Reaction product of D-Glucopyranoside, methyl; esterified with octadecanoic acid, methyl ester

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Under the conditions of a screening study involving an analogue test item, no test material-related effects on F0 males and female fertility and mating indices, F0 male copulation index, female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicitywas considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 30, 100, 250, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level (OECD 422).

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 April 2015 to 11 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

various deviations with no impact on results or integrity of the study (aee Appendix A, attached)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

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 Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. The animal model, the Crl:CD(SD) rat, is recognised as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, WIL Research has reproductive historical control data in the Crl:CD(SD) rat.
- The number of animals selected for this study (15 [Groups 1 and 5] or 10 rats/sex/group [Groups 2-4]) was based on the OECD Guidelines for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 22-Mar-1996, which recommends evaluation of at least 8 pregnant females per group.
- Given the possibility of non-gravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, 10 animals/sex/group was an appropriate number of animals to obtain a sample size of 8 at termination. In addition, 5 animals/sex in the control and high-dose
groups were necessary to assess recovery, persistence, or progression of any toxic effects following the cessation of dosing.
- Crl:CD(SD) rats (66 males and 66 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 28-Apr-2015. The animals were approximately 61 days old upon receipt. Each animal was examined by a qualified biologist on the day of receipt. Two days following receipt, all animals were weighed and clinical observations were recorded. Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period. The animals were housed for an acclimation period of 9 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behaviour, and the testes were palpated at least once for all males.

ANIMAL HOUSING
- Following receipt and until pairing, all F0 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 analysed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research.
- During cohabitation, breeding phase rats (10/sex/group) were paired in a solid-bottom cage with bedding material. Following the breeding period, the breeding phase males and females were individually housed in solid-bottom cages with bedding material. The dams and their litters were housed in these cages until euthanasia on lactation day 4. Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until delivery or post-mating day 25. The 5 rats/sex in the control and high-dose groups that were assigned to the recovery phase were not paired for mating and remained housed in groups of 2-3 in clean solid-bottom cages until
euthanasia.
- 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 oral health of the animals, and were sanitised 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. Feeders were changed and sanitised 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, with the following exception. All males and recovery phase females were fasted prior to clinical pathology blood collection when food, but not water, was withheld.

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 70.3 °F to
72.5 °F (21.3 °C to 22.5 °C) and mean daily relative humidity ranged from 46.7 % to 66.4 % 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.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- At the conclusion of the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was selected for use in the computerized randomisation procedure based on body weight stratification in a block design. At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMSTM. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated. The animals then were arranged into groups according to the printout. Animals not assigned to study were transferred to the WIL Research colony.
- The experimental design consisted of 4 test item-treated groups and 1 control group composed of 10 rats/sex/group. An additional 5 rats/sex in the control and high-dose groups were selected to be evaluated following a 14-day recovery period; animals assigned to the recovery period were not evaluated for reproductive toxicity. At the initiation of dose administration (study day 0), the males and females were approximately 10 weeks old. Male body weights ranged from 335 g to 414 g and female body weights ranged from 216 g to 265 g on study day 0. The animals were approximately 12 weeks old when paired on study day 13; female body weights ranged from 228 g to 288 g on gestation day 0.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

Lot numbers 2EA0347 and 2EA0218; expiry date 31 January 2016

Details on exposure:

TEST ITEM PREPARATION
- The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group and stored refrigerated (2 °C to 8 °C), protected from light. The vehicle was mixed throughout the sampling and dose administration procedures. On each dosing day, aliquots were stirred at room temperature for at least 30 minutes prior to dispensation for dosing.
- Dosing formulations were prepared at the test item concentrations indicated in the table below.
- The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated, protected from light. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. On each dosing day, aliquots were stirred at room temperature for at least 30 minutes prior to dispensation for dosing. The first test item dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- Study group assignments are shown in the table below.
- The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily.
- The males selected for pairing were dosed during study days 0-28 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 29 doses.
- The females selected for pairing were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-43 doses.
- Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses.
- The extra 5 rats/sex in the control and high-dose groups were not used for mating, but were treated on a comparable regimen beginning on study day 0; following 29 doses for males and 39 doses for females, these animals remained on study for a 14-day recovery period.
- 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.
- Dosage levels were selected based on the results of a previous 28-day study in rats (Haas, 2015, WIL-168233). In that study, the test item was administered to male and female rats for 28 consecutive days at dosage levels of 30, 100, 250, and 1000 mg/kg/day. There were no significant clinical signs noted at any dosage level. In addition, body weights and food consumption were unaffected by treatment. The selected route of administration for this study was oral (gavage) because this is a potential route of human exposure. Historically, this route has been used extensively for studies of this nature.

DATA ACQUISITION AND ANALYSIS
- Details of acquisition, analysis and reporting of data are attached.

Details on mating procedure:

BREEDING PROCEDURES
- The 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females.
- A breeding record containing the male and female identification numbers and the start date of cohabitation was 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 following a vaginal lavage and verified by a second biologist.
- Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages.
- For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The test substance in the vehicle was determined to be stable at concentrations of 5 and 230 mg/mL following 8 days of refrigerated storage in a previous study (Bystry, 2015, WIL-168232). In addition, resuspension homogeneity was assessed at concentrations of 60 to 200 mg/mL in a previous 28-day toxicity study (Haas, 2015, WIL-168233).
- Samples for homogeneity and/or concentration determinations were collected from the top, middle, and bottom strata of the first, fourth, fifth, sixth, seventh, and last test item dosing formulations and from the middle stratum of the first, fourth, fifth, sixth, seventh, and last control group dosing formulations. Due to difficulty with the analytical method, beginning with the fourth preparation, samples from all preparations were collected and stored refrigerated. To establish 17-day refrigerated stability, small batch sizes were prepared at the low and high concentrations used in this study. Samples were collected from the top, middle, and bottom strata of these formulations for determination of homogeneity and, following 17 days of refrigerated storage, stability. One set of samples from study weeks 0, 4, 5, and 7 was subjected to the appropriate analyses. All remaining samples were stored refrigerated (2 °C to 8 °C) as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a method validated on a previous study (Bystry, 2015, WIL-168232), as well as an alternate method that was cross-validated in this study.

