[No public or meaningful name is available]

Endpoint summary

Description of key information

Treatment at dosages of up to 1000 mg/kg bw/day was well tolerated and the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day (OECD 422).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

08 June 2016 to 27 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

no guideline required

Principles of method if other than guideline:

Test item in the vehicle arachis oil was administered to four groups of male/female rats orally by gavage once daily for 14 days. The objective of the study was to determine the dosage levels to be evaluated in a potential combined repeated dose toxicity study with reproductive/developmental screening.

GLP compliance:

no

Limit test:

no

Specific details on test material used for the study:

- The purity of the test item was 100 %
- The test material was stored at room temperature (18 to 24 °C) protected from light and was considered stable under these conditions.

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMAL RECEIPT AND ACCLIMATION
- Crl:CD(SD) rats (28 males and 28 females) were received in good health from Charles River Laboratories Inc, Saint Constant, QC, Canada on 05 July 2016.
- The animals were approximately 63 days old upon receipt.
- Each animal was examined by a qualified biologist on the day of receipt.
- On the day 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 dorsocapular region during the acclimation period.
- The animals were housed for 8 days for acclimation purposes.
- During the acclimation period, the rats were observed twice daily for mortality and general changes in appearance and behaviour.

ANIMAL HOUSING
- Upon arrival, all rats were house two per cage 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 and analyses were provided. No contaminants were present in the bedding at concentrations sufficient to interfere with the outcome of the study.
- Assignment of individual animals to social groups was recorded.
- 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. The devices were sanitised weekly.
DIET, DRINKING WATER AND MAINTENANCE
- The basal diet used in the study (PMI Nutrition International LLC Certified Rodent LabDiet 5002) was a certified feed with appropriate analyses performed by the manufacturer.
- Feed lots used during the investigation 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 testing house SOPs.
- The results of the diet and water analyses were retained.
- No contaminants were present in the animal feed or water at concentrations sufficient to interfere with the objectives of the study.
- Reverse osmosis-purified (on-site) drinking water, delivered by an automatic watering system, and the basal diet were provided ad libitum throughout the acclimation period and during the study.

ENVIRONMENTAL CONDITIONS
- All rats were housed throughout the acclimation period and during the study in an environmentally controlled room.
- Controls were set to maintain room temperature at 73 ± 5 °F (22 ± 3 °C and relative humidity at 50 ± 20 %. 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 72.3 to 72.6 °F (22.4 to 22.6 °C) during the study.
- Mean daily relative humidity ranged from 41.5 to 52.2 % during the study.
- Fluorescent lighting provided illumination for a 12-hour light (06:00 hours to 18:00 hours) and 12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes.
- Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Route of administration:

oral: gavage

Details on route of administration:

ORGANISATION OF TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- The vehicle and test item formulations were administered orally by gavage via appropriately sized, disposable plastic, feeding tubes.
- Administration took place once daily during study days 0 to 13.
- The dose 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 bw/day dose.
- All animals were dosed at approximately the same time each day.
- Dosage levels were selected by the sponsor and were intended to cover a wide range of dosage levels to assess tolerability up to the limit dose of 1000 mg/kg bw/day.
- The dosage levels selected were not expected to produce mortality but were intended to provide dose-response information to be used in subsequent studies.
- The selected route of administration was oral (gavage) because this is a potential route of exposure for humans and historically the oral route has been used extensively for studies of this nature.

Vehicle:

arachis oil

Remarks:

Lot number 2EL0356 (expiry date 31 December 2016)

Details on oral exposure:

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 at room temperature (18 °C to 24 °C) protected from light.
- The vehicle was mixed throughout the dose administration procedures.
- Dosing formulations were prepared at the test item concentrations indicated in Table 2 (below).
- Dosing formulations were not adjusted for purity.
- Test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18 °C to 24 °C) protected from light.
- The test item formulations were stirred continuously throughout the preparation and dose administration procedures.
- The first test item dosing formulations were visually inspected by the study director and were found to be visibly homogeneous and acceptable for administration.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

- Analyses to demonstrate the homogeneity, concentration and stability of the test item formulations were not conducted as part of this non-GLP study.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

ASSIGNMENT OF ANIMALS TO TREATMENT GROUPS
- At the conclusion of the acclimation period, all available animals were weighed and examined in detail for physical abnormalities.
- At the discretion of the study director, each animal judged to be in good health and meeting acceptable body weight requirements was selected for use in a computerised randomisation procedure. At that time the individual body weights and corresponding animal identification numbers were entered into WTDMS.
- A printout containing the animal numbers, corresponding body weights, and individual group assignments was generated based on body weight stratification in a block design. The animals were then arranged into groups according to the printout.
- Animals not assigned to the study were transferred to the testing house rat colony.
- The experimental design consisted of four test item-treated groups and one control group, composed of 5 rats/sex/group.
- The selected animals were approximately 10 weeks old at the initiation of dose administration.
- Individual body weights ranged from 317 g to 367 g (males) and from 214 g to 271 g (females) on study day 0.

DATA ACQUISITION AND REPORTING
- Critical computerised systems are summarised in Table 5 (attached).

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND SURVIVAL
- All rats were observed twice daily, once in the morning and once in the afternoon, for morbundity and mortality.
- Individual clinical observations were recorded daily (prior to dose administration) during the treatment period.
- Animals were also observed for signs of toxicity approximately one hour after 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.
- The social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal and positive findings, if any, were recorded.

BODY WEIGHTS
- Individual body weights were recorded on study days 0, 4, 7, 11 and 14. Group mean body weights were calculated for each of those days.
- Mean body weight changes were calculated for each corresponding interval and also for study days 0 to 14.

FOOD CONSUMPTION
- Individual food consumption was recorded on study days 0, 4, 7, 11 and 14.
- Food consumption was measured on a per cage basis for the corresponding body weight intervals.
- Food consumption was normalised for the number of animals/cage and was reported as g/animal/day.

