without associated rib anomalies. These skeletal malformations consisted of absent rib, arches, and centra; extra arches and ribs; malproportioned arches; malpositioned centra, arches, and ribs; fused ribs and arches; and forked ribs. In addition, 1 fetus in the 250 mg/kg/day group had sternoschisis (sternal band nos. 4 through 6 not joined) and 1 fetus in this same group had a costal cartilage anomaly (fused and malpositioned); costal cartilage anomalies were also noted for 6 fetuses in the control group. Because these skeletal malformations were noted for single fetuses, were observed similarly in the concurrent control group, and the mean litter proportions were not statistically significantly different from the concurrent control group and/or were within the WIL Research historical control data, they were not considered to be test substance-related.
- Test substance-related skeletal developmental variations were noted in the 500 and 1000 mg/kg/day groups. Increased mean litter proportions of 14th rudimentary rib(s), reduced ossification of the skull, reduced ossification of the vertebral arches, unossification of sternebra(e) nos. 5 and/or 6, unossification of sternebra(e) nos. 1, 2, 3, and/or 4, bent rib(s), and reduced ossification of the ribs and decreased mean litter proportions of ossification of cervical centrum no. 1 were noted in the 500 and 1000 mg/kg/day groups compared to the concurrent control group; the differences were generally significant (p<0.01) and the mean litter proportions were outside of the WIL Research historical control data ranges. Additionally, increased mean litter proportions of unossified hyoid, 14th full rib(s), 7th cervical rib(s), unossification of the entire sternum, pubis, ischium, and vertebral centra, and bent scapula(e) were noted at 1000 mg/kg/day when compared to the concurrent control group; all values were significant (p<0.01) with the exception of 14th full rib(s), 7th cervical rib(s), and vertebral centra unossified, and the mean litter proportions of these findings exceeded the maximum mean values in the WIL Research historical control data ranges. The majority of the test substance-related skeletal developmental variations noted in the 500 and 1000 mg/kg/day groups consisting of reduced ossification or unossified bones were indicative of developmental delay and considered secondary to the reduced fetal weights noted at these dosage levels. Table 5 (attached) summarizes the absolute number of fetuses and mean litter proportions of the test substance-related skeletal developmental variations noted in the 500 and 1000 mg/kg/day groups.
- Other skeletal developmental variations observed in the test substance-treated groups consisted of 27 presacral vertebrae, sternebra(e) malaligned (slight or moderate), and reduced ossification of the 13th rib(s). These findings occurred infrequently or at a frequency similar to the concurrent control group, did not occur in a dose-related manner, and/or the mean litter proportions were within the WIL Research historical control data range, and therefore were not considered to be test substance-related.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

VISCERAL MALFORMATIONS AND VARIATIONS
- Visceral malformations were noted for 0(0), 2(2), 0(0), and 12(8) fetuses (litters) in the control, 250, 500, and 1000 mg/kg/day groups, respectively. A test substance-related visceral malformation noted in the 1000 mg/kg/day group consisted of a right-sided aortic arch (aortic arch and descending aorta course to the right of the vertebral column; right carotid and right subclavian arteries arose independently from the aortic arch [no brachiocephalic trunk] left carotid and left subclavian arteries arose from a common vessel from the aortic arch). Although the mean litter proportion of this finding was not statistically significantly different from the concurrent control group; the value exceeded the maximum mean value in the WIL Research historical control data. No other test substance-related visceral malformations were noted for fetuses in this study.
- In the 1000 mg/kg/day group, coarctation of the pulmonary trunk was noted for fetus nos. 23982-11 and 24012-08 (also noted with transposition of the great vessels), cartilaginous bands were absent from the entire length of the trachea for fetus no. 23972-17, and vestigial aortic arch was noted for fetus no. 23994-03. Interventricular septal defect was noted for 1 and 2 fetuses in the 250 and 1000 mg/kg/day groups, respectively, and coarctation of the aortic arch was noted for 1 fetus each in these same groups. Also in the 250 mg/kg/day group, fetus no. 24034-12 had lobular dysgenesis of the lungs (1 lobe present) and situs inversus (the liver, stomach, pancreas, spleen, kidneys, adrenals, and intestine were laterally transposed). These visceral malformations were noted for 1-2 fetuses per group, the mean litter proportions were not statistically significantly different from the concurrent control group, and/or the mean litter proportions of these findings were within the WIL Research historical control data ranges. Therefore, these visceral malformations were not considered to be test substance-related.
- Test substance-related visceral developmental variations (see Table 3, attached) noted in the 1000 mg/kg/day group include higher mean litter proportions of small and pale spleen and major blood vessel variation (right carotid and right subclavian arteries arose independently from the aortic arch [no brachiocephalic trunk]) compared to the concurrent control group. Although the mean litter proportions of these findings were not statistically significantly different from the concurrent control group; they exceeded the maximum mean values in the WIL Research historical control data and were therefore attributed to maternal test substance administration.
- All other visceral developmental variations noted in the test substance-treated groups were noted at similar frequencies in the concurrent control group, did not occur in a dose-related manner, and/or the mean litter proportions of the findings were not statistically significantly different from the concurrent control group and were within the WIL Research historical control data ranges and therefore were not considered test substance-related.
- Renal papilla(e) not fully developed (Woo and Hoar Grade 1) were noted for 1, 3, and 2 fetuses in the 250, 500, and 1000 mg/kg/day groups, respectively. In addition, 1 fetus (no. 23967-12) in the 500 mg/kg/day group had a swollen liver (all lobes). These findings were not classified as either malformations or developmental variations, were not included on the summary tables, and were not considered to be test substance-related because they occurred infrequently and/or in a manner that was not dose-related.