Duration of treatment / exposure:

- Males: Total of 29 doses (throughout the mating period through 1 day prior to euthanasia)
- Females: Total of 39 to 43 doses (14 days prior to pairing with dosing continued until lactation Day 3)

Frequency of treatment:

Daily

Details on study schedule:

- A flow chart summarising the study design is attached.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- Vehicle control: 15 males and 15 females
- Test item administered at 30, 100 and 250 mg/kg bw/day: 10 males and 10 females
- Test item administered at 1000 mg/kg bw/day: 15 males and 15 females

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
- All rats were observed twice daily, once in the morning and once in the afternoon, for appearance, behaviour, moribundity, mortality, and signs of overt toxicity.
- Individual detailed physical examinations were recorded weekly (prior to dose administration during the treatment period).
- Each male and female was also 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
- Individual male body weights were recorded weekly throughout the study and prior to the scheduled euthanasia. Individual female body weights were recorded weekly until evidence of copulation was observed or until euthanasia (for females assigned to recovery phase). Mean weekly body weights and body weight changes were presented for each interval.
- In addition, cumulative mean body weight changes were presented for the pre-mating period (males and females) and for the entire dosing period (males and recovery phase females). Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible), 1, and 4. Mean gestation body weights and corresponding mean body weight changes were presented for these intervals and for the overall gestation interval (days 0-20); mean lactation body weight changes are presented for lactation days 1-4.
- Weekly body weights for females with no evidence of mating were presented in the individual report tables until delivery.
- When body weights could not be determined for an animal during a given interval (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
- Individual food consumption was recorded on the corresponding weekly body weight days until pairing (for animals paired for breeding) or euthanasia (for animals assigned to the recovery period). - Food consumption was measured on a per cage basis for the corresponding body weight intervals. Food consumption was normalised to the number of animals/cage and was reported in g/animal/day. Food intake was not recorded during the breeding period for animals selected for pairing.
- Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4; food consumption was reported as g/anima/day and g/kg/day during gestation and lactation.
- Following the breeding period, food consumption for females with no evidence of mating was measured on a weekly basis until delivery.
- Weekly food consumption for females with no evidence of mating was presented in the individual report tables until necropsy.
- Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage.
- When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, 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.

FOB ASSESSMENTS
- FOB assessments were recorded for 5 animals/sex/group following approximately 28 days of dose administration (study week 4; males selected for pairing) and on lactation day 4 (females).
- The FOB used at WIL Research is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988, Moser et al., 1991; and O’Donoghue, 1989).
- FOB testing was performed by the same biologists, to the extent possible, without knowledge of the animal’s group assignment.
- The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB.
- All animals were observed for the parameters as listed in the table below.
- Forelimb and hindlimb grip strength were measured using a device similar to the one described by Meyer et al (1979). The animal was allowed to grip a T-shaped grip bar with its forepaws and was pulled back gently along a platform until its grip was broken. As the backward locomotion continues, the animal’s hindpaws reach a T-shaped rearlimb grip bar, which it is allowed to grasp and then forced to release by continued pulling. Mark-10 series EG digital force gauges (Mark-10 Corporation, Copiague, NY) were used to record the maximum strain required to break forelimb and hindlimb grip. The average of 3 valid measurements was taken as the animal’s score for each grip strength measure.

MOTOR ACTIVITY
- Motor activity was assessed for 5 animals/sex/group following approximately 28 days of dose administration (study week 4, males selected for pairing) or on lactation day 4 (females). Motor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilises a series of infrared photobeams surrounding an amber plastic, rectangular cage to quantify each animal’s motor activity.
- Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. Each animal was tested separately. Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation.
- Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).

CLINICAL PATHOLOGY
- Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected from 5 animals/sex/group at the primary necropsies (study week 4 for males and lactation day 4 for females selected for pairing) and from 5 animals/sex in the control and high-dose groups at the recovery necropsy (following a 14-day recovery period; study day 42 for males and study day 52 for females). All males and recovery phase females were fasted overnight prior to blood collection.
- Blood for serum chemistry and haematology was collected from the retro-orbital sinus following isoflurane anaesthesia.
- Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing K2EDTA (haematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry).
- Parameters evaluated were as shown in the tables below.

Litter observations:

F1 LITTER VIABILITY AND DEATHS
- Each litter was examined daily for survival, and all deaths were recorded.
- All pups were individually identified by application of tattoo markings on the digits following completion of parturition.
- A daily record of litter size was maintained.
- Intact offspring that were found dead were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984).
- The carcass of each pup was then discarded.

F1 CLINICAL OBSERVATIONS
- Litters were examined daily for survival and any adverse changes in appearance or behaviour.
- Each pup received a clinical examination on PND 1 and 4.
- Any abnormalities in nesting and nursing behaviour were recorded.

F1 BODY WEIGHTS
- Pups were individually weighed on PND 1 and 4.
- Mean pup weights were presented by sex for each litter and by dose group.
- When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods a given pup was not weighed were left blank or designated as “NA” on the individual report tables.

F1 SEX DETERMINATION
- Pups were individually sexed on PND 0 and 4.

Postmortem examinations (parental animals):

MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all F0 parental animals at the scheduled termination.
- All F0 adults were euthanised by carbon dioxide inhalation.
- Males were euthanised following completion of the mating period or following the 14-day recovery period. Females that delivered were euthanized on lactation day 4; the number of former implantation sites and corpora lutea were recorded.
- Females that failed to deliver were euthanised on post-mating day 25 (females with evidence of mating). Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).
- Females not selected for pairing were euthanised following the 14-day recovery period.
- Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. Clinical findings that were verified at necropsy were designated CEO.
- At the time of necropsy, the tissues and organs were placed in 10% neutral-buffered formalin (except as noted in the table below).

ORGANS WEIGHED AT NECROPSY
- 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.

HISTOLOGY AND MICROSCOPIC EXAMINATIONS
- 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 SOPs, mounted on glass microscope slides, and stained with haematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.
- Microscopic examination was performed on all tissues from all animals in the control and 1000 mg/kg/day groups at the primary necropsy and on gross lesions from all animals in all groups at the primary necropsy.
- Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section, or other reasons as appropriate. 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):

F1 SCHEDULED EUTHANASIA
- On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded.

Statistics:

See below

Reproductive indices:

MATING, FERTILITY AND COPULATION INDICES
- Mating fertility and copulation.conception indices were calculated using the following equations:
(i) Male (female) mating index (%) = [Number of males (females) with evidence of mating (or confirmed pregnant) / total number of males (females) used for mating] * 100
(ii) Male fertility index (%) = [Number of males siring a litter / total number of males used for mating] * 100
(iii) Male copulation index (%) = [Number of males siring a litter / number of males with evidence of mating (or females with confirmed pregnancy] * 100
(iv) Female fertility index (%) = [Number of females with confirmed pregnancy / total number of females used for mating] * 100
(v) Female conception index (%) = [Number of females with confirmed pregnancy / number of females with evidence or mating (or confirmed pregnancy)] * 100

Offspring viability indices:

F1 CALCULATION OF LITTER PARAMETERS
- Litter parameters were defined as shown in the equations below:
(i) Mean live litter size = Total number of viable pups on PND 0 / number of litters with viable pups on PND 0
(ii) Postnatal survival between birth and PND 0 or PND 4 (% per litter) = Sum of (viable pups per litter on PND 0 or PND 4 / Number of pups born per litter) / Number of litters per group * 100.
(iii) 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 * 100 where N = PND 0–1 and 1–4.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- No test item-related clinical findings were noted at the detailed physical examinations or approximately 1 hour following dose administration.
- Clinical findings observed in the test item-treated groups, including hair loss and red or clear material on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

no mortality observed

Description (incidence):

