Registration Dossier

Administrative data

Description of key information

Under the conditions of the screening study, based on the lack of adverse effects at any dosage level, 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 F0 systemic toxicity when test item was administered orally by gavage to Crl:CD(SD) rats (OECD 422).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 April 2017 to 03 January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
various (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 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, 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 [Groups 2 to 4] rats/sex/group) 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 12–13 females/group. Females were evaluated for oestrous cyclicity during the pre-test period, and any female that failed to exhibit normal 4–5 day oestrous cycles (e.g., EDDDE), repeatedly during the pre-test period, was 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 test substance-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 (65 males and 75 females) were received in good health from Charles River Laboratories, Inc., Raleigh, NC on 25 Apr 2017. The animals were approximately 47 days old upon receipt. Each animal was examined by a qualified technician 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 microchip (BMDS system) which was implanted subcutaneously in the dorsoscapular region during the acclimation period. The animals were housed for an acclimation period of 23 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 rats/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 until the scheduled necropsy. Following positive evidence of mating, the females were individually housed until euthanasia on Lactation Day 14. Females with no evidence of mating or that failed to deliver were individually housed until Post-cohabitation 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 with bedding material until euthanasia.
- Animals were maintained in accordance with the Guide for the Care and Use of Laboratory
Animals. The animal facilities at Charles River Ashland are accredited by AAALAC International. Enrichment devices were provided to all animals as appropriate throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were sanitised 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 sanitised once per week (see Appendix 1; Deviations to study protocol).
- 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, with the following exception. All males and females (including those 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 °F to 78 °F (20 °C 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. Actual mean daily temperature ranged from 72.2 °F to 72.6 °F (22.3 °C to 22.6 °C) and mean daily relative humidity ranged from 37.8 % to 57.5 % during the study.
- Fluorescent lighting provided illumination for a 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod (see Appendix 1; Deviations to study protocol). 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, meeting acceptable body weight requirements, and exhibiting normal 4 to 5 day oestrous cycles (females) was selected for use in the computerised 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 or euthanised by carbon dioxide inhalation and discarded.
- 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 (see Appendix 1; Deviations to study protocol).
- At the initiation of dose administration (Study Day 0), the males and females were approximately 10 weeks old. Male body weights ranged from 319 g to 422 g and female body weights ranged from 186 g to 258 g on Study Day 0 (see Appendix 1; Deviations to study protocol). The animals were approximately 12 weeks old when paired on Study Day 13 (see Appendix 1; Deviations to study protocol); female body weights ranged from 202 g to 289 g on Gestation Day 0.
Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Lot numbers 1GB0631 (expiry date 31 January 2018) and 2GE0196 (expiry date 31 March 2018)
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 °C to 24 °C), protected from light. The vehicle was mixed throughout the preparation, sampling, and dose administration procedures.
- The test substance formulations were prepared as shown in the table below. Prior to formulation, the test substance was melted in an incubator set to approximately 60 °C. The test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature (18 °C to 24 °C), protected from light. On each day of dose administration, dosing formulation aliquots from each concentration were placed in a water bath set at approximately 35 °C prior to dosing. 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.

TEST GROUPS, DOSAGE LEVELS AND TREATMENT REGIMEN
- Study group assignment is shown in the table below.
- The vehicle and test substance formulations were administered orally by gavage, via appropriately sized flexible, disposable, plastic feeding tubes (Instech Laboratories, Inc., 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 49-63 doses.
- Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Post-mating or Post-cohabitation Day 25) for a total of 41 or 52 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
SAMPLING AND ANALYSIS
- Test substance formulations had been previously shown to be stable at concentrations of 50 and 200 mg/mL for 11 days at room temperature. Therefore, stability of test substance formulations
was not assessed on this study.
- Samples for homogeneity and/or concentration determination were collected from the top, middle, and bottom strata of the first 70, 150, and 200 mg/mL dosing formulations and from the middle stratum of the first control and 100 mg/mL dosing formulations.
- In addition, samples for re-suspension homogeneity were collected from the top and bottom strata of an aliquot taken from the first 70, 150, and 200 mg/mL dosing suspensions following room temperature storage for 10 days; aliquots were mixed for a minimum of 30 minutes prior to sample collection.
- Samples for concentration analysis were also collected from the middle stratum of the first, third, and last dosing formulations (including the control group) prepared during the study.
- 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 ultraviolet absorbance detection.
Duration of treatment / exposure:
- Males received 14 daily doses prior to mating and 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 49–63 doses.
- Females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Postcohabitation Day 25 or Postmating Day 25, respectively) for a total of 41 or 52 doses.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
350 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
- 10 males and 10 females (350, 500 and 750 mg/kg bw/day groups)
- 15 males and 15 females (vehicle control and 1000 mg/kg bw/day groups)
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 computerised 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.
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 detailed physical examinations were conducted weekly (prior to dose administration during the treatment period) (see Appendix 1; Deviations to study protocol).
- Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration (see Appendix 1; Deviations to study protocol).
- 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. 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).
- Weekly body weights for females with no evidence of mating were presented on the individual report tables until necropsy.
- 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
- 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 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. 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 a weighing error, scheduled euthanasia, 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
- All animals were observed for the parameters listed in the table below.
- FOB assessments were recorded for 5 animals/sex/group during the last week of dose administration (Study Day 27, males selected for pairing) or on Lactation Day 13 (females).
- The FOB used at Charles River is based on previously developed protocols.4-9 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 60 dB to 80 dB.
- 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 anima’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 during the last week of dose administration (Study Day 27, 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 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 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).