Details on embryotoxic / teratogenic effects:

SUMMARY OF EXTERNAL, VISCERAL AND SKELETAL EXAMINATIONS
- The numbers of fetuses (litters) available for morphological evaluation were 377(25), 348(25), 373(25), and 318(24) in the control, 250, 500, and 1000 mg/kg/day groups, respectively. Malformations were observed in 6(3), 6(6), 2(2), and 72(20) fetuses (litters) in these same respective dose groups.
- A significantly (p<0.01) higher total mean litter proportion of fetal malformations was observed in the 1000 mg/kg/day group (26.1% per litter) compared to the control group (1.7% per litter). This was due to the higher percent per litter of external (19.2% per litter), soft tissue (3.4% per litter), and skeletal (8.1% per litter) malformations noted at 1000 mg/kg/day compared to the concurrent control group values (0.0%, 0.0%, and 1.7% per litter, respectively); only the difference for external malformations was significant (p<0.05). Higher incidences of external (fetal anasarca, cleft palate, and localized fetal edema), visceral (right-sided aortic arch), and skeletal (rib anomaly, interrupted ossification of the rib[s], and bent limb bone[s]) fetal malformations were noted in the 1000 mg/kg/day group.
- Significantly (p<0.01) higher total mean litter proportions of fetal developmental variations were observed in the 500 and 1000 mg/kg/day groups (78.4% and 100.0%, respectively) compared to the concurrent control group (45.3% per litter). This was due to the significantly (p<0.01) higher percent per litter of skeletal variations in the 500 and 1000 mg/kg/day groups (77.7% and 100.0% per litter, respectively). Test substance-related increased incidences of skeletal developmental variations (ossification of 14th rudimentary rib[s], reduced ossification of the skull, reduced ossification of the vertebral arches, unossification of sternebra[e] nos. 5 and/or 6, unossification of sternebra[e] nos. 1, 2, 3, and/or 4, bent rib[s], and reduced ossification of the rib[s]) and decreased incidences of ossification of cervical centrum no. 1 were noted at 500 and 1000 mg/kg/day. Additionally, increased incidences of skeletal developmental variations consisting of unossified hyoid, 14th full rib(s), unossification of the entire sternum, 7th cervical rib(s), unossified pubis, unossified ischium, unossified vertebral centra, and bent scapula(e) were noted at 1000 mg/kg/day. The skeletal developmental variations noted in the 500 and 1000 mg/kg/day groups consisting of reduced ossification or unossified bones were indicative of developmental delay and considered secondary to the reduced fetal weights noted at these dosage levels. Additionally, test substance-related visceral developmental variations consisting of increase in the mean litter proportions of small and pale spleen and major blood vessel variation were noted at 1000 mg/kg/day.
- No test substance-related fetal malformations were noted in the 250 or 500 mg/kg/day groups and no test substance-related developmental variations were noted in the 250 mg/kg/day group.

Dose descriptor:

NOAEL

Remarks:

maternal dose

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

skeletal malformations

visceral malformations

Abnormalities:

effects observed, treatment-related

Localisation:

other: see discussion below

Developmental effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

ANALYSES OF DOSING FORMULATIONS

- The mean concentrations of the initial 50, 100, and 200 mg/mL formulations prepared on 08-Aug-2014 and the mean concentration of the final 50 mg/mL formulation prepared on 21-Aug-2014 were between 71.5% and 83.9% of the target concentration; all other samples were between 90.5% and 92.8% of the target concentration.

- The results of these analyses were consistent with the results obtained during the method validation 70-day storage assessment; i.e., formulations at lower prepared concentrations have lower mean percent recovery following prolonged storage (Haas, 2015, WIL-168211).