- All males and females in the control, 30, 100, 250, and 1000 mg/kg/day groups survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Males: Mean body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day group males were unaffected by test item administration throughout the study. A significantly (p < 0.05) lower mean body weight gain was noted for males in the 250 mg/kg/day group during study days 13 20 and significantly (p < 0.05) higher mean body weight gains were noted for males in the 100 and 1000 mg/kg/day groups for the pre-mating period (study days 0-13). However, these results did not occur in a dose-related manner and did not affect mean absolute body weights. No other differences from the control group were statistically significant.
- Females (pre-mating): Mean body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day group females were unaffected by test item administration during the pre-mating period (females selected for breeding) or the entire study (females selected for recovery phase). Significantly (p < 0.05) higher mean body weight gains were noted for females in the 1000 mg/kg/day group during study days 34-38 and 46-51. However, these results were transient and did not affect mean absolute body weights. No other differences from the control group were statistically significant.
- Females (gestation): Mean body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.
- Females (lactation): Mean body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- Males: Mean food consumption in the 30, 100, 250, and 1000 mg/kg/day group males was similar to that in the control group throughout the study. No statistically significant differences were observed.
- Females (pre-mating): Mean food consumption in the 30, 100, 250, and 1000 mg/kg/day group females was unaffected by test item administration during the pre-mating period (females selected for breeding) or the entire study (females selected for recovery phase). None of the differences from the control group were statistically significant.
- Females (gestation): Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 30, 100, 250, and 1000 mg/kg/day groups was unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.
- Females (lactation): Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 30, 100, 250, and 1000 mg/kg/day groups was unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related alterations in haematology and coagulation parameters at the scheduled necropsies. However, some statistically significant (p < 0.05) differences were observed when the control and test substance-treated groups were compared.
- Lower absolute basophil values were noted for females in the 100 and 1000 mg/kg/day groups at the primary necropsy. There was no dose-response relationship, values were of minimal magnitude difference, and were similar to the control group at the recovery necropsy; the differences were attributed to biological variability.
- Higher prothrombin time (PT) values were noted in the 1000 mg/kg/day group males at the recovery necropsy. Values were of minimal magnitude change and were similar to control and 1000 mg/kg/day group values at the primary necropsy; the finding was attributed to biological variability and not related to administration of test item.
- Statistically significant (p < 0.05) findings that involved percentage leukocyte differential counts were not itemized above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related alterations in serum chemistry parameters at the scheduled necropsies.
- However, some statistically significant (p < 0.01 or p < 0.05) differences were observed when the control and test item-treated groups were compared.
- Lower serum chloride values were noted in the 1000 mg/kg/day group males at the primary necropsy. Other serum electrolyte values were similar to the control group, the difference was of minimal magnitude, and there were no histologic correlates; the finding was attributed to biological variability.
- Higher serum urea nitrogen values were noted in the 1000 mg/kg/day group males and higher serum triglyceride values were noted in the 1000 mg/kg/day group females at the recovery necropsy. Both values were similar to the control group at the primary necropsy and were of minimal magnitude difference; the Findings were attributed to biological variability and not related to administration of test item.
- Other statistically significant differences lacked a dose-response relationship, and were attributed to biological variability.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
- Home cage observations: Home cage parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Handling observations: Handling parameters were unaffected by test item administration. A significantly (p < 0.05) higher number of males in the 250 mg/kg/day group had slight resistance to being handled; however, this did not occur in a dose-related manner. There were no other statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Open field observations: Open field parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Sensory observations: Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Neuromuscular observations: Neuromuscular parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Physiological observations: Physiological parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

MOTOR ACTIVITY
- Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test item administration at all concentrations when evaluated during study week 4 (males) or on lactation day 4 (females). Values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the WIL historical control data.
- Differences from the control group were slight, not statistically significant when analysed by a repeated measures analysis, within the WIL historical control data ranges and/or did not occur in a dose-related manner. No remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated during study week 4 (males) or on lactation day 4 (females).

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related histologic changes at the primary necropsy. Histologic changes noted 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.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE
- F0 male and female reproductive parameters are presented in the table attached.
- No test item-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test item-treated groups. Single males in each of the 100 and 1000 mg/kg/day groups did not sire a litter. Single females in each of the 100 and 1000 mg/kg/day groups had evidence of mating but did not deliver.
- The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.

GESTATION LENGTH AND PARTURITION
- Mean gestation lengths in the 30, 100, 250, and 1000 mg/kg/day groups were similar to those in the control group.
- No statistically significant differences were noted.
- No signs of dystocia were noted in these groups.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

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 was unaffected by parental administration of the test item.
- Pups that were found dead numbered 1, 0, 3, 3, and 0 in the control, 30, 100, 250, and 1000 mg/kg/day groups, respectively.
- One, 1, 0, 1, and 3 pups in the same respective groups was missing and presumed to have been cannibalised.

Mortality / viability:

no mortality observed

Description (incidence and severity):

PND 0 LITTER DATA AND POSTNATAL SURVIVAL
- The mean number of pups born, live litter size and the percentage of males at birth in the 30, 100, 250, and 1000 mg/kg/day groups were similar to the control group values.
- Postnatal survival in the 30, 100, 250, and 1000 mg/kg/day groups was unaffected by test item administration.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

OFFSPRING BODY WEIGHTS
- Mean male and female pup body weights and body weight changes during PND 1-4 in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by parental administration of the test item.
- No statistically significant differences from the control group were noted.

Gross pathological findings:

no effects observed

Description (incidence and severity):

NECROPSIES OF PUPS FOUND DEAD
- The numbers of pups (litters) found dead during PND 0-4 numbered 1(1), 0(0), 3(2), 3(2), and 0(0) in the control, 30, 100, 250, and 1000 mg/kg/day groups, respectively.
- Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

viability

Key result

Reproductive effects observed:

no

ANALYSES OF DOSING FORMULATIONS

- Results of the analyses of dosing formulation are summarised in the tables attached.

- The analysed dosing formulations were within WIL Research SOP range for suspensions (85 % to 115 %), were homogeneous, and were stable following 17 days of refrigerated storage, with the following exceptions. The 6 mg/mL formulation prepared on 06 May 2015 was 114 % of target concentration with 12 % RSD. In addition, a peak eluting at the same time as the analyte was detected in the control group formulation prepared on 06-May-2015. The peak was believed to be caused by the initial injections of the vehicle (peanut oil), with vehicle interference carryover of the peak detected in the Group 2 formulations, forcing the higher RSD value. This was corrected by cross-validation of the method to alternate instrument settings and HPLC column. When analysed with the alternate instrumentation method, the dosing formulations were within WIL Research SOP range and no test item was detected in the analysed vehicle administered to the control group. Therefore, this did not affect the quality or integrity of the study.