Sacrifice and pathology:

SCHEDULED NECROPSY
- Gross necropsy was conducted on all animals at the scheduled euthanasia on study day 14.
- All animals were euthanised by carbon dioxide inhalation.
- Necropsy included examination of the external body surface, all orifices, and the abdominal, cranial, pelvic and thoracic cavities, including viscera.
- The following organs were weighed from all surviving animals at the scheduled necropsy: adrenal glands, brain, epididymides (paired organs weighed separately), heart, kidneys, liver, ovaries with oviducts, spleen, testes (paired organs weighed separately), thymus gland and thyroids with parathyroids (fixed in 10 % neutral-buffered formalin prior to weighing).

Statistics:

STATISTICAL ANALYSES
- All statistical analyses were performed using WTDMS unless otherwise noted.
- Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1 % and 5 %, comparing each test item-treated group to the control group.
- Each mean was presented with the standard deviation (SD), standard error (SE) and the number of animals (N) used to calculate the mean.
- Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations and standard errors on the summary and individual tables may have differed slightly. The use of reported individual values to calculate subsequent parameters or means may therefore, in some instances, yield minor variations from those listed in the report tables.
- Body weights, body weight changes, food consumption plus absolute and relative organ weights were subjected to a parametric one-way ANOVA to determine intergroup differences.
- If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett’s test was used to compare the test item-treated groups to the control group.

Clinical signs:

no effects observed

Description (incidence and severity):

- No remarkable clinical findings were noted at the daily examinations or one hour following dose administration.

Mortality:

no mortality observed

Description (incidence):

- All animals in the control, 100, 300, 500 and 1000 mg/kg bw/day groups survived to the scheduled necropsy on study day 14.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Mean body weights and body weight gains in the mails and females in the 100, 300, 500 and 1000 mg/kg bw/day groups were similar to that in the control group.
- Differences from the control group were slight and not statistically significant with the exception of significantly (p < 0.05) higher body weight gain for the female 100 mg/kg bw/day group during study days 7-11 compared to the control group. However, tis did not occur in a dose-related manner.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- Mean food consumption, evaluated as g/animal/day, was similar to that in the control group for the male and female 100, 300, 500 and 1000 mg/kg bw/day groups.
- Differences from the control group were slight and not statistically significant.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- Mean male and female final body weights in the test item-treated groups were similar to the control group.
- Significantly (p < 0.05) lower absolute left and right epididymis weights and right epididymis weight relative to brain weight were noted for the male 500 mg/kg bw/day group compared to the control group. However, this did not occur in a dose-related manner.
- No other noteworthy changes in organ weights were noted for males and females at any dosage level.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No remarkable internal findings were observed at the scheduled necropsy on study day 14 for dosage levels of 100, 300, 500 and 1000 mg/kg bw/day.
- Macroscopic findings observed in the test item-treated groups occurred infrequently, at similar frequencies in the control group and/or in a manner that was not dose-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

No effects on survival, body weight, body weight gains, food consumption, organ weight and necropsy observations were noted for males and females in the 100, 300, 500 and 1000 mg/kg bw/day groups. Therefore dosage levels of 100, 300, 500 and 1000 mg/kg bw/day were selected for a combined 28-day repeated dose oral toxicity study with reproduction/developmental toxicity screening in the rat.

Executive summary:

OBJECTIVE

The objective of the study was to determine dosage levels of test material to be evaluated in a potential combined repeated dose toxicity study with reproduction/developmental screening (OECD 422) in rats.

STUDY DESIGN

The test item, in the vehicle arachis (peanut) oil, was administered orally by gavage once daily to four groups of Crl:CD(SD) rats, each group consisting of 5 males and 5 females, once daily for 14 consecutive days. Dosage levels were 100, 300, 500 and 1000 mg/kg bw/day administered at a dose volume of 5 mL/kg. A concurrent control group composed of 5 rats/sex received the vehicle on a comparable regimen. The animals were approximately 10 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights and food consumption were recorded at appropriate intervals. Complete necropsies were conducted on all animals on study day 14 and selected organs were weighed.

RESULTS

All males and females in the control, 100, 300, 500 and 1000 mg/kg bw/day groups survived to the scheduled necropsy on study day 14. No remarkable clinical observations were noted for any of the test item-treated group. Mean body weight, body weight gains and food consumption for the male and female 100, 300, 500 and 1000 mg/kg bw/day groups were similar to the control group throughout the study. No remarkable macroscopic finding or organ weight alterations were observed for males or females at any dosage level.

CONCLUSION

No effects on survival, body weight, body weight gains, food consumption, organ weight and necropsy observations were noted for males and females in the 100, 300, 500 and 1000 mg/kg bw/day groups. Therefore dosage levels of 100, 300, 500 and 1000 mg/kg bw/day were selected for a combined 28-day repeated dose oral toxicity study with reproduction/developmental toxicity screening in the rat.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 August 2016 to 08 May 2017

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 quality or integrity of data or study outcome (see Appendix 1, 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
- Overall design of the study is shown in the diagram attached.
- 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 recognized as appropriate for reproductive toxicity studies and has been proven to be susceptible to the effects of reproductive toxicants. In addition, Charles River Ashland 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 Guideline for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016, which recommends that evaluation of each group be initiated with at least 10 males and 12 to 13 females/group.
- Females were evaluated for oestrous cyclicity during the pre-test period and any females that failed to exhibit normal 4–5 day oestrous cycles (e.g., EDDDE), repeatedly during the pre-test period, were excluded from the study; therefore, the extra females were included to yield at least 10 females/group. Given the possibility of nongravid animals, unexpected deaths, total litter losses, or treatment-related moribundity and/or mortality, this was an appropriate number of animals to obtain a sample size of 8 at termination.
- Crl:CD(SD) rats (66 males and 78 females) were received in good health from Charles River Labouratories, Inc., Raleigh, NC on 25 Aug 2016. The animals were approximately 51 days old upon receipt. Each animal was examined by a qualified biologist on the day of receipt. On the day 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 21 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/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 Charles River.
- During cohabitation, breeding phase rats (10/sex/group) were paired in a solid-bottom cage with bedding material.
- Following the breeding period, the males were individually housed in solid-bottom cages until the scheduled necropsy. Following positive evidence of mating, the females were individually housed in solid-bottom cages with bedding material. The dams and their litters were housed in these cages until euthanasia on PND 13 or Lactation Day 14. Females that failed to deliver were housed in solid-bottom cages with bedding material until 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.
- Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining oral health of animals, and were sanitized weekly.