CLINICAL PATHOLOGY
- Parameters listed in the tables below were evaluated.
- Blood samples for clinical pathology evaluations (haematology, coagulation, and serum chemistry) were collected from 5 animals/sex/group at the scheduled necropsies (Study Day 28 for males and Lactation Day 14 for females selected for pairing) and from all males in the control and high-dose groups at the recovery necropsy (following a 15-day recovery period; Study Day 42).
- The animals were fasted overnight prior to blood collection.
- Blood for serum chemistry and haematology was collected via the jugular vein using the hand-held restraint method for males and via the retro-orbital sinus following isoflurane anesthesia for females (see Appendix 1; Deviations to study protocol).
- 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).

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 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 possible future thyroid hormone analysis on the day prior to euthanasia.
- Females that failed to deliver were euthanised on Post-mating Day 25 (females with evidence of mating) or Post-cohabitation Day 25 (females with no 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.
- Females with total litter loss were euthanised within 24 hours of litter 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 (see Appendix 1; Deviations to study protocol).
- 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.
- At the time of necropsy, tissues and organs were placed in 10% neutral-buffered formalin unless otherwise noted.

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.

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.
- Gross lesions were examined from all animals in all groups. 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 (see Appendix 1; Deviations to study protocol).
Sacrifice and pathology:
SCHEDULED EUTHANASIA
- On PND 13, surviving F1 rats were euthanised via an intraperitoneal injection of sodium pentobarbital.
- Blood samples were collected for thyroid hormone analysis immediately prior to euthanasia from 1 pup/sex/litter; gross necropsies were conducted and the thyroids (with parathyroids, if present) were placed in 10% neutral-buffered formalin for possible histopathological examination. Remaining pups (not used for blood collection) were discarded without examination.
Statistics:
See below
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Noteworthy clinical observations were noted approximately 1 hour after dose administration, and included clear and/or red material around the nose and/or mouth and red material around the nose in the in the 500, 750, and 1000 mg/kg/day group males and the 750 and 1000 mg/kg/day group females. The red material findings were also noted at the weekly detailed physical examinations primarily for males and females in the 750 and 1000 mg/kg/day groups, albeit to a much lesser extent than noted during the postdosing period. These observations were noted sporadically throughout the treatment period, but were considered non-adverse in the absence of other signs of toxicity at these dosage levels and the lack of persistence to the daily examinations the following day.
- There were no other noteworthy clinical observations noted at the weekly detailed physical examinations, daily examinations, or approximately 1 hour after dose administration for males or females at any dosage level. Observations noted in the treated groups, including hair loss on various body surfaces and yellow material around the urogenital area 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):
- There were no effects on survival at any dosage level. Female No. 6380 in the 750 mg/kg/day group was euthanised due to a total litter loss on Lactation Day 0. The nesting box that contained this female was found to be backwards in the cage rack, which resulted in no access to water overnight during the process of parturition. This female was noted with red material around the eyes, mouth, and nose for 2 days prior to and including the day of euthanasia at the
detailed physical examinations and/or approximately 1 hour after dose administration. In addition, a body weight loss (44 g) and reduced food consumption (6 g/day) were noted for this female during Gestation Days 17–20. All other F0 males and females survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Males: Mean body weights and body weight gains in the 350, 500, 750, and 1000 mg/kg/day group males were unaffected throughout the study. Significantly (p < 0.05 or p < 0.01) lower mean body weight gains compared to the control group were noted in the 500 mg/kg/day group males during Study Days 7–13 and the 750 mg/kg/day group males during Study Days 0–7 and when the entire pre-mating dosing period (Study Days 0–13) was evaluated; however, the changes were transient, did not occur in a dose-responsive manner, and/or were not sufficient magnitude to affect absolute mean body weights. No other statistically significant differences were noted at any dosage level.
- Females (weekly): Mean body weights and body weight gains in the 350, 500, 750, and 1000 mg/kg/day group females were unaffected throughout the study. A significantly (p < 0.05) higher mean body weight gain was noted in the 750 mg/kg/day group females during Study Days 0–7; however, the change was transient, did not occur in a dose-responsive manner, and was not of sufficient magnitude to affect absolute mean body weights. No other statistically significant differences were noted at any dosage level.
- Females (gestation): Mean body weights and body weight gains in the 350, 500, 750, and 1000 mg/kg/day groups were unaffected during gestation. None of the differences from the control group were statistically significant.
- Females (lactation): Mean body weights and body weight gains in the 350, 500, 750, and 1000 mg/kg/day groups were unaffected during lactation. None of the differences from the control group were statistically significant.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Males: Mean food consumption, evaluated as g/animal/day, in the 350, 500, 750, and 1000 mg/kg/day group males was unaffected throughout the study. None of the differences from the control group were statistically significant.
- Females (weekly): Mean food consumption, evaluated as g/animal/day, in the 350, 500, 750, and 1000 mg/kg/day group females was unaffected throughout the study. None of the differences from the control group were statistically significant.
- Females (gestation): Mean maternal food consumption, evaluated as g/animal/day, in the 350, 500, 750, and 1000 mg/kg/day groups was unaffected during gestation. Significantly (p < 0.05) higher mean food consumption was noted for the 1000 mg/kg/day group compared to the control group during Gestation Days 11–14 and when the entire gestation treatment period (Gestation Days 0–20) was evaluated; however, these transient differences were slight and did not correspond to effects on mean body weight gain at this dosage level. No other statistically significant differences were noted at any dosage level.