Conclusions:

Under the conditions of this screening study, no test item-related effects on F0 males and female fertility and mating indices, F0 male copulation index, female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 30, 100, 250, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.

Executive summary:

GUIDELINE

This study was designed to evaluate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition, and early postnatal development. The protocol was designed in general accordance with the OECD Guidelines for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (22March1996).

 

METHODS

The test item, in the vehicle (arachis [peanut] oil), was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and mid-dose groups (Groups 2-4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 30, 100, 250, and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dosage volume was 5 mL/kg for all groups. Males and females were approximately 10 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to

mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 29 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-43 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 29 doses for males and 39 doses for females, these animals were assigned to a 14-day non-dosing recovery period.

 

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following approximately 28 days of dose administration and for 5 females/group on lactation day 4. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (haematology and serum chemistry) were performed on 5 F0 animals/sex/group at necropsy. F0 males were euthanized following completion of the mating period or 14-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered, post-mating day 25 for females that failed to deliver, or following the 14-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups at the primary necropsy.

 

RESULTS

All F0 males and females in the control, 30, 100, 250, and 1000 mg/kg/day groups survived to the scheduled necropsies. No test item-related clinical findings were noted for males or females at the detailed physical examinations or approximately 1 hour following dose administration at any dosage level.

 

Mean F0 male and female body weights, body weight gains, and food consumption in the test item groups were unaffected by test item administration throughout the study. There were no test item-related effects noted during the FOB or motor activity evaluations for F0 animals at any dosage level.

Mean male and female mating, fertility, copulation, and conception indices in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration. The mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration.

 

There were no test item-related gross necropsy observations, microscopic findings, or effects on organ weights noted for F0 males and females at any dosage level during the dosing and recovery periods. No test item-related alterations in clinical pathology parameters (haematology, coagulation, and serum chemistry) were noted for F0 males and females in the 30, 100, 250, and 1000 mg/kg/day groups during the dosing and recovery evaluations.

 

Mean numbers of former implantation sites and F1 pups born, live litter size, percentage of males at birth, postnatal survival, the general physical condition of the F1 pups, and pup body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration.

 

CONCLUSION

Under the conditions of this screening study, no test item-related effects on F0 males and female fertility and mating indices, F0 male copulation index, female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 30, 100, 250, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Justification for type of information:

See read-across justification attached in Section 13

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

viability

Key result

Reproductive effects observed:

no

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

An analogue test item, in the vehicle (arachis [peanut] oil), was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and mid-dose groups (Groups 2-4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 30, 100, 250, and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dosage volume was 5 mL/kg for all groups. Males and females were approximately 10 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 29 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-43 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 29 doses for males and 39 doses for females, these animals were assigned to a 14-day non-dosing recovery period.

 

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following approximately 28 days of dose administration and for 5 females/group on lactation day 4. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (haematology and serum chemistry) were performed on 5 F0 animals/sex/group at necropsy. F0 males were euthanized following completion of the mating period or 14-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered, post-mating day 25 for females that failed to deliver, or following the 14-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups at the primary necropsy.

 

All F0 males and females in the control, 30, 100, 250, and 1000 mg/kg/day groups survived to the scheduled necropsies. No test item-related clinical findings were noted for males or females at the detailed physical examinations or approximately 1 hour following dose administration at any dosage level.

 

Mean F0 male and female body weights, body weight gains, and food consumption in the test item groups were unaffected by test item administration throughout the study. There were no test item-related effects noted during the FOB or motor activity evaluations for F0 animals at any dosage level.

Mean male and female mating, fertility, copulation, and conception indices in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration. The mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration.

 

There were no test item-related gross necropsy observations, microscopic findings, or effects on organ weights noted for F0 males and females at any dosage level during the dosing and recovery periods. No test item-related alterations in clinical pathology parameters (haematology, coagulation, and serum chemistry) were noted for F0 males and females in the 30, 100, 250, and 1000 mg/kg/day groups during the dosing and recovery evaluations.

 

Mean numbers of former implantation sites and F1 pups born, live litter size, percentage of males at birth, postnatal survival, the general physical condition of the F1 pups, and pup body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration.

 

Under the conditions of this screening study, no test item-related effects on F0 males and female fertility and mating indices, F0 male copulation index, female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 30, 100, 250, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.

Effects on developmental toxicity

Description of key information

Under the conditions of a screening study involving an analogue test item, no test material-related effects on F0 males and female fertility and mating indices, F0 male copulation index, female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicitywas considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 30, 100, 250, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level (OECD 422).

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 April 2015 to 11 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Screening Test)

Deviations:

yes

Remarks:

various deviations with no impact on results or integrity of the study (see Appendix A, attached)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST SYSTEM, ANIMAL RECEIPT AND ACCLIMATION PERIOD
- Sexually mature male and virgin female Sprague Dawley [Crl:CD(SD)] rats were used as the test system on this study. The animal model, the Crl:CD(SD) rat, is recognised as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, WIL Research has reproductive historical control data in the Crl:CD(SD) rat.
- The number of animals selected for this study (15 [Groups 1 and 5] or 10 rats/sex/group [Groups 2-4]) was based on the OECD Guidelines for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 22-Mar-1996, which recommends evaluation of at least 8 pregnant females per group.
- Given the possibility of non-gravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, 10 animals/sex/group was an appropriate number of animals to obtain a sample size of 8 at termination. In addition, 5 animals/sex in the control and high-dose
groups were necessary to assess recovery, persistence, or progression of any toxic effects following the cessation of dosing.
- Crl:CD(SD) rats (66 males and 66 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 28-Apr-2015. The animals were approximately 61 days old upon receipt. Each animal was examined by a qualified biologist on the day of receipt. Two days following receipt, all animals were weighed and clinical observations were recorded. Each animal was uniquely identified using a programmable microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period. The animals were housed for an acclimation period of 9 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behaviour, and the testes were palpated at least once for all males.

ANIMAL HOUSING
- Following receipt and until pairing, all F0 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 analysed by the manufacturer for contaminants. Analyses of the bedding material were provided by the manufacturer. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study. The results of these analyses are maintained at WIL Research.
- During cohabitation, breeding phase rats (10/sex/group) were paired in a solid-bottom cage with bedding material. Following the breeding period, the breeding phase males and females were individually housed in solid-bottom cages with bedding material. The dams and their litters were housed in these cages until euthanasia on lactation day 4. Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until delivery or post-mating day 25. The 5 rats/sex in the control and high-dose groups that were assigned to the recovery phase were not paired for mating and remained housed in groups of 2-3 in clean solid-bottom cages until
euthanasia.
- 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 oral health of the animals, and were sanitised 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. Feeders were changed and sanitised 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, with the following exception. All males and recovery phase females were fasted prior to clinical pathology blood collection when food, but not water, was withheld.

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 70.3 °F to
72.5 °F (21.3 °C to 22.5 °C) and mean daily relative humidity ranged from 46.7 % to 66.4 % 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.