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 Charles River. Feed lots used during the study were documented in the study records. Feeders were changed and sanitized once per week (see deviations to study protocol in Appendix 1, attached).
- Municipal water supplying the facility was sampled for contaminants according to Charles River SOPs. The results of the diet and water analyses are maintained at Charles River. 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 (see deviations to study protocol in Appendix 1, attached), with the following exception. All F0 males and females (including animals not selected for clinical pathology evaluation) 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 68 to 78 °F (20 to 26 °C) and 30 % to 70 %, respectively.
- Room temperature and relative humidity data were monitored continuously and were scheduled for automatic collection on an hourly basis (see deviations to study protocol in Appendix 1, attached).
- Actual mean daily temperature ranged from 71.3 to 72.7 °F (21.8 to 22.6 °C) and mean daily relative humidity ranged from 37.8 % to 57.7 % during the study.
- Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod. The light status (on or off) was recorded once every 15 minutes. Air handling units were set to provide a minimum of 10 fresh air changes per hour.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

Lot 2EL0356; expiry date 31 December 2016

Details on oral exposure:

TEST ITEM PREPARATION
- The vehicle was dispensed approximately weekly for administration to the control group (Group 1) and for preparation of the test substance formulations.
- Aliquots were prepared for daily dispensation to the control group and stored at room temperature (18 to 24 °C), protected from light.
- The vehicle was mixed throughout the sampling and dose administration procedures.
- Dosing formulations were prepared as indicated in the dose formulation concentration table (below).
- Test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18 to 24 °C), protected from light.
- The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.
- The first test substance dosing formulations were visually inspected by the Study Director and were found to be visibly homogeneous and acceptable for administration.

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, meeting acceptable body weight requirements, and exhibiting normal 4–5 day oestrous cycles (females) was selected for use in the computerized randomization 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 WTDMS. 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 Charles River rat colony.
- The experimental design consisted of 4 test substance-treated groups and 1 control group composed of 10 rats/sex/group. An additional 5 rats/sex in the control and high-dose group were selected to be evaluated following a 15-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 313 g to 417 g and female body weights ranged from 206 g to 272 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 299 g on Gestation Day 0.

TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- Vehicle and test substance formulations were administered orally by gavage, via appropriately sized flexible, disposable, plastic feeding tubes (Instech Solomon, Plymouth Meeting, PA) once daily. - The males selected for pairing were dosed during Study Days 0–27 (14 days prior to pairing through 1 day prior to scheduled euthanasia) for a total of 28 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 13) for a total of 49–54 doses.
- Females that failed to deliver were dosed through the day prior to euthanasia (Post-Mating Day 25) for a total of 39–40 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 28 doses for males and 49 doses for females, these animals remained on study for a 15-day recovery period.
- The dose volume for all groups was 5 mL/kg. Individual dosages were based on the most recently recorded body weights to provide the correct mg/kg/day dose. All animals were dosed at approximately the same time each day.
- Study group assignment is shown in the table below. Dosage levels were selected based on the results of a previous 14-day range-finding study in which male and female rats were administered the test substance at dosage levels of 100, 300, 500, and 1000 mg/kg/day.
- In the previous study, there were no significant clinical observations noted at any dosage level. In addition, mean body weights and food consumption were unaffected by treatment. As a result, the same dosage levels were chosen for the current study.
- The selected route of administration for this study was oral (gavage) because this a potential route of exposure for humans. Historically, this route has been used extensively for studies of thi

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

SAMPLING AND ANALYSIS
- Homogeneity and stability of the test substance in the vehicle following 6 and 11 days of room temperature storage at concentrations ranging from 10 to 200 mg/mL were established in a previous study. Therefore, stability assessments were not conducted in the current study.
- Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first test substance dosing formulations and from the middle stratum of the first control dosing formulations. Samples for concentration analysis were also collected from the middle stratum of the last dosing formulations (including the control group) prepared during the study (see study deviations discussed in Appendix 1, attached).
- One set of samples from each collection was subjected to the appropriate analyses. All remaining samples were stored at room temperature as back-up. All analyses were conducted by the Charles River Analytical Chemistry Department using a validated high performance liquid chromatography method with charged aerosol detection.

Duration of treatment / exposure:

- Males: 14 days prior to mating and throughout the mating period through 1 day prior to euthanasia for a total of 28 doses.
- Females: 14 days prior to pairing and through Lactation Day 13 for a total of 49-54 doses.
- Females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39–40 doses.
- The extra 5 males and 5 females in the control and high-dose groups not used for mating were treated beginning on Study Day 0 and, following 28 doses for males and 49 doses for females, these animals were assigned to a 15-day nondosing recovery period.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

- Low and mid-dose groups: 10 rats/sex
- High dose group: 15 rats/sex

Control animals:

yes, concurrent vehicle

Details on study design:

DATA ACQUISITION AND ANALYSIS
- The major computer systems used on this study include, but are not limited to, the systems shown in the table attached.
- All computerized systems used for data collection during the conduct of this study have been validated (with the exception of Microsoft Office); when a particular system has not satisfied all requirements, appropriate administration and procedural controls were implemented to assure the quality and integrity of the data.