- Females (lactation): Mean maternal food consumption, evaluated as g/animal/day, in the 350, 500, 750, and 1000 mg/kg/day groups was unaffected during lactation. Significantly (p < 0.01) higher mean food consumption was noted for the 1000 mg/kg/day group compared to the control group during Lactation Days 4–7 and when the entire gestation period (Lactation Days 1–13) was evaluated; however these transient differences were slight and did not correspond to effects on mean body weight gain at this dosage level. No other statistically significant differences were noted at any dosage level.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- Significantly (p < 0.05 or p < 0.01) higher mean percentage of neutrophils and a lower mean percentage of lymphocytes in the 750 and 1000 mg/kg/day group males and a higher mean absolute neutrophil count in the 1000 mg/kg/day group males were noted compared to the control group.
- The higher mean percentage of neutrophils, lower mean percentage of lymphocytes, and higher absolute neutrophil count were considered non-adverse because there was no correlation to changes in organ weights or pathology data, and the changes were not noted for the 1000 mg/kg/day group during the recovery period.
- No other effects on haematology and coagulation parameters were noted for the males during the dosing and recovery periods or for the females during the dosing period.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- In the 350, 500, 750, and 1000 mg/kg/day group males, higher mean alanine aminotransferase (ALT) levels (57, 82, 75, and 84 U/L, respectively) was noted compared to the concurrent control group (45 U/L). Although the difference was only significant (p < 0.05) in the 1000 mg/kg/day group, the values in all treated groups exceeded the maximum mean value in the Charles River Ashland historical control data (52 U/L; version 3.6).
- These differences were considered non-adverse because there was no correlation to changes in organ weights or pathology data and the ALT level in the 1000 mg/kg/day recovery male group (47 U/L) was similar to the concurrent control recovery male group (53 U/L). No other effects on serum chemistry were noted for the males during the dosing and recovery periods, or for females during the dosing period.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Home cage parameters were unaffected. There were no statistically significant differences for the treated groups when compared to the control group on Study Day 27 (males) or on Lactation Day 13 (females).
- Handling parameters were unaffected. There were no statistically significant differences for the treated groups when compared to the control group on Study Day 27 (males) or on Lactation Day 13 (females).
- Open field parameters were unaffected. There were no statistically significant differences for the treated groups when compared to the control group on Study Day 27 (males) or on Lactation Day 13 (females).
- Sensory parameters were unaffected. There were no statistically significant differences for the treated groups when compared to the control group on Study Day 27 (males) or on Lactation Day 13 (females).
- Neuromuscular parameters were unaffected. There were no statistically significant differences for the treated groups when compared to the control group on Study Day 27 (males) or on Lactation Day 13 (females).
- Physiological parameters were unaffected. There were no statistically significant differences for the treated groups when compared to the control group on Study Day 27 (males) or on Lactation Day 13 (females).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- There were no remarkable alterations in organ weights at the scheduled necropsies. However, some significant (p < 0.05) differences were observed when the control and treated groups were compared.
- Higher mean liver weights (relative to final body weight) were noted in the 500 and 1000 mg/kg/day group females at the primary necropsy. Higher mean liver weights lacked a dose-response relationship and had no microscopic correlate.
- In addition, mean liver weights (absolute and relative to final body and brain weights) from control and treated groups were higher than the Charles River Ashland historical control database range.
- Higher mean liver weights (absolute and relative to brain weight) were also noted in the 1000 mg/kg/day group females at the recovery necropsy.
- Higher mean liver weights were attributed to biological variability associated with pregnancy/lactation.
- A higher mean heart weight (relative to final body weight) was noted in the 1000 mg/kg/day group males at the recovery necropsy; however, absolute weight was similar to the control group, the difference was of minimal magnitude, weights were within the Charles River Ashland historical control database range, and a difference in mean heart weights was not noted at the primary necropsy; therefore, the difference in mean heart weight was attributed to biological variability.
- Statistically significant differences in organ weights restricted to low- and mid-dose groups were considered to be due to biological variability because of lack of dose-response relationships and absence of significant differences in high-dose groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
- No remarkable internal findings were observed at any dosage level in females that failed to deliver, the female with total litter loss, or in males and females at the scheduled necropsies.
- Macroscopic findings observed in the 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 350, 500, 750, and 1000 mg/kg/day groups were similar to the control group values.
Neuropathological findings:
no effects observed
Description (incidence and severity):
MOTOR ACTIVITY
- Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected at all concentrations when evaluated on Study Day 27 (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 (version 2016.01).
- 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 treated groups when the F0 animals were evaluated at Study Day 27 or Lactation Day 13.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
- There were no remarkable histologic changes noted at the primary necropsy.
- Histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation. There was no remarkable alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
THYROID HORMONES
- There were no effects on thyroid hormone values in the F0 males at any dosage level.
- A significantly (p < 0.05) lower mean T4 value was noted in the 500 mg/kg/day group males during the dosing period compared to the control group; however, this did not occur in a dose-responsive manner.
- All other treated group male thyroid hormone values were similar to the control group.
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:
clinical signs
Key result
Critical effects observed:
no