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- At the conclusion of the acclimation period, all available males and females were weighed and examined in detail for physical abnormalities. At the discretion of the Study Director, each animal judged to be in good health and meeting acceptable body weight requirements was selected for use in the computerized randomisation procedure based on body weight stratification in a block design. At that time, the individual body weights and corresponding animal identification numbers were entered into WTDMSTM. A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated. The animals then were arranged into groups according to the printout. Animals not assigned to study were transferred to the WIL Research colony.
- The experimental design consisted of 4 test item-treated groups and 1 control group composed of 10 rats/sex/group. An additional 5 rats/sex in the control and high-dose groups were selected to be evaluated following a 14-day recovery period; animals assigned to the recovery period were not evaluated for reproductive toxicity. At the initiation of dose administration (study day 0), the males and females were approximately 10 weeks old. Male body weights ranged from 335 g to 414 g and female body weights ranged from 216 g to 265 g on study day 0. The animals were approximately 12 weeks old when paired on study day 13; female body weights ranged from 228 g to 288 g on gestation day 0.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

Lot numbers 2EA0347 and 2EA0218; expiry date 31 January 2016

Details on exposure:

TEST ITEM PREPARATION
- The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group and stored refrigerated (2 °C to 8 °C), protected from light. The vehicle was mixed throughout the sampling and dose administration procedures. On each dosing day, aliquots were stirred at room temperature for at least 30 minutes prior to dispensation for dosing.
- Dosing formulations were prepared at the test item concentrations indicated in the table below.
- The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated, protected from light. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. On each dosing day, aliquots were stirred at room temperature for at least 30 minutes prior to dispensation for dosing. The first test item dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- Study group assignments are shown in the table below.
- The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily.
- The males selected for pairing were dosed during study days 0-28 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 29 doses.
- The females selected for pairing were dosed during study days 0 through the day prior to euthanasia (14 days prior to pairing through lactation day 3) for a total of 39-43 doses.
- Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses.
- The extra 5 rats/sex in the control and high-dose groups were not used for mating, but were treated on a comparable regimen beginning on study day 0; following 29 doses for males and 39 doses for females, these animals remained on study for a 14-day recovery period.
- 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.
- Dosage levels were selected based on the results of a previous 28-day study in rats (Haas, 2015, WIL-168233). In that study, the test item was administered to male and female rats for 28 consecutive days at dosage levels of 30, 100, 250, and 1000 mg/kg/day. There were no significant clinical signs noted at any dosage level. In addition, body weights and food consumption were unaffected by treatment. The selected route of administration for this study was oral (gavage) because this is a potential route of human exposure. Historically, this route has been used extensively for studies of this nature.

DATA ACQUISITION AND ANALYSIS
- Details of acquisition, analysis and reporting of data are attached.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The test substance in the vehicle was determined to be stable at concentrations of 5 and 230 mg/mL following 8 days of refrigerated storage in a previous study (Bystry, 2015, WIL-168232). In addition, resuspension homogeneity was assessed at concentrations of 60 to 200 mg/mL in a previous 28-day toxicity study (Haas, 2015, WIL-168233).
- Samples for homogeneity and/or concentration determinations were collected from the top, middle, and bottom strata of the first, fourth, fifth, sixth, seventh, and last test item dosing formulations and from the middle stratum of the first, fourth, fifth, sixth, seventh, and last control group dosing formulations. Due to difficulty with the analytical method, beginning with the fourth preparation, samples from all preparations were collected and stored refrigerated. To establish 17-day refrigerated stability, small batch sizes were prepared at the low and high concentrations used in this study. Samples were collected from the top, middle, and bottom strata of these formulations for determination of homogeneity and, following 17 days of refrigerated storage, stability. One set of samples from study weeks 0, 4, 5, and 7 was subjected to the appropriate analyses. All remaining samples were stored refrigerated (2 °C to 8 °C) as back-up. All analyses were conducted by the WIL Research Analytical Chemistry Department using a method validated on a previous study (Bystry, 2015, WIL-168232), as well as an alternate method that was cross-validated in this study.

Details on mating procedure:

BREEDING PROCEDURES
- The 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females.
- A breeding record containing the male and female identification numbers and the start date of cohabitation was 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 following a vaginal lavage and verified by a second biologist.
- Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in plastic maternity cages.
- For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

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

Duration of treatment / exposure:

- Males: Total of 29 doses (throughout the mating period through 1 day prior to euthanasia)
- Females: Total of 39 to 43 doses (14 days prior to pairing with dosing continued until lactation Day 3)

Frequency of treatment:

Daily

Duration of test:

- A flow chart summarising the study design is attached.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

- Vehicle control: 15 males and 15 females
- Test item administered at 30, 100 and 250 mg/kg bw/day: 10 males and 10 females
- Test item administered at 1000 mg/kg bw/day: 15 males and 15 females

Control animals:

yes, concurrent vehicle

Maternal examinations:

CLINICAL OBSERVATIONS AND SURVIVAL
- All rats were observed twice daily, once in the morning and once in the afternoon, for appearance, behaviour, moribundity, mortality, and signs of overt toxicity.
- Individual detailed physical examinations were recorded weekly (prior to dose administration during the treatment period).
- Each male and female was also 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
- Individual male body weights were recorded weekly throughout the study and prior to the scheduled euthanasia. Individual female body weights were recorded weekly until evidence of copulation was observed or until euthanasia (for females assigned to recovery phase). Mean weekly body weights and body weight changes were presented for each interval.
- In addition, cumulative mean body weight changes were presented for the pre-mating period (males and females) and for the entire dosing period (males and recovery phase females). Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible), 1, and 4. Mean gestation body weights and corresponding mean body weight changes were presented for these intervals and for the overall gestation interval (days 0-20); mean lactation body weight changes are presented for lactation days 1-4.
- Weekly body weights for females with no evidence of mating were presented in the individual report tables until delivery.
- When body weights could not be determined for an animal during a given interval (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
- Individual food consumption was recorded on the corresponding weekly body weight days until pairing (for animals paired for breeding) or euthanasia (for animals assigned to the recovery period). - Food consumption was measured on a per cage basis for the corresponding body weight intervals. Food consumption was normalised to the number of animals/cage and was reported in g/animal/day. Food intake was not recorded during the breeding period for animals selected for pairing.
- Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4; food consumption was reported as g/anima/day and g/kg/day during gestation and lactation.
- Following the breeding period, food consumption for females with no evidence of mating was measured on a weekly basis until delivery.
- Weekly food consumption for females with no evidence of mating was presented in the individual report tables until necropsy.
- Calculation of the comprehensive intervals excludes all erroneous values such as total food spillage.
- When food consumption could not be determined for an animal during a given interval (due to a weighing error, food spillage, 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.