STATISTICAL ANALYSES
- Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was two or less. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means, standard deviations, and standard errors on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
- Analyses conducted by Charles River: All statistical tests were performed using WTDMS unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1 % and 5 %, comparing each test substance-treated group to the control group by sex. Parental mating, fertility, conception, and copulation indices were analysed using the Chi-square test with Yates’ correction factor. Parental body weights (weekly, gestation, and lactation), body weight changes, and food consumption, offspring body weights and body weight changes, oestrous cycle length, pre-coital intervals, gestation length, numbers of former implantation sites, and unaccounted-for sites, number of pups born, live litter size on PND 0, absolute and relative organ weights, clinical pathology values, thyroid hormone values, anogenital distance (absolute and relative to the cube root of body weight), number of nipples/areolae, and FOB data values were subjected to a parametric one-way ANOVA17 to determine intergroup differences. If the ANOVA revealed significant (p < 0.05) intergroup variance, Dunnett's test was used to compare the test substance-treated groups to the control group. FOB parameters that yield scalar or descriptive data in the test substance-treated groups were compared to the control group using Fisher’s Exact test. Mean litter proportions (percent per litter) of males at birth and postnatal survival were subjected to the Kruskal-Wallis nonparametric ANOVA to determine intergroup differences. If the nonparametric ANOVA revealed significant (p < 0.05) intergroup variance, Dunn’s test was used to compare the test substance-treated groups to the control group.
- Analyses conducted by BioSTAT Consultants Inc: All analyses were conducted using SAS version 9.2,22 or higher, software. Ambulatory and total counts measured during motor activity assessments were analysed by sex and session, with a RMANOVA. Factors in the model included treatment group (TRT), time interval (TIME) and the interaction of time interval and treatment group (TRT*TIME). The SAS procedure PROC MIXED was used for analysis with the random effect of animal included as the repeated measurement. The covariance structure across time was selected by comparing Akaike’s Information Criterion (AIC). The monotonic dose-response relationship was evaluated using sequential linear trend tests based on ordinal spacing of dosage levels. The linear dose by time interaction (LinTrt*Time) was evaluated, and if significant at the 0.05 level, trend tests on treatment means were performed at the 0.05 level for each time interval. If the linear dose by time interaction was not significant, the trend test was conducted across the pooled time intervals by session only. Nonmonotonic dose responses were evaluated whenever no significant linear trends were detected but TRT and/or TRT*TIME interaction was significant at the 0.01 level. Within the framework of the RMANOVA, pair-wise comparisons were made for each individual treated group with the control group through linear contrasts. If TRT*TIME was significant, the comparisons were conducted for each time interval. If only the TRT effect was significant, the comparisons were conducted across the pooled time intervals of the entire session. These nonmonotonic dose response comparisons were conducted at the 0.01 significance level.

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND SURVIVAL
- All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.
- Individual clinical observations were recorded daily and individual detailed physical examinations were recorded weekly (prior to dose administration during the treatment period) (see deviations to study protocol in Appendix 1, attached).
- 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 labour, delayed labour) or other difficulties (see deviations to study protocol in Appendix 1, attached). In addition, the social groups were observed at the appropriate intervals for findings that could not be attributed to a single animal; only positive findings were recorded.

BODY WEIGHTS
- Individual male body weights were recorded weekly throughout the study and prior to the scheduled euthanasia.
- Individual female body weights were recorded weekly until evidence of copulation was observed 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 dosing 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, 4, 7, 10, 13, and 14 (fasted).
- Body weight changes were presented for each of these intervals and for the entire gestation and lactation intervals (Days 0–20 and 1–13, respectively).
- When body weights could not be determined for an animal during a given interval (due to an unscheduled death, as females enter gestation, weighing error, etc.), group mean values were calculated for that interval using the available data. The time periods when body weight values were unavailable for a given animal were left blank or designated as “NA” on the individual report tables.

FOOD CONSUMPTION
- 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 normalized to the number of animals/cage and was reported as 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, 4, 7, 10, and 13.
- Following the breeding period, individual food consumption for females with no evidence of mating and for all males was measured on a weekly basis until the scheduled euthanasia. Calculation of the
comprehensive intervals excludes all erroneous values such as total food spillage.
- When food consumption could not be determined for a given interval (due to an unscheduled death, weighing error, food spillage, etc.), group mean values were calculated for that interval using the available data. The time periods when food consumption values were unavailable 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 3; males selected for pairing) or on Lactation Day 13 (females).
- The FOB used at Charles River is based on previously developed protocols. FOB testing was performed by the same biologists, to the extent possible, without knowledge of the animal group assignments.
- 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 following parameters shown in the table below.
- Forelimb and hindlimb grip strength were measured using a device similar to the one described by Meyer. 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 day of dose administration (Study Week 3; males selected for pairing) or on Lactation Day 13 (females).
- Motor activity, recorded after the completion of the FOB, was measured automatically using a personal computer-controlled system that utilizes 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 60 dB to 80 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).

Sacrifice and pathology:

MACROSCOPIC EXAMINATION
- A complete necropsy was conducted on all F0 parental animals euthanised in extremis or at the scheduled termination. All surviving F0 adults were euthanised by carbon dioxide inhalation.
- Males were euthanised following completion of the mating period or following the 15-day recovery period; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia. - Females that delivered were euthanised on Lactation Day 14; blood samples were collected for thyroid hormone analysis immediately prior to euthanasia.
- 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.
- For females that delivered, the numbers of former implantation sites (the attachment site of the placenta to the uterus) were recorded. The number of unaccounted-for sites was calculated for each female that delivered by subtracting the number of pups born from the number of former implantation sites observed. Numbers of corpora lutea were also recorded for females with macroscopic evidence of implantation and for females necropsied during gestation through Lactation Day 4.
- Females not selected for pairing were euthanised following the 15-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, 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.
- When organ weights could not be determined for an animal (due to weighing error, lost or damaged organ, etc.), group mean values were calculated using the available data. The organs for which weights could not be determined were designated as “NA” on the individual report tables.