ANALYSES OF DOSING FORMULATIONS

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

- The analysed dosing formulations were within Charles River SOP range for suspensions (85 % to 115 %) and were homogeneous. The test substance was not detected in the analysed vehicle formulation that was administered to the control group (Group 1).

Conclusions:
Under the conditions of this screening study, based on the lack of adverse effects at any dosage level, 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 F0 reproductive toxicity, F0 systemic toxicity, and F1 neonatal toxicity of test item when administered orally by gavage to Crl:CD(SD) rats.
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 (corn oil), was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and mid-dose groups (Groups 24) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 350, 500, 750, 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 4963 doses; females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Post-cohabitation Day 25 or Post-mating Day 25, respectively) for a total of 41 or 52 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 during the last week of dose administration (Study Day 27) 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 (at least 2/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 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 at the primary and recovery necropsies and from F0 females on the day of the last dose (Lactation Day 13); only male samples collected at the primary necropsy 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 13 for females that delivered, Post-cohabitation or 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 in the control and high-dose groups at the primary necropsy.

 

RESULTS

There were no effects on survival. Non-adverse findings (clear and/or red material around the nose and/or mouth) were noted throughout the treatment period approximately 1 hour after dose administration, and to a lesser extent at the weekly detailed physical examinations, in the 500, 750, and/or 1000 mg/kg/day group males and/or the 750 and 1000 mg/kg/day group females. No other noteworthy clinical observations were noted at the daily examinations, detailed physical examinations, or approximately 1 hour after dose administration at any dosage level. No effects on F0 body weight and food consumption parameters were noted for males and females at any dosage level. No effects were noted during the FOB or motor activity evaluations at any dosage level in the F0 generation. No effects were noted for F0 gestation length, oestrous cycle length, reproductive performance (mating, fertility, and copulation/conception indices), and the process of parturition at any dosage level. There were no effects on organ weights, or noteworthy macroscopic and microscopic findings for F0 males and females at any dosage level at the primary and/or recovery necropsies.