- For logistical reasons (i.e., sample shipment and method development times) sample analyses in this study were conducted approximately 26 to 45 days after formulation and dosing. However, because all formulations were used for dosing within 8 days of preparation, it is believed that the animals on this study received the intended dose concentration/dosage levels.

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

- Analyses of dosing formulations are summarised in Table 1 (attached).

Conclusions:

Maternal toxicity was evidenced at 1000 mg/kg/day by lower mean body weight gains, body weights, and food consumption. No evidence of maternal toxicity was noted at 250 or 500 mg/kg/day. Based on these results, a dosage level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Developmental toxicity was noted at 500 and 1000 mg/kg/day as evidenced by lower mean fetal body weights, which, in conjunction with a lower number of viable fetuses, correlated with a lower mean gravid uterine weight in the 1000 mg/kg/day group. Higher mean litter proportion of postimplantation loss with a corresponding lower mean number and litter proportion of viable fetuses were also noted at 1000 mg/kg/day. In addition, test substance-related fetal malformations were noted at 1000 mg/kg/day and test substance-related developmental variations were noted at 500 and 1000 mg/kg/day. Intrauterine growth in the 250 mg/kg/day group and survival in the 250 and 500 mg/kg/day groups were unaffected by test substance administration. Based on these results, a dosage level of 250 mg/kg/day was considered to be the NOAEL for embryo/fetal development when test item was administered orally by gavage to bred Crl:CD(SD) rats.

Executive summary:

GUIDELINE

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

 

METHODS

The test substance in the vehicle (peanut oil) was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 250, 500, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

 

RESULTS

All females in the control, 250, 500, and 1000 mg/kg/day groups survived to the scheduled necropsy on gestation day 20. Test substance-related, nonadverse increased incidences of clear and red material around the mouth were noted in the 250, 500, and 1000 mg/kg/day group approximately 1 hour following dose administration and increased incidences of yellow material around the ventral trunk and urogenital areas were noted in the 1000 mg/kg/day group generally throughout the treatment period. There were no other test substance-related clinical findings noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.

 

Test substance-related lower mean body weight gains with corresponding lower mean food consumption were noted in the 1000 mg/kg/day group throughout the treatment period. In addition, lower mean body weights, net body weight, and net body weight gain were observed at this dosage level.

 

In the 500 mg/kg/day, mean body weight gains were similar to the control group during gestation days 6-9 and then lower than the control group during the remainder of the treatment period (gestation days 9-12, 12-15, and 15-20). However, the lower mean body weight gains noted in this group were not of sufficient magnitude to affect the overall treatment period (gestation days 6-20) or mean body weights and therefore were not considered to be adverse. There were no test substance-related effects on mean body weight gains at 250 mg/kg/day or mean body weights, net body weights, net body weight gains, food consumption, or gravid uterine weights at 250 and 500 mg/kg/day.

 

There were no test substance-related macroscopic findings noted at any dosage level.

 

Mean fetal body weights in the 500 and 1000 mg/kg/day groups were up to 15.8% and 35.9% lower, respectively, than the control group. The lower mean fetal weights corresponded to the lower mean gravid uterine weight in the 1000 mg/kg/day group; mean gravid uterine weight in the 500 mg/kg/day group was similar to the control group. In the 1000 mg/kg/day group, a higher mean litter proportion of postimplantation loss and a corresponding lower mean number and mean litter proportion of viable fetuses were noted compared to the control group. Intrauterine growth at 250 mg/kg/day and survival at 250 and 500 mg/kg/day were unaffected by maternal test substance administration.

 

In the 1000 mg/kg/day groups, test substance-related higher incidences of external (fetal anasarca, cleft palate, and localized fetal edema around the neck and/or thorax), visceral (right-sided aortic arch), and skeletal (rib anomaly, interrupted ossification of the rib[s], and bent limb bone[s]) fetal malformations and visceral developmental variations (small and pale spleen and major blood vessel variation) were noted compared to the control group. In addition, test substance-related skeletal developmental variations were noted at 500 and 1000 mg/kg/day. Higher incidences of 14th rudimentary rib(s), reduced ossification of the skull and vertebral arches, unossification of sternebra(e) nos. 5 and/or 6 and sternebra(e) nos. 1, 2, 3, and/or 4, bent rib(s), and reduced ossification of the rib(s), in addition to lower incidences of ossification of cervical centrum no. 1, were noted in the 500 and 1000 mg/kg/day groups when compared to the control group. In addition, higher incidences of 14th full rib(s), unossification of the hyoid, entire sternum, pubis, ischium, and vertebral centra, 7th cervical rib(s), and bent scapula(e) were noted at 1000 mg/kg/day. The aforementioned skeletal developmental variations were considered test substance-related and secondary to the reduced fetal weights noted at these dosage levels. No test substance-related fetal malformations were noted at 250 and 500 mg/kg/day and no test substance-related developmental variations were noted at 250 mg/kg/day.