Other examinations:

CLINICAL PATHOLOGY
- Blood samples for clinical pathology evaluations (haematology, coagulation, and serum chemistry) were collected from 5 animals/sex/group at the scheduled/primary necropsies (Study Day 28 for males and Lactation Day 14 for females selected for pairing) and from 5 animals/sex in the control and high-dose groups at the recovery necropsy (following a 15-day recovery period; Study Day 42 for males and Study Day 63 for females).
- The animals were fasted overnight prior to blood collection. Blood for serum chemistry and haematology was collected from the jugular vein or retro-orbital sinus following isoflurane anesthesia. 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 (coagulation), or no anticoagulant (serum chemistry). Parameters evaluated were as shown in the tables below.

HISTOLOGY AND MICROSCOPIC EXAMINATIONS
- After fixation, protocol-specified tissues were trimmed according to Charles River SOPs and the protocol. Trimmed tissues were processed into paraffin blocks, sectioned according to Charles
River SOPs, mounted on glass microscope slides, and stained with hematoxylin and eosin. In addition, PAS staining was used for the testes and epididymides.
- Microscopic examination was performed on all tissues listed from all animals in the control and 1000 mg/kg/day groups at the primary necropsy and from all animals euthanised in extremis prior to the scheduled necropsy.
- Gross lesions were examined 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.

Statistics:

See above

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

CLINICAL OBSERVATIONS
- Test substance-related clinical observations noted in the 500 and 1000 mg/kg/day group males at the daily examinations were limited to red material around the nose. These observations were noted sporadically during the treatment period, and therefore in the absence of other sign of toxicity the material findings were considered non-adverse. There were no test substance-related clinical observations noted at the daily examinations for F0 females at any dosage level. There were no test substance-related clinical observations noted at the weekly detailed physical examinations or approximately 1 hour following dose administration for males and females at any dosage level. Observations noted in the test substance-treated groups, including hair loss, and scabbing, occurred infrequently, at similar frequencies in the control group, and/or in a manner that was not dose-related. Clinical observations noted during the recovery period in the 1000 mg/kg/day group, including hair loss on various body surfaces and red material around the nose, occurred infrequently and/or at similar frequencies in the control group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

SURVIVAL
- There was no test substance-related effect on survival at any dosage level. Female No. 2303 in the 1000 mg/kg/day group was euthanised in extremis on Lactation Day 0. Clinical observations noted for this female at the daily examinations on the day of euthanasia included prostrate posture, decreased respiration rate, clear discharge from both eyes, and yellow material in the urogenital area. Female No. 2303 delivered 2 viable pups prior to euthanasia and was noted with 12 viable foetuses in utero. Test substance-related minimal hepatocellular hypertrophy was noted in this female; the finding was considered adaptive and non-adverse. In the 500 mg/kg/day group, Female No. 2338 was euthanised in extremis on Gestation Day 20. Clinical observations noted for this female at the daily examinations on the day of euthanasia included decreased defecation, hypoactivity, splayed hind limbs, and a cool body. Macroscopic findings noted for Female No. 2338 included depressed areas on the kidneys and dark red areas on the stomach; this female had 16 viable foetuses in utero. No test substance-related macroscopic findings were noted in either female that was euthanised in extremis. The moribundity at 500 and 1000 mg/kg/day was attributed to dystocia that was not considered test substance-related because it was limited to 1 female in each group, both females were noted with viable foetuses, and there was no effect on the mean gestation lengths in either group. All other animals survived to the scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

BODY WEIGHTS
- Males: Mean body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day group males were unaffected by test substance administration throughout the study. None of the differences from the control group were statistically significant.
- Females: Mean body weights and body weight gains in the 100, 300, 500, and 1000 mg/kg/day group females were unaffected by test substance administration during the pre-mating period and during the entire dosing and recovery periods. None of the differences from the control group were statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

FOOD CONSUMPTION
- Males: Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, 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 (weekly): Mean food consumption, evaluated as g/animal/day, in the 100, 300, 500, and 1000 mg/kg/day group females was unaffected by test substance administration during the pre-mating period and during the entire dosing and recovery periods. 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):

HAEMATOLOGY
- Haematology and coagulation: There were no test substance-related effects on haematology and coagulation parameters. Significantly (p < 0.05) higher mean percent and absolute large unstained cells (LUC) values were noted in the 1000 mg/kg/day group males on Study Day 42 compared to the control group. On Lactation Day 14, a higher mean absolute LUC value was noted in the 300 mg/kg/day group females; the difference from the control group was significant (p < 0.05). Significantly (p < 0.05) higher mean red cell distribution width and hemoglobin distribution width were noted in the 1000 mg/kg/day group females on Study Day 63. The aforementioned differences did not occur in a dose-related manner, were only observed in 1 sex, and/or were of minimal magnitude; therefore, the alterations in haematology and coagulation parameters were not considered test substance-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

CLINICAL BIOCHEMISTRY
- Serum chemistry: There were no test substance-related effects on serum chemistry. A significantly (p < 0.01) higher mean cholesterol value was noted in the 1000 mg/kg/day group males on Study Day 42 compared to the control group. On Lactation Day 14, a significantly (p < 0.05) higher phosphorus value was noted in the 500 mg/kg/day group females compared to the control group. Significantly (p < 0.05) lower albumin and albumin/globulin ratio values were noted in the 1000 mg/kg/day group females on Study Day 63. The alterations in serum chemistry parameters were not observed in a dose-related manner, were only observed in 1 sex, and/or were of minimal magnitude, and therefore were considered the result of individual animal variation and not test substance-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
- Home cage observations: Home cage parameters were unaffected by test substance administration. Differences between the test substance-treated groups and the control group during Study Week 3 (males) or on Lactation Day 13 (females) were slight and not statistically significant, with the following exceptions. During the Study Week 3 home cage observations, a significantly (p < 0.05) higher number of males in the 500 mg/kg/day group were sitting or standing normally. On Lactation Day 13, a higher number of females in the 1000 mg/kg/day group were noted with fecal pellets and consequently, a lower number of females in this group were noted without fecal pellets; differences from the control group were significant (p < 0.05). These findings were considered normal home cage observations for rats, and therefore were not test substance-related.
- Handling observations: Handling parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 3 (males) or on Lactation Day 13 (females).
- Open field observations: Open field parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 3 (males) or on Lactation Day 13 (females).
- Sensory observations: Sensory parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 3 (males) or on Lactation Day 13 (females).
- Neuromuscular observations: Neuromuscular parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 3 (males) or on Lactation Day 13 (females).
- Physiological observations: Physiological parameters were unaffected by test substance administration. There were no statistically significant differences for the test substance-treated groups when compared to the control group during Study Week 3 (males) or on Lactation Day 13 (females).