FOB ASSESSMENTS
- FOB assessments were recorded for 5 animals/sex/group following approximately 28 days of dose administration (study week 4; males selected for pairing) and on lactation day 4 (females).
- The FOB used at WIL Research is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988, Moser et al., 1991; and O’Donoghue, 1989).
- FOB testing was performed by the same biologists, to the extent possible, without knowledge of the animal’s group assignment.
- The FOB was performed in a sound-attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB.
- All animals were observed for the parameters as listed in the table below.
- Forelimb and hindlimb grip strength were measured using a device similar to the one described by Meyer et al (1979). The animal was allowed to grip a T-shaped grip bar with its forepaws and was pulled back gently along a platform until its grip was broken. As the backward locomotion continues, the animal’s hindpaws reach a T-shaped rearlimb grip bar, which it is allowed to grasp and then forced to release by continued pulling. Mark-10 series EG digital force gauges (Mark-10 Corporation, Copiague, NY) were used to record the maximum strain required to break forelimb and hindlimb grip. The average of 3 valid measurements was taken as the animal’s score for each grip strength measure.

MOTOR ACTIVITY
- Motor activity was assessed for 5 animals/sex/group following approximately 28 days of dose administration (study week 4, males selected for pairing) or on lactation day 4 (females). Motor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilises a series of infrared photobeams surrounding an amber plastic, rectangular cage to quantify each animal’s motor activity.
- Four-sided black plastic enclosures were used to surround the transparent plastic boxes and decrease the potential for distraction from extraneous environmental stimuli or activity by biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams. The motor activity assessment was performed in a sound-attenuated room equipped with a white-noise generator set to operate at 70 ± 10 dB. Each animal was tested separately. Data were collected in 5-minute epochs (print intervals) and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation.
- Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).

CLINICAL PATHOLOGY
- Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected from 5 animals/sex/group at the primary necropsies (study week 4 for males and lactation day 4 for females selected for pairing) and from 5 animals/sex in the control and high-dose groups at the recovery necropsy (following a 14-day recovery period; study day 42 for males and study day 52 for females). All males and recovery phase females were fasted overnight prior to blood collection.
- Blood for serum chemistry and haematology was collected from the retro-orbital sinus following isoflurane anaesthesia.
- Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing K2EDTA (haematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry).
- Parameters evaluated were as shown in the tables below.

MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all F0 parental animals at the scheduled termination.
- All F0 adults were euthanised by carbon dioxide inhalation.
- Males were euthanised following completion of the mating period or following the 14-day recovery period. Females that delivered were euthanized on lactation day 4; the number of former implantation sites and corpora lutea were recorded.
- Females that failed to deliver were euthanised on post-mating day 25 (females with evidence of mating). Uteri with no macroscopic evidence of implantation were opened and subsequently placed in 10 % ammonium sulfide solution for detection of early implantation loss (Salewski, 1964).
- Females not selected for pairing were euthanised following the 14-day recovery period.
- Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera. Clinical findings that were verified at necropsy were designated CEO.
- At the time of necropsy, the tissues and organs were placed in 10% neutral-buffered formalin (except as noted in the table below).

ORGANS WEIGHED AT NECROPSY
- 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.

HISTOLOGY AND MICROSCOPIC EXAMINATIONS
- 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 SOPs, mounted on glass microscope slides, and stained with haematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.
- Microscopic examination was performed on all tissues from all animals in the control and 1000 mg/kg/day groups at the primary necropsy and on gross lesions from all animals in all groups at the primary necropsy.
- Missing tissues were identified as not found at necropsy, lost at necropsy, lost during processing, not in plane of section, or other reasons as appropriate. 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.

Fetal examinations:

F1 LITTER VIABILITY AND DEATHS
- Each litter was examined daily for survival, and all deaths were recorded.
- All pups were individually identified by application of tattoo markings on the digits following completion of parturition.
- A daily record of litter size was maintained.
- Intact offspring that were found dead were necropsied using a fresh dissection technique, which included examination of the heart and major vessels (Stuckhardt and Poppe, 1984).
- The carcass of each pup was then discarded.

F1 CLINICAL OBSERVATIONS
- Litters were examined daily for survival and any adverse changes in appearance or behaviour.
- Each pup received a clinical examination on PND 1 and 4.
- Any abnormalities in nesting and nursing behaviour were recorded.

F1 BODY WEIGHTS
- Pups were individually weighed on PND 1 and 4.
- Mean pup weights were presented by sex for each litter and by dose group.
- When body weights could not be determined for a pup during a given interval (due to an unscheduled death, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods a given pup was not weighed were left blank or designated as “NA” on the individual report tables.

F1 SEX DETERMINATION
- Pups were individually sexed on PND 0 and 4.

F1 SCHEDULED EUTHANASIA
- On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital and discarded.

Statistics:

See below

Indices:

MATING, FERTILITY AND COPULATION INDICES
- Mating fertility and copulation.conception indices were calculated using the following equations:
(i) Male (female) mating index (%) = [Number of males (females) with evidence of mating (or confirmed pregnant) / total number of males (females) used for mating] * 100
(ii) Male fertility index (%) = [Number of males siring a litter / total number of males used for mating] * 100
(iii) Male copulation index (%) = [Number of males siring a litter / number of males with evidence of mating (or females with confirmed pregnancy] * 100
(iv) Female fertility index (%) = [Number of females with confirmed pregnancy / total number of females used for mating] * 100
(v) Female conception index (%) = [Number of females with confirmed pregnancy / number of females with evidence or mating (or confirmed pregnancy)] * 100

F1 CALCULATION OF LITTER PARAMETERS
- Litter parameters were defined as shown in the equations below:
(i) Mean live litter size = Total number of viable pups on PND 0 / number of litters with viable pups on PND 0
(ii) Postnatal survival between birth and PND 0 or PND 4 (% per litter) = Sum of (viable pups per litter on PND 0 or PND 4 / Number of pups born per litter) / Number of litters per group * 100.
(iii) 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 * 100 where N = PND 0–1 and 1–4.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- No test item-related clinical findings were noted at the detailed physical examinations or approximately 1 hour following dose administration.
- Clinical findings observed in the test item-treated groups, including hair loss and red or clear material on various body surfaces, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related.