 

CONCLUSIONS

Maternal toxicity was evidenced at 1000 mg/kg/day by lower mean body weight gains, body weights, and food consumption. No evidence of maternal toxicity was noted at 250 or 500 mg/kg/day. Based on these results, a dosage level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Developmental toxicity was noted at 500 and 1000 mg/kg/day as evidenced by lower mean fetal body weights, which, in conjunction with a lower number of viable fetuses, correlated with a lower mean gravid uterine weight in the 1000 mg/kg/day group. Higher mean litter proportion of postimplantation loss with a corresponding lower mean number and litter proportion of viable fetuses were also noted at 1000 mg/kg/day. In addition, test substance-related fetal malformations were noted at 1000 mg/kg/day and test substance-related developmental variations were noted at 500 and 1000 mg/kg/day. Intrauterine growth in the 250 mg/kg/day group and survival in the 250 and 500 mg/kg/day groups were unaffected by test substance administration. Based on these results, a dosage level of 250 mg/kg/day was considered to be the NOAEL for embryo/fetal development when test item was administered orally by gavage to bred Crl:CD(SD) rats.

Reference 2

Endpoint:

developmental toxicity

Type of information:

other: Expert opinion

Adequacy of study:

supporting study

Study period:

03 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: valid and conclusive toxicological opinion

Qualifier:

no guideline required

Principles of method if other than guideline:

- Expert opinion was provided concerning the results of the rat prenatal development toxicity study (WIL-168214)conducted at WIL Research between July 2014 and February 2015.
- The author of the report was not employed by WIL Research at the time the investigation was performed and was not involved in study design, conduct or interpretation.

Developmental effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

no

Any development effects noted in these fetuses should be interpreted with caution as they were noted in the presence of, and are likely secondary to, frank maternal toxicity.

Conclusions:

Based on detailed review of the study data, fetal skeletal variations noted at 500 and 1000 mg/kg/day occurred in the presence of maternal toxicity and fetal development delay and should be considered secondary. Given the excessive reductions in maternal body weight at 1000 mg/kg/day and consequent drastic reductions in fetal weight (up to 35.9 % versus controls), the dose was excessively high. Thus, any development effects noted in these fetuses should be interpreted with caution as they were noted in the presence of, and are likely secondary to, frank maternal toxicity.

Executive summary:

STUDY DESIGN

WIL-168214 was a prenatal development toxicity study of the test item in rats and was conducted in accordance with the United States EPA Health Effects Test Guidelines OPPTS 870.3700 and the OECD 414 Guideline for Testing of Chemicals (January 2001). Maternal dosage levels of test item were 250, 500 and 1000 mg/kg/day with a concurrent control group receiving the vehicle (peanut oil) on a comparable regimen.

 

INTERPRETATION OF RESULTS

250 mg/kg/day

There were no significant test substance-related effects noted for maternal rats or the developing fetus at the lowest dose tested (250 mg/kg/day).

 

500 mg/kg/day

Slightly lower mean maternal body weights, without corresponding effects on food consumption, were observed at this dosage level. These effects were considered non-adverse because there was no effect on absolute mean maternal body weights, which were generally comparable to the control group throughout gestation. There were no significant maternal macroscopic findings and intrauterine survival was unaffected by exposure to the test item.

 

At the scheduled necropsy, test substance-related effects on interuterine growth were observed at this dosage level. Lower mean fetal weights (up to 15.8 % versus controls) were noted. Although there were no test substance-related fetal development malformations noted at this dosage level, the incidence of fetal skeletal variations was significantly higher (78.4 %) compared to the concurrent control group (45.3 %). Noted variations consisted of increases in the incidence of unossified cervical centrum number 1 and reduced ossifaction of the skull vertebral arches and sternebra(e) numbers 5 and/or 6.

 

The occurrence of skeletal ossification-related fetal development variations is often suggestive of an organism exhibiting development delay and conventional expert opinions do not view these to be a sign of overt development toxicity. Development variations are alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity. Although they may potentially occur at high incidence rates, variations represent only slight deviations from normal. Supporting data for this opinion is provided in the discussion of effects that were noted at the upper dosage level of the study.