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

ORGAN WEIGHTS
- Test item-related higher mean liver weights (absolute and relative to brain and final body weights) were noted in the 300, 500, and 1000 mg/kg/day group males at the primary necropsy (PN), with dose-response relationship; absolute weights were 11.1 %, 14.4 % and 24.6 % higher than the control group mean, respectively. Higher liver weights reached statistical significance in the 300 (relative to final body weight), 500 (relative to final body and brain weights) and 1000 (absolute and relative to final body and brain weights) mg/kg/day group males. Higher mean liver weights were additionally noted in the 1000 mg/kg/day group females, with statistically significantly higher liver weight relative to body weight; absolute weight was 13.4 % higher than the control group mean. Higher liver weights correlated microscopically with hepatocellular hypertrophy in the 1000 mg/kg/day groups; the finding was considered adaptive and non-adverse.
- There were no other test item-related alterations in organ weights at the primary necropsy.
- No test item-related organ weight alterations were noted at the recovery necropsy.
- Mean liver weights in the 1000 mg/kg/day group males and females were similar to the control group (6.2 % and 8.5 % higher than the control group, respectively, consistent with recovery). However, statistically significantly lower mean pituitary gland weights (absolute and relative to body and brain weights) were noted in the 1000 mg/kg/day group females. Lower weights were not noted at the primary necropsy, and weights were within the historical control database range; the finding was attributed to biological variability.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

MACROSCOPIC EXAMINATIONS
- There were no unscheduled deaths attributed to administration of test item. However, two test item-treated group females (500 mg/kg/day group Female No. 2338 and 1000 mg/kg/day group Female No. 2303) were euthanised in extremis (EE) on Gestation Day 20 and Lactation Day 0, respectively; the moribundity was attributed to dystocia in each of the EE females. No test item-related macroscopic findings were noted in either EE female. Live foetuses with no apparent malformations were observed in each EE female.
- Test item-related minimal hepatocellular hypertrophy was noted in the 1000 mg/kg/day group female (No. 2303); the finding was considered adaptive and non-adverse.
- Review of the gross necropsy observations at the scheduled necropsies revealed no observations that were considered to be associated with administration of test item. The mean numbers of implantation sites and unaccounted for sites in the 100, 300, 500, 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 substance administration at all dosage levels when evaluated on Study Week 3 (males) or Lactation Day 13 (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 Charles River Ashland historical control data. Differences from the control group were slight, not statistically significant when analysed by a repeated measures analysis, within the Charles River Ashland 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 substance-treated groups when the F0 animals were evaluated at Study Week 3 (males) or Lactation Day 13 (females).

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

MICROSCOPIC EXAMINATIONS
- Test substance-related hepatocellular hypertrophy was noted in the 1000 mg/kg/day groups at the primary necropsy. The finding was noted in two 1000 mg/kg/day group males (Nos. 2244 and
2252) and a single 1000 mg/kg/day group female (No. 2341), and correlated with higher individual liver weights. Hepatocellular hypertrophy was considered adaptive, and non-adverse.
- There were no other test item-related histologic changes. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than administration of the test substance. There was no test substance related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

ANALYSES OF DOSING FORMULATIONS
- Results of the analyses of dosing formulations are summarised in the table attached.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse toxic effects at any dosage level

Key result

Critical effects observed:

no

Conclusions:

Under the conditions of the screening study, no test substance-related effects were observed on reproductive performance at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. There were no adverse test substance-related clinical observations or effects on mean body weights, body weight gains, and food consumption noted at any dosage level. Furthermore, there were no adverse effects on organ weights, clinical pathology parameters, thyroid hormones (males only) or macroscopic/microscopic examination in the F0 males and females at any dosage level. Therefore, the NOAEL for F0 systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for F1 neonatal toxicity was 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.

Executive summary:

GUIDELINE

The objectives of the study were to investigate the potential toxic effects of the test substance when administered to rats for a minimum of 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behaviour, and conception through day 13 of postnatal life. The protocol was designed in general accordance with the OECD Guideline for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016.

 

METHODS

The test substance, 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 100, 300, 500 and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Males and females were approximately 10 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through Lactation Day 13 for a total of 49-54 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39–40 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 28 doses for males and 49 doses for females, these animals were assigned to a 15-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 13. All F0 females selected for pairing were allowed to deliver and rear their pups until Lactation Day 13. F1 clinical observations, body weights, and sexes were recorded at appropriate intervals and anogenital distance was recorded on PND 1. To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4; blood samples for possible thyroid hormone analysis were collected from the culled pups (2 pups/litter). All F1 male pups were evaluated for areolae/nipple anlagen on PND 13. Remaining F1 pups were euthanised on PND 13; blood samples for thyroid hormone analysis were collected and selected organs were weighed from 1 pup/sex/litter. Clinical pathology evaluations (haematology, coagulation, and serum chemistry) were performed on 5 F0 animals/sex/group at the primary necropsy and 5 animals/sex in the control and high-dose groups at the recovery necropsy. Blood samples for thyroid hormone analysis were collected from F0 males and females at the primary (Lactation Day 14 for females) and recovery necropsies; only primary necropsy male samples were analysed. F0 males were euthanised following completion of the mating period or 15-day recovery period and F0 females were euthanised on Lactation Day 14 for females that delivered, post-mating day 25 for females that failed to deliver, or following the 15-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 euthanised in extremis and all F0 animals in the control and high-dose groups at the primary necropsy.