Mortality:

no mortality observed

Description (incidence):

- All males and females in the control, 30, 100, 250, and 1000 mg/kg/day groups survived to the scheduled necropsies.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Males: Mean body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day group males were unaffected by test item administration throughout the study. A significantly (p < 0.05) lower mean body weight gain was noted for males in the 250 mg/kg/day group during study days 13 20 and significantly (p < 0.05) higher mean body weight gains were noted for males in the 100 and 1000 mg/kg/day groups for the pre-mating period (study days 0-13). However, these results did not occur in a dose-related manner and did not affect mean absolute body weights. No other differences from the control group were statistically significant.
- Females (pre-mating): Mean body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day group females were unaffected by test item administration during the pre-mating period (females selected for breeding) or the entire study (females selected for recovery phase). Significantly (p < 0.05) higher mean body weight gains were noted for females in the 1000 mg/kg/day group during study days 34-38 and 46-51. However, these results were transient and did not affect mean absolute body weights. No other differences from the control group were statistically significant.
- Females (gestation): Mean body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.
- Females (lactation): Mean body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- Males: Mean food consumption in the 30, 100, 250, and 1000 mg/kg/day group males was similar to that in the control group throughout the study. No statistically significant differences were observed.
- Females (pre-mating): Mean food consumption in the 30, 100, 250, and 1000 mg/kg/day group females was unaffected by test item administration during the pre-mating period (females selected for breeding) or the entire study (females selected for recovery phase). None of the differences from the control group were statistically significant.
- Females (gestation): Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 30, 100, 250, and 1000 mg/kg/day groups was unaffected by test item administration during gestation. None of the differences from the control group were statistically significant.
- Females (lactation): Mean maternal food consumption, evaluated as g/animal/day and g/kg/day, in the 30, 100, 250, and 1000 mg/kg/day groups was unaffected by test item administration during lactation. None of the differences from the control group were statistically significant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related alterations in haematology and coagulation parameters at the scheduled necropsies. However, some statistically significant (p < 0.05) differences were observed when the control and test substance-treated groups were compared.
- Lower absolute basophil values were noted for females in the 100 and 1000 mg/kg/day groups at the primary necropsy. There was no dose-response relationship, values were of minimal magnitude difference, and were similar to the control group at the recovery necropsy; the differences were attributed to biological variability.
- Higher prothrombin time (PT) values were noted in the 1000 mg/kg/day group males at the recovery necropsy. Values were of minimal magnitude change and were similar to control and 1000 mg/kg/day group values at the primary necropsy; the finding was attributed to biological variability and not related to administration of test item.
- Statistically significant (p < 0.05) findings that involved percentage leukocyte differential counts were not itemized above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related alterations in serum chemistry parameters at the scheduled necropsies.
- However, some statistically significant (p < 0.01 or p < 0.05) differences were observed when the control and test item-treated groups were compared.
- Lower serum chloride values were noted in the 1000 mg/kg/day group males at the primary necropsy. Other serum electrolyte values were similar to the control group, the difference was of minimal magnitude, and there were no histologic correlates; the finding was attributed to biological variability.
- Higher serum urea nitrogen values were noted in the 1000 mg/kg/day group males and higher serum triglyceride values were noted in the 1000 mg/kg/day group females at the recovery necropsy. Both values were similar to the control group at the primary necropsy and were of minimal magnitude difference; the Findings were attributed to biological variability and not related to administration of test item.
- Other statistically significant differences lacked a dose-response relationship, and were attributed to biological variability.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
- Home cage observations: Home cage parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Handling observations: Handling parameters were unaffected by test item administration. A significantly (p < 0.05) higher number of males in the 250 mg/kg/day group had slight resistance to being handled; however, this did not occur in a dose-related manner. There were no other statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Open field observations: Open field parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Sensory observations: Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Neuromuscular observations: Neuromuscular parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).
- Physiological observations: Physiological parameters were unaffected by test item administration. There were no statistically significant differences for the test item-treated groups when compared to the control group during study week 4 (males) or on lactation day 4 (females).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related alterations in final body weights or organ weights at the scheduled necropsies. However, some statistically significant (p < 0.05) differences were observed when the control and test item-treated groups were compared.
- Lower absolute kidney weights were noted in the 1000 mg/kg/day group males at the recovery necropsy. Values were similar to the control group when corrected for body weight; the finding was
not considered to be related to administration of test item.
- Higher thyroid/parathyroid gland weights (absolute and relative to body and brain weights) were noted in the 1000 mg/kg/day group females at the recovery necropsy. Weights were similar between control and 1000 mg/kg/day group females at the primary necropsy, and the magnitude of change was minimal; the findings were attributed to biological variability and not related to administration of test item.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

MACROSCOPIC EXAMINATIONS
- No test item-related internal findings were observed at any dosage level in females that failed to deliver or males and females at the scheduled necropsies.
- Macroscopic findings observed in the test item-treated groups occurred infrequently and/or in a manner that was not dose-related.
- The mean numbers of unaccounted-for sites and implantation sites in the 30, 100, 250, and 1000 mg/kg/day groups were similar to the control group values.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

MOTOR ACTIVITY
- Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test item administration at all concentrations when evaluated during study week 4 (males) or on lactation day 4 (females). Values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50 and 51-60 minutes) and the overall 60-minute test session values were comparable to the concurrent control values and the WIL historical control data.
- Differences from the control group were slight, not statistically significant when analysed by a repeated measures analysis, within the WIL historical control data ranges and/or did not occur in a dose-related manner. No remarkable shifts in the pattern of habituation occurred in any of the test item-treated groups when the F0 animals were evaluated during study week 4 (males) or on lactation day 4 (females).

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related histologic changes at the primary necropsy. Histologic changes noted 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.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): GESTATION LENGTH AND PARTURITION
- Mean gestation lengths in the 30, 100, 250, and 1000 mg/kg/day groups were similar to those in the control group.
- No statistically significant differences were noted.
- No signs of dystocia were noted in these groups.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

REPRODUCTIVE PERFORMANCE
- F0 male and female reproductive parameters are presented in the table attached.
- No test item-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test item-treated groups. Single males in each of the 100 and 1000 mg/kg/day groups did not sire a litter. Single females in each of the 100 and 1000 mg/kg/day groups had evidence of mating but did not deliver.
- The mean numbers of days between pairing and coitus in the test item-treated groups were similar to the control group value. None of these differences were statistically significant.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

maternal abnormalities

Remarks on result:

other: no test item-related effects reported at any dose level

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): OFFSPRING BODY WEIGHTS
- Mean male and female pup body weights and body weight changes during PND 1-4 in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by parental administration of the test item.
- No statistically significant differences from the control group were noted.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

PND 0 LITTER DATA AND POSTNATAL SURVIVAL
- The mean number of pups born, live litter size and the percentage of males at birth in the 30, 100, 250, and 1000 mg/kg/day groups were similar to the control group values.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

- Postnatal survival in the 30, 100, 250, and 1000 mg/kg/day groups was unaffected by test item administration.

Visceral malformations:

no effects observed

Description (incidence and severity):

NECROPSIES OF PUPS FOUND DEAD
- The numbers of pups (litters) found dead during PND 0-4 numbered 1(1), 0(0), 3(2), 3(2), and 0(0) in the control, 30, 100, 250, and 1000 mg/kg/day groups, respectively.
- Aside from the presence or absence of milk in the stomach, no other internal findings were noted.