 

1000 mg/kg/day

At the highest dose level tested, significant test substance-related effects were observed for maternal animals and included reduced mean maternal body weight gains and food consumption generally throughout the treatment period, which corresponded with mean body weights that were up to 11.8 % lower compared to the control group. Compared to controls, lower mean gravid uterine weights, net body weight, and net body weight gain were also noted. At scheduled necropsy, test substance-related effects on interuterine growth and survival was observed at this dosage level. A higher mean litter proportion of postimplantation loss (early and late resorptions), with a correspondingly lower mean number and litter proportion of viable fetuses was accompanied by lower mean fetal body weights (up to 35.9 % compared to the control group).

 

Test substance-related effects on fetal morphology were also observed at this dosage level. Higher total mean litter proportion of fetal malformations was observed (26.1 % per litter) compared to the control group (1.7 % per litter). Externally, these malformations included fetal anasarca (generalized edema), cleft palate and localized edema (neck and/or thorax). Viscerally, the only test substance-related finding was right-sided aortic arch and, skeletally, noted malformations included rib anomaly, interrupted ossification of the ribs and bent limb bone(s). A higher total mean litter proportion of fetal developmental variations (100 %) compared to the concurrent control group (45.3 %) was also observed at this dosage level. Viscerally, these variations consisted of higher mean litter proportions of small and pale spleen and major blood vessel variation. Skeletally, the most commonly noted findings included reduced ossification of the skull, vertebral arches and rib(s), plus unossified hyoid, pubis, ischium, vertebral centra, cervical centrum number 1, sternebra(e) numbers 1, 2, 3 and/or 4 or 5 and/or 6 (or entire sternum unossified). In addition, there was a higher incidence of 14^thfull and rudimentary rib(s), 7^thcervical rib(s), bent rib(s) and bent scapula(e).

 

Delayed (incomplete) ossification of developing bones and wavy (bent) ribs are the two most commonly noted skeletal variations noted in regulatory guideline developmental toxicity studies [1]. Reduced maternal food consumption, accompanied by reduced gestational body weight gain, have previously been shown, in food restriction studies, to cause fetal weight reductions and reduced ossification, specifically in the number/delays in sternebral ossification [2, 3], even in fetuses examined on gestation day 21 (versus the current study where fetuses were examined on gestation day 20). Moreover, the incidence and severity of delayed ossification has been shown to be related to food restriction that continued until term. In fact, restricting feed during the last week of gestation (GD 15-21) in pregnant rats was just as effective in delaying fetal growth as restricting feed throughout gestation [4]. Moreover, maternal malnutrition can result in reduced placental blood flow, which can cause fetal development delay [5]. Therefore, since skeletal ossification is highly dependent upon maternal physiological factors, such as utero-placental blood flow, nutritional status etc and increases in fetal skeletal variations such as ossification rates can be altered by maternal toxicity [1].

 

It is widely accepted that ossification delays may not be developmentally adverse. For instance, delays in ossification and wavy (bent) ribs are not mechanistically linked to malformations and have been shown to resolve during postnatal skeletal remodelling [1]. Additionally, examination of the incidence of wavy ribs between fetuses and post-weaning pups has concluded that the severity of the findings generally reduced with aging through the lactation period such that the ribs were almost normalized at weaning [6]. Interestingly, the same study revealed that the wave (bend) in ribs was initially noted as a delay in ossification in the middle of the ribs, which followed by calcification of the curves caused the “appearance” of wavy (bent) ribs. Lastly, in a review article by Kimmel et al (2014) [7], the incidence of bent long bones and bent scapulae was discussed and, similar to bent ribs, these findings were noted to generally occur in the presence of maternal toxicity and fetal development delay and hence are considered secondary. Both findings, similar to wavy ribs, have been shown to be resolved postnatally and these development variations are considered to have no significant biological effect on animal health or body conformity.

 

EXPERT OPINION

Based on detailed review of the study data, fetal skeletal variations noted at 500 and 1000 mg/kg/day occurred in the presence of maternal toxicity and fetal development delay and should be considered secondary. Given the excessive reductions in maternal body weight at 1000 mg/kg/day and consequent drastic reductions in fetal weight (up to 35.9 % versus controls), the dose was excessively high. Thus, any development effects noted in these fetuses should be interpreted with caution as they were noted in the presence of, and are likely secondary to, frank maternal toxicity.

 

REFERENCES

1. Carney EW and Kimmel CA. Interpretation of Skeletal Variations for Human Risk Assessment: Delayed Ossification and Wavy Ribs. Birth Defects Research (Part B) 80: 473-496 (2007).

2. Fleeman TL, Cappon GD, Chapin RE and Hurtt ME. Effects of Feed Restriction During Organogenesis on Embryo-Fetal Development in the Rat. Birth Defects Research (Part B) 74: 442-449 (2005).

3. Khera KS. Common fetal aberrations and their teratologic significance: a review. Fundamental and Applied Toxicology 1: 13-18 (1981).