 

RESULTS

There was no test substance-related effect on survival at any dosage level. One female each in the 500 and 1000 mg/kg/day groups were euthanised in extremis on Gestation Day 20 and Lactation Day 0 (after delivering 2 viable pups), respectively. Clinical observations noted for these females at the daily examinations on the day of euthanasia included prostrate posture, decreased respiration rate, clear discharge from both eyes, yellow material in the urogenital area, decreased defecation, hypoactivity, splayed hind limbs, and/or a cool body. No test substance-related macroscopic findings were noted in either female that was euthanised in extremis and both were noted with live foetuses with no apparent malformations in utero. Test substance-related minimal hepatocellular hypertrophy was noted in the 1000 mg/kg/day group female and was considered adaptive and non-adverse. The moribundity at 500 and 1000 mg/kg/day was attributed to dystocia that was not considered test substance-related because it was limited to 1 female in each group, both females were noted with viable foetuses and/or pups, and there was no effect on the mean gestation lengths in either group. All other animals survived to the scheduled necropsy. Test substance-related clinical observations noted for males in the 500 and 1000 mg/kg/day groups consisted of red material around the nose at the daily examinations. Although these observations were dose-responsive, they were sporadic, and occurred in the absence of other signs of systemic toxicity (body weight and food consumption decrements), and therefore were considered non-adverse. There were no other test substance-related clinical observations at the daily examinations, detailed physical examinations, or approximately 1 hour following dose administration at any dosage level.

 

No test substance-related effects on F0 body weight and food consumption parameters were noted for males and females at any dosage level.

 

No test substance-related effects were noted during the FOB or motor activity evaluations at any

dosage level in the F0 generation.

 

F0 gestation length, oestrous cycle length, reproductive performance (mating, fertility, and copulation/conception indices), and the process of parturition in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by test substance administration.

 

Test substance-related hepatocellular hypertrophy was noted in the 1000 mg/kg/day group (2 males and 1 female) at the primary necropsy; the finding correlated with higher (24.6 % for males and 13.4 % for females) liver weights. Higher liver weights were also noted in the 300 and 500 mg/kg/day group males. Hepatocellular hypertrophy was additionally noted in one 1000 mg/kg/day group female that was euthanised in extremis on Lactation Day 0. Hepatic findings were consistent with test substance-related hepatocellular microsomal enzyme induction, and were considered adaptive and non-adverse.1 Liver weights were similar to the control groups at the recovery necropsy. The similarity of liver weights in control and high-dose recovery animals is consistent with reversibility, and supports the interpretation of adaptive change. There were no other test substance-related changes in organ weights or microscopic findings, and no test substance-related macroscopic observations were noted at the scheduled necropsies.

 

There were no test substance-related alterations in haematology, coagulation, and serum chemistry parameters for F0 males and females. T4 levels in F0 males were unaffected by test substance administration.

 

Mean numbers of 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 100, 300, 500, and 1000 mg/kg/day groups were unaffected by parental test substance administration. There were no test substance-related effects noted on F1 anogenital distance or areolae/nipple anlagen (males only) in the 100, 300, 500, and 1000 mg/kg/day groups. No necropsy findings were noted for F1 pups that were euthanised on PND 13. There were no test substance-related changes in mean serum T4 levels or mean thyroid/parathyroid weights in F1 males or females on PND 13.

 

CONCLUSION

Under the conditions of the screening study, no test substance-related effects were observed on reproductive performance at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. There were no adverse test substance-related clinical observations or effects on mean body weights, body weight gains, and food consumption noted at any dosage level. Furthermore, there were no adverse effects on organ weights, clinical pathology parameters, thyroid hormones (males only) or macroscopic/microscopic examination in the F0 males and females at any dosage level. Therefore, the NOAEL for F0 systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for F1 neonatal toxicity was 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral route

Range-finding study

In a supporting 14-day non-GLP range-finding study,no effects on survival, body weight, body weight gains, food consumption, organ weight and necropsy observations were noted for males and females in the 100, 300, 500 and 1000 mg/kg bw/day groups. Therefore dosage levels of 100, 300, 500 and 1000 mg/kg bw/day were selected for a combined 28-day repeated dose oral toxicity study with reproduction/developmental toxicity screening in the rat.

Key study

The objectives of the key study were to investigate the potential toxic effects of the test substance when administered to rats for a minimum of 28 days and to evaluate the potential of the test substance to affect male and female reproductive performance such as gonadal function, mating behaviour, and conception through day 13 of postnatal life. The protocol was designed in general accordance with the OECD Guideline for Testing of Chemicals, Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, 29 Jul 2016.

The test substance, 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 100, 300, 500, and 1000 mg/kg/day. A concurrent control group of 15 rats/sex received the vehicle on a comparable regimen. The dose volume was 5 mL/kg for all groups. Males and females were approximately 10 weeks of age at the beginning of test substance administration. Males received 14 daily doses prior to mating. Males were dosed throughout the mating period through 1 day prior to euthanasia for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through Lactation Day 13 for a total of 49-54 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 39–40 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 28 doses for males and 49 doses for females, these animals were assigned to a 15-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 13. All F0 females selected for pairing were allowed to deliver and rear their pups until Lactation Day 13. F1 clinical observations, body weights, and sexes were recorded at appropriate intervals and anogenital distance was recorded on PND 1. To reduce variability among the litters, 8 pups/litter, 4 pups/sex when possible, were randomly selected on PND 4; blood samples for possible thyroid hormone analysis were collected from the culled pups (2 pups/litter). All F1 male pups were evaluated for areolae/nipple anlagen on PND 13. Remaining F1 pups were euthanised on PND 13; blood samples for thyroid hormone analysis were collected and selected organs were weighed from 1 pup/sex/litter. Clinical pathology evaluations (haematology, coagulation, and serum chemistry) were performed on 5 F0 animals/sex/group at the primary necropsy and 5 animals/sex in the control and high-dose groups at the recovery necropsy. Blood samples for thyroid hormone analysis were collected from F0 males and females at the primary (Lactation Day 14 for females) and recovery necropsies; only primary necropsy male samples were analysed. F0 males were euthanised following completion of the mating period or 15-day recovery period and F0 females were euthanised on Lactation Day 14 for females that delivered, post-mating day 25 for females that failed to deliver, or following the 15-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 euthanised in extremis and all F0 animals in the control and high-dose groups at the primary necropsy.