Other effects:

no effects observed

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 was unaffected by parental administration of the test item.
- Pups that were found dead numbered 1, 0, 3, 3, and 0 in the control, 30, 100, 250, and 1000 mg/kg/day groups, respectively.
- One, 1, 0, 1, and 3 pups in the same respective groups was missing and presumed to have been cannibalised.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

other: absence of test item-related effects at any maternal dose level

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

ANALYSES OF DOSING FORMULATIONS

- Results of the analyses of dosing formulation are summarised in the tables attached.

- The analysed dosing formulations were within WIL Research SOP range for suspensions (85 % to 115 %), were homogeneous, and were stable following 17 days of refrigerated storage, with the following exceptions. The 6 mg/mL formulation prepared on 06 May 2015 was 114 % of target concentration with 12 % RSD. In addition, a peak eluting at the same time as the analyte was detected in the control group formulation prepared on 06-May-2015. The peak was believed to be caused by the initial injections of the vehicle (peanut oil), with vehicle interference carryover of the peak detected in the Group 2 formulations, forcing the higher RSD value. This was corrected by cross-validation of the method to alternate instrument settings and HPLC column. When analysed with the alternate instrumentation method, the dosing formulations were within WIL Research SOP range and no test item was detected in the analysed vehicle administered to the control group. Therefore, this did not affect the quality or integrity of the study.

Conclusions:

Under the conditions of this screening study, no test item-related effects on F0 males and female fertility and mating indices, F0 male copulation index, female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 30, 100, 250, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.

Executive summary:

GUIDELINE

This study was designed to evaluate the potential toxic effects of the test item when administered to rats for 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition, and early postnatal development. The protocol was designed in general accordance with the OECD Guidelines for the Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (22March1996).

 

METHODS

The test item, in the vehicle (arachis [peanut] oil), was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and mid-dose groups (Groups 2-4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 30, 100, 250, and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dosage volume was 5 mL/kg for all groups. Males and females were approximately 10 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to

mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 29 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-43 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 29 doses for males and 39 doses for females, these animals were assigned to a 14-day non-dosing recovery period.

 

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following approximately 28 days of dose administration and for 5 females/group on lactation day 4. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (haematology and serum chemistry) were performed on 5 F0 animals/sex/group at necropsy. F0 males were euthanized following completion of the mating period or 14-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered, post-mating day 25 for females that failed to deliver, or following the 14-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups at the primary necropsy.

 

RESULTS

All F0 males and females in the control, 30, 100, 250, and 1000 mg/kg/day groups survived to the scheduled necropsies. No test item-related clinical findings were noted for males or females at the detailed physical examinations or approximately 1 hour following dose administration at any dosage level.

 

Mean F0 male and female body weights, body weight gains, and food consumption in the test item groups were unaffected by test item administration throughout the study. There were no test item-related effects noted during the FOB or motor activity evaluations for F0 animals at any dosage level.

Mean male and female mating, fertility, copulation, and conception indices in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration. The mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration.

 

There were no test item-related gross necropsy observations, microscopic findings, or effects on organ weights noted for F0 males and females at any dosage level during the dosing and recovery periods. No test item-related alterations in clinical pathology parameters (haematology, coagulation, and serum chemistry) were noted for F0 males and females in the 30, 100, 250, and 1000 mg/kg/day groups during the dosing and recovery evaluations.

 

Mean numbers of former implantation sites and F1 pups born, live litter size, percentage of males at birth, postnatal survival, the general physical condition of the F1 pups, and pup body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration.

 

CONCLUSION

Under the conditions of this screening study, no test item-related effects on F0 males and female fertility and mating indices, F0 male copulation index, female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 30, 100, 250, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Justification for type of information:

See read-across justification attached in Section 13

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

maternal abnormalities

Remarks on result:

other: no test item-related effects reported at any dose level

Key result

Abnormalities:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

maternal dose

Sex:

male/female

Basis for effect level:

other: absence of test item-related effects at any maternal dose level

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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

An analogue test item, in the vehicle (arachis [peanut] oil), was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and mid-dose groups (Groups 2-4) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 30, 100, 250, and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dosage volume was 5 mL/kg for all groups. Males and females were approximately 10 weeks of age at the beginning of test item administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 29 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 3 for a total of 39-43 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 29 doses for males and 39 doses for females, these animals were assigned to a 14-day non-dosing recovery period.

 

All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. FOB and motor activity data were recorded for 5 males/group following approximately 28 days of dose administration and for 5 females/group on lactation day 4. All F0 females selected for pairing were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Surviving pups were euthanized and discarded on PND 4. Clinical pathology evaluations (haematology and serum chemistry) were performed on 5 F0 animals/sex/group at necropsy. F0 males were euthanized following completion of the mating period or 14-day recovery period and F0 females were euthanized on lactation day 4 for females that delivered, post-mating day 25 for females that failed to deliver, or following the 14-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals in the control and high-dose groups at the primary necropsy.

 

All F0 males and females in the control, 30, 100, 250, and 1000 mg/kg/day groups survived to the scheduled necropsies. No test item-related clinical findings were noted for males or females at the detailed physical examinations or approximately 1 hour following dose administration at any dosage level.

 

Mean F0 male and female body weights, body weight gains, and food consumption in the test item groups were unaffected by test item administration throughout the study. There were no test item-related effects noted during the FOB or motor activity evaluations for F0 animals at any dosage level.

Mean male and female mating, fertility, copulation, and conception indices in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration. The mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration.

 

There were no test item-related gross necropsy observations, microscopic findings, or effects on organ weights noted for F0 males and females at any dosage level during the dosing and recovery periods. No test item-related alterations in clinical pathology parameters (haematology, coagulation, and serum chemistry) were noted for F0 males and females in the 30, 100, 250, and 1000 mg/kg/day groups during the dosing and recovery evaluations.

 

Mean numbers of former implantation sites and F1 pups born, live litter size, percentage of males at birth, postnatal survival, the general physical condition of the F1 pups, and pup body weights and body weight gains in the 30, 100, 250, and 1000 mg/kg/day groups were unaffected by test item administration.

 

Under the conditions of this screening study, no test item-related effects on F0 males and female fertility and mating indices, F0 male copulation index, female conception index, gestation length, parturition, or reproductive organs were noted at any dosage level. Therefore, a dosage level of 1000 mg/kg/day, the highest dosage level evaluated, was considered to be the no-observed-adverse-effect level (NOAEL) for reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 1000 mg/kg/day based on the lack of adverse test item-related effects in the 30, 100, 250, and 1000 mg/kg/day groups. The NOAEL for neonatal toxicity was 1000 mg/kg/day based the absence of test item-related effects on pup clinical condition, postnatal survival, and pup body weights and body weight gains at any dosage level.

Justification for classification or non-classification

No adverse treatment-related effects on P0 or F1 generations were reported at dose levels of 30, 100, 250 and 1000 mg/kg bw when an analogue substance was investigated (OECD 422). Classification of the target substance for reproductive or developmental toxicity is therefore unnecessary under the criteria of Regulation (EC) No 1272/2008 and subsequent amendments.