 

Non-adverse higher mean ALT levels in the 1000 mg/kg/day group males, a higher mean percentage of neutrophils and a lower mean percentage of lymphocytes in the 750 and 1000 mg/kg/day group males, and higher mean absolute neutrophil counts in the 1000 mg/kg/day group males were noted compared to the control group. There were no other noteworthy alterations in serum chemistry, haematology, and coagulation parameters for the males during the dosing or recovery periods or females during the dosing period. Thyroid hormone (T4) levels in F0 males were unaffected.

 

There were no effects on 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 at any dosage level. There were no effects noted on F1 anogenital distance or areolae/nipple anlagen (males only) in the 350, 500, 750, and 1000 mg/kg/day groups. No

necropsy findings were noted for F1 pups that were euthanised on PND 13. There were no remarkable changes in mean serum T4 levels in F1 males or females on PND 13.

 

CONCLUSION

 

Under the conditions of this screening study, based on the lack of adverse effects at any dosage level, 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 F0 reproductive toxicity, F0 systemic toxicity, and F1 neonatal toxicity of test item when administered orally by gavage to Crl:CD(SD) rats.

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

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.

 

The test substance, in the vehicle (corn oil), was administered orally by gavage once daily to 4 groups of Crl:CD(SD) rats. The low- and mid-dose groups (Groups 24) each consisted of 10 rats/sex and the high-dose group (Group 5) consisted of 15 rats/sex. Dosage levels were 350, 500, 750, 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 4963 doses; females with no evidence of mating or that failed to deliver were dosed through the day prior to euthanasia (Post-cohabitation Day 25 or Post-mating Day 25, respectively) for a total of 41 or 52 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 during the last week of dose administration (Study Day 27) 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 (at least 2/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 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 at the primary and recovery necropsies and from F0 females on the day of the last dose (Lactation Day 13); only male samples collected at the primary necropsy 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 13 for females that delivered, Post-cohabitation or 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 in the control and high-dose groups at the primary necropsy.

 

There were no effects on survival. Non-adverse findings (clear and/or red material around the nose and/or mouth) were noted throughout the treatment period approximately 1 hour after dose administration, and to a lesser extent at the weekly detailed physical examinations, in the 500, 750, and/or 1000 mg/kg/day group males and/or the 750 and 1000 mg/kg/day group females. No other noteworthy clinical observations were noted at the daily examinations, detailed physical examinations, or approximately 1 hour after dose administration at any dosage level. No effects on F0 body weight and food consumption parameters were noted for males and females at any dosage level. No effects were noted during the FOB or motor activity evaluations at any dosage level in the F0 generation. No effects were noted for F0 gestation length, oestrous cycle length, reproductive performance (mating, fertility, and copulation/conception indices), and the process of parturition at any dosage level. There were no effects on organ weights, or noteworthy macroscopic and microscopic findings for F0 males and females at any dosage level at the primary and/or recovery necropsies.

 

Non-adverse higher mean ALT levels in the 1000 mg/kg/day group males, a higher mean percentage of neutrophils and a lower mean percentage of lymphocytes in the 750 and 1000 mg/kg/day group males, and higher mean absolute neutrophil counts in the 1000 mg/kg/day group males were noted compared to the control group. There were no other noteworthy alterations in serum chemistry, haematology, and coagulation parameters for the males during the dosing or recovery periods or females during the dosing period. Thyroid hormone (T4) levels in F0 males were unaffected.

 

There were no effects on 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 at any dosage level. There were no effects noted on F1 anogenital distance or areolae/nipple anlagen (males only) in the 350, 500, 750, and 1000 mg/kg/day groups. No

necropsy findings were noted for F1 pups that were euthanised on PND 13. There were no remarkable changes in mean serum T4 levels in F1 males or females on PND 13.

 

Under the conditions of this screening study, based on the lack of adverse effects at any dosage level, 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 F0 reproductive toxicity, F0 systemic toxicity, and F1 neonatal toxicity of test item when administered orally by gavage to Crl:CD(SD) 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 waxy solid with determined vapour pressure of 1.75 x 10E-03 Pa at 25 °C and 1.21 Pa at 154 °C and a high onset boiling point (decomposition from approximately 232 °C at 101 kPa). Based on evaluation of the life cycle of the substance, it is therefore expected that inhalation exposure will be low and that the most likely route of exposure for workers and consumers is the dermal route. A repeated dose toxicity study via the inhalation route is, therefore, not appropriate.

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.

Justification for classification or non-classification

No treatment-related effects were reported at dose levels of 350, 500, 750 and 1000 mg/kg bw (OECD 422). 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.