4. Anderson GD, Ahokas RA, Lipshitz J, Dilts PV Jr. Effect of maternal dietary restriction during pregnancy on maternal weight gain and fetal birth weight in the rat. Journal of Nutrition 110(5): 883-890 (1980).

5. Ahokas RA, Anderson GD, Lipshitz J. Effect of dietary restriction during the last week only or throughout gestation, on cardiac output and uteroplacental blood flow in pregnant rats. Journal of Nutrition 113(9): 1766-1776 (1982).

6. Nishimura M, Iizuka M, Iwaki S, Kast A. Repairability of drug-induced “wavy ribs” in rat offspring. Arzneimittelforschung 32 (12): 1518-1522 (1982).

7. Kimmel CA, Garry MR, DeSesso JM. Relationship Between Bent Long Bones, Bent Scapulae and Wavy Ribs: Malformations or Variations? Birth Defects Research (Part B) 101: 379-392 (2014).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Key study

GUIDELINE

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

 

METHODS

The test substance in the vehicle (peanut oil) was administered orally by gavage to 3 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 250, 500, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 25 bred females receivedthe vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and thenumbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

 

RESULTS

All females in the control, 250, 500, and 1000 mg/kg/day groups survived to the scheduled necropsy on gestation day 20. Test substance-related, nonadverse increased incidences of clear and red material around the mouth were noted in the 250, 500, and 1000 mg/kg/day group approximately 1 hour following dose administration and increased incidences of yellow material around the ventral trunk and urogenital areas were noted in the 1000 mg/kg/day group generally throughout the treatment period. There were no other test substance-related clinical findings noted at the daily examinations or approximately 1 hour following dose administration at any dosage level.

 

Test substance-related lower mean body weight gains with corresponding lower mean food consumption were noted in the 1000 mg/kg/day group throughout the treatment period. In addition, lower mean body weights, net body weight, and net body weight gain were observed at this dosage level.

 

In the 500 mg/kg/day, mean body weight gains were similar to the control group during gestation days 6-9 and then lower than the control group during the remainder of the treatment period (gestation days 9-12, 12-15, and 15-20). However, the lower mean body weight gains noted in this group were not of sufficient magnitude to affect the overall treatment period (gestation days 6-20) or mean body weights and therefore were not considered to be adverse. There were no test substance-related effects on mean body weight gains at 250 mg/kg/day or mean body weights, net body weights, net body weight gains, food consumption, or gravid uterine weights at 250 and 500 mg/kg/day.

 

There were no test substance-related macroscopic findings noted at any dosage level.

 

Mean fetal body weights in the 500 and 1000 mg/kg/day groups were up to 15.8% and 35.9% lower, respectively, than the control group. The lower mean fetal weights corresponded to the lower mean gravid uterine weight in the 1000 mg/kg/day group; mean gravid uterine weight in the 500 mg/kg/day group was similar to the control group. In the 1000 mg/kg/day group, a higher mean litter proportion of postimplantation loss and a corresponding lower mean number and mean litter proportion of viable fetuses were noted compared to the control group. Intrauterine growth at 250 mg/kg/day and survival at 250 and 500 mg/kg/day were unaffected by maternal test substance administration.

 

In the 1000 mg/kg/day groups, test substance-related higher incidences of external (fetal anasarca, cleft palate, and localized fetal edema around the neck and/or thorax), visceral (right-sided aortic arch), and skeletal (rib anomaly, interrupted ossification of the rib[s], and bent limb bone[s]) fetal malformations and visceral developmental variations (small and pale spleen and major blood vessel variation) were noted compared to the control group. In addition, test substance-related skeletal developmental variations were noted at 500 and 1000 mg/kg/day. Higher incidences of 14th rudimentary rib(s), reduced ossification of the skull and vertebral arches, unossification of sternebra(e) nos. 5 and/or 6 and sternebra(e) nos. 1, 2, 3, and/or 4, bent rib(s), and reduced ossification of the rib(s), in addition to lower incidences of ossification of cervical centrum no. 1, were noted in the 500 and 1000 mg/kg/day groups when compared to the control group. In addition, higher incidences of 14th full rib(s), unossification of the hyoid, entire sternum, pubis, ischium, and vertebral centra, 7th cervical rib(s), and bent scapula(e) were noted at 1000 mg/kg/day. The aforementioned skeletal developmental variations were consideredtest substance-related and secondary to the reduced fetal weights noted at these dosage levels. No test substance-related fetal malformations were noted at 250 and 500 mg/kg/day and no test substance-related developmental variations were noted at 250 mg/kg/day.