There was no test substance-related effect on survival at any dosage level. One female each in the 500 and 1000 mg/kg/day groups were euthanised in extremis on Gestation Day 20 and Lactation Day 0 (after delivering 2 viable pups), respectively. Clinical observations noted for these females at the daily examinations on the day of euthanasia included prostrate posture, decreased respiration rate, clear discharge from both eyes, yellow material in the urogenital area, decreased defecation, hypoactivity, splayed hind limbs, and/or a cool body. No test substance-related macroscopic findings were noted in either female that was euthanised in extremis and both were noted with live foetuses with no apparent malformations in utero. Test substance-related minimal hepatocellular hypertrophy was noted in the 1000 mg/kg/day group female and was considered adaptive and non-adverse. The moribundity at 500 and 1000 mg/kg/day was attributed to dystocia that was not considered test substance-related because it was limited to 1 female in each group, both females were noted with viable foetuses and/or pups, and there was no effect on the mean gestation lengths in either group.

All other animals survived to the scheduled necropsy. Test substance-related clinical observations noted for males in the 500 and 1000 mg/kg/day groups consisted of red material around the nose at the daily examinations. Although these observations were dose-responsive, they were sporadic, and occurred in the absence of other signs of systemic toxicity (body weight and food consumption decrements), and therefore were considered non-adverse. There were no other test substance-related clinical observations at the daily examinations, detailed physical examinations, or approximately 1 hour following dose administration at any dosage level.

 

No test substance-related effects on F0 body weight and food consumption parameters were noted for males and females at any dosage level.

No test substance-related effects were noted during the FOB or motor activity evaluations at any dosage level in the F0 generation.

 

F0 gestation length, oestrous cycle length, reproductive performance (mating, fertility, and copulation/conception indices), and the process of parturition in the 100, 300, 500, and 1000 mg/kg/day groups were unaffected by test substance administration.

Test substance-related hepatocellular hypertrophy was noted in the 1000 mg/kg/day group (2 males and 1 female) at the primary necropsy; the finding correlated with higher (24.6 % for males and 13.4 % for females) liver weights. Higher liver weights were also noted in the 300 and 500 mg/kg/day group males. Hepatocellular hypertrophy was additionally noted in one 1000 mg/kg/day group female that was euthanised in extremis on Lactation Day 0. Hepatic findings were consistent with test substance-related hepatocellular microsomal enzyme induction, and were considered adaptive and non-adverse.1 Liver weights were similar to the control groups at the recovery necropsy. The similarity of liver weights in control and high-dose recovery animals is consistent with reversibility, and supports the interpretation of adaptive change. There were no other test substance-related changes in organ weights or microscopic findings, and no test substance-related macroscopic observations were noted at the scheduled necropsies.

There were no test substance-related alterations in haematology, coagulation, and serum chemistry parameters for F0 males and females. T4 levels in F0 males were unaffected by test substance administration.

 

Mean numbers of 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 100, 300, 500, and 1000 mg/kg/day groups were unaffected by parental test substance administration. There were no test substance-related effects noted on F1 anogenital distance or areolae/nipple anlagen (males only) in the 100, 300, 500, and 1000 mg/kg/day groups. No necropsy findings were noted for F1 pups that were euthanised on PND 13. There were no test substance-related changes in mean serum T4 levels or mean thyroid/parathyroid weights in F1 males or females on PND 13.

Under the conditions of the screening study, no test substance-related effects were observed on reproductive performance at any dosage level. Therefore, a dosage level of 1000 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for F0 reproductive toxicity of test item when administered orally by gavage to Crl:CD(SD) rats. There were no adverse test substance-related clinical observations or effects on mean body weights, body weight gains, and food consumption noted at any dosage level. Furthermore, there were no adverse effects on organ weights, clinical pathology parameters, thyroid hormones (males only) or macroscopic/microscopic examination in the F0 males and females at any dosage level. Therefore, the NOAEL for F0 systemic toxicity was considered to be 1000 mg/kg/day. The NOAEL for F1 neonatal toxicity was 1000 mg/kg/day based on the absence of effects on F1 offspring at all dosage levels.

Dermal route

According to REACH, Annex VIII, Section 8.6.1, short-term repeated dose toxicity should be investigated using one species and the most likely route of human exposure. However, no evidence of systemic toxicity was demonstrated during an acute study via the dermal route and, in order to minimise the number of vertebrate animals tested, oral dosing by gavage was considered most appropriate for a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) in rats.

Inhalation route

According to REACH, Annex VIII, Section 8.6.1, short-term repeated dose toxicity should be investigated using one species and the most likely route of human exposure. The substance is a liquid with high onset boiling point (286 °C at 101 kPa) and a determined vapour pressure of 8.46 x 10E-03 Pa at 25 °C. It is therefore expected that inhalation exposure will be low under general use conditions at ambient temperature. Lack of systemic toxicity when the substance is administered via the oral and dermal routes suggests that determined vapour pressures of 1.19 x 10E-02 Pa at 40 °C and 6.81 x 10E-02 Pa at 100 °C also give no cause for concern. The inhalation route is therefore not the most applicable method of investigating repeated dose toxicity.

Justification for classification or non-classification

No treatment-related effects were reported at dose levels of 100, 300, 500 and 1000 mg/kg bw (OECD 422) and classification for Specific Target Organ Toxicity (STOT RE category 1 or 2) is not required under the terms of Regulation (EC) No 1272/2008 and subsequent amendments.