 

CONCLUSIONS

Maternal toxicity was evidenced at 1000 mg/kg/day by lower mean body weight gains, body weights, and food consumption. No evidence of maternal toxicity was noted at 250 or 500 mg/kg/day. Based on these results, a dosage level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Developmental toxicity was noted at 500 and 1000 mg/kg/day as evidenced by lower mean fetal body weights, which, in conjunction with a lower number of viable fetuses, correlated with a lower mean gravid uterine weight in the 1000 mg/kg/day group. Higher mean litter proportion of postimplantation loss with a corresponding lower mean number and litter proportion of viable fetuses were also noted at 1000 mg/kg/day. In addition,test substance-related fetal malformations were noted at 1000 mg/kg/day and test substance-related developmental variations were noted at 500 and 1000 mg/kg/day. Intrauterine growth in the 250 mg/kg/day group and survival in the 250 and 500 mg/kg/day groups were unaffected by test substance administration. Based on these results, a dosage level of 250 mg/kg/day was considered to be the NOAEL for embryo/fetal development when test item was administered orally by gavage to bred Crl:CD(SD) rats.

Expert opinion

STUDY DESIGN

WIL-168214 was a prenatal development toxicity study of the test item in rats and was conducted in accordance with the United States EPA Health Effects Test Guidelines OPPTS 870.3700 and the OECD 414 Guideline for Testing of Chemicals (January 2001). Maternal dosage levels of test item were 250, 500 and 1000 mg/kg/day with a concurrent control group receiving the vehicle (peanut oil) on a comparable regimen.

 

INTERPRETATION OF RESULTS

250 mg/kg/day

There were no significant test substance-related effects noted for maternal rats or the developing fetus at the lowest dose tested (250 mg/kg/day).

 

500 mg/kg/day

Slightly lower mean maternal body weights, without corresponding effects on food consumption, were observed at this dosage level. These effects were considered non-adverse because there was no effect on absolute mean maternal body weights, which were generally comparable to the control group throughout gestation. There were no significant maternal macroscopic findings and intrauterine survival was unaffected by exposure to the test item.

 

At the scheduled necropsy, test substance-related effects on interuterine growth were observed at this dosage level. Lower mean fetal weights (up to 15.8 % versus controls) were noted. Although there were no test substance-related fetal development malformations noted at this dosage level, the incidence of fetal skeletal variations was significantly higher (78.4 %) compared to the concurrent control group (45.3 %). Noted variations consisted of increases in the incidence of unossified cervical centrum number 1 and reduced ossifaction of the skull vertebral arches and sternebra(e) numbers 5 and/or 6.

 

The occurrence of skeletal ossification-related fetal development variations is often suggestive of an organism exhibiting development delay and conventional expert opinions do not view these to be a sign of overt development toxicity. Development variations are alterations in anatomic structure that are considered to have no significant biological effect on animal health or body conformity. Although they may potentially occur at high incidence rates, variations represent only slight deviations from normal. Supporting data for this opinion is provided in the discussion of effects that were noted at the upper dosage level of the study.

 

1000 mg/kg/day

At the highest dose level tested, significant test substance-related effects were observed for maternal animals and included reduced mean maternal body weight gains and food consumption generally throughout the treatment period, which corresponded with mean body weights that were up to 11.8 % lower compared to the control group. Compared to controls, lower mean gravid uterine weights, net body weight, and net body weight gain were also noted. At scheduled necropsy, test substance-related effects on interuterine growth and survival was observed at this dosage level. A higher mean litter proportion of postimplantation loss (early and late resorptions), with a correspondingly lower mean number and litter proportion of viable fetuses was accompanied by lower mean fetal body weights (up to 35.9 % compared to the control group).

 

Test substance-related effects on fetal morphology were also observed at this dosage level. Higher total mean litter proportion of fetal malformations was observed (26.1 % per litter) compared to the control group (1.7 % per litter). Externally, these malformations included fetal anasarca (generalized edema), cleft palate and localized edema (neck and/or thorax). Viscerally, the only test substance-related finding was right-sided aortic arch and, skeletally, noted malformations included rib anomaly, interrupted ossification of the ribs and bent limb bone(s). A higher total mean litter proportion of fetal developmental variations (100 %) compared to the concurrent control group (45.3 %) was also observed at this dosage level. Viscerally, these variations consisted of higher mean litter proportions of small and pale spleen and major blood vessel variation. Skeletally, the most commonly noted findings included reduced ossification of the skull, vertebral arches and rib(s), plus unossified hyoid, pubis, ischium, vertebral centra, cervical centrum number 1, sternebra(e) numbers 1, 2, 3 and/or 4 or 5 and/or 6 (or entire sternum unossified). In addition, there was a higher incidence of 14^thfull and rudimentary rib(s), 7^thcervical rib(s), bent rib(s) and bent scapula(e).

 

Delayed (incomplete) ossification of developing bones and wavy (bent) ribs are the two most commonly noted skeletal variations noted in regulatory guideline developmental toxicity studies [1]. Reduced maternal food consumption, accompanied by reduced gestational body weight gain, have previously been shown, in food restriction studies, to cause fetal weight reductions and reduced ossification, specifically in the number/delays in sternebral ossification [2, 3], even in fetuses examined on gestation day 21 (versus the current study where fetuses were examined on gestation day 20). Moreover, the incidence and severity of delayed ossification has been shown to be related to food restriction that continued until term. In fact, restricting feed during the last week of gestation (GD 15-21) in pregnant rats was just as effective in delaying fetal growth as restricting feed throughout gestation [4]. Moreover, maternal malnutrition can result in reduced placental blood flow, which can cause fetal development delay [5]. Therefore, since skeletal ossification is highly dependent upon maternal physiological factors, such as utero-placental blood flow, nutritional status etc and increases in fetal skeletal variations such as ossification rates can be altered by maternal toxicity [1].

 

It is widely accepted that ossification delays may not be developmentally adverse. For instance, delays in ossification and wavy (bent) ribs are not mechanistically linked to malformations and have been shown to resolve during postnatal skeletal remodelling [1]. Additionally, examination of the incidence of wavy ribs between fetuses and post-weaning pups has concluded that the severity of the findings generally reduced with aging through the lactation period such that the ribs were almost normalized at weaning [6]. Interestingly, the same study revealed that the wave (bend) in ribs was initially noted as a delay in ossification in the middle of the ribs, which followed by calcification of the curves caused the “appearance” of wavy (bent) ribs. Lastly, in a review article by Kimmel et al (2014) [7], the incidence of bent long bones and bent scapulae was discussed and, similar to bent ribs, these findings were noted to generally occur in the presence of maternal toxicity and fetal development delay and hence are considered secondary. Both findings, similar to wavy ribs, have been shown to be resolved postnatally and these development variations are considered to have no significant biological effect on animal health or body conformity.

 

EXPERT OPINION

Based on detailed review of the study data, fetal skeletal variations noted at 500 and 1000 mg/kg/day occurred in the presence of maternal toxicity and fetal development delay and should be considered secondary. Given the excessive reductions in maternal body weight at 1000 mg/kg/day and consequent drastic reductions in fetal weight (up to 35.9 % versus controls), the dose was excessively high. Thus, any development effects noted in these fetuses should be interpreted with caution as they were noted in the presence of, and are likely secondary to, frank maternal toxicity.

 

REFERENCES

1. Carney EW and Kimmel CA. Interpretation of Skeletal Variations for Human Risk Assessment: Delayed Ossification and Wavy Ribs. Birth Defects Research (Part B) 80: 473-496 (2007).

2. Fleeman TL, Cappon GD, Chapin RE and Hurtt ME. Effects of Feed Restriction During Organogenesis on Embryo-Fetal Development in the Rat. Birth Defects Research (Part B) 74: 442-449 (2005).

3. Khera KS. Common fetal aberrations and their teratologic significance: a review. Fundamental and Applied Toxicology 1: 13-18 (1981).

4. Anderson GD, Ahokas RA, Lipshitz J, Dilts PV Jr. Effect of maternal dietary restriction during pregnancy on maternal weight gain and fetal birth weight in the rat. Journal of Nutrition 110(5): 883-890 (1980).

5. Ahokas RA, Anderson GD, Lipshitz J. Effect of dietary restriction during the last week only or throughout gestation, on cardiac output and uteroplacental blood flow in pregnant rats. Journal of Nutrition 113(9): 1766-1776 (1982).

6. Nishimura M, Iizuka M, Iwaki S, Kast A. Repairability of drug-induced “wavy ribs” in rat offspring. Arzneimittelforschung 32 (12): 1518-1522 (1982).

7. Kimmel CA, Garry MR, DeSesso JM. Relationship Between Bent Long Bones, Bent Scapulae and Wavy Ribs: Malformations or Variations? Birth Defects Research (Part B) 101: 379-392 (2014).

Justification for classification or non-classification

No effects on F0 and F1 reproductive performance were reported at any dosage level in a two-generation study at the highest dosage level tested (400 mg/kg bw/day) and expert opinion considers that developmental effects were secondary to maternal toxicity at 500 mg/kg bw/day or 1000 mg/kg bw/day. Classification for reproductive/developmental toxicity is therefore unnecessary under the criteria of Regulation (EC) No 1272/2008 and subsequent amendments.