Registration Dossier

Administrative data

Description of key information

Repeated dose - oral toxicity, OECD 422 screening study: NOAEL for systemic toxicity was considered to be 125 mg/kg/day based on lower body weights, body weight gains, and reduced food consumption in the 300 mg/kg/day group males and females. The NOAEL for neonatal toxicity was 50 mg/kg/day based on lower pup body weights and body weight gains at 125 and/or 300 mg/kg/day.

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:
2016-05-16 - 2017-02-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
Deviations did not impact the quality or integrity of the data or the outcome of the study.
Deviations:
yes
Remarks:
Temp.during stirring; parturition surveillance checks day 36; F0 males remained 15 d without treatment; morning parturition check not performed for No. 6317 (control, day 37); 2 bw were recorded for all F0 m.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Details on species / strain selection:
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 has reproductive historical control data in the Crl:CD(SD) rat.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, received on 17 May 2016
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: ~ 10 weeks
- Weight at study initiation: male: 310 g to 402 g
female: 195 g to 249 g
- Housing: F0 animals: 2-3 per cage by sex in clean, solid-bottom cages with bedding material
after breeding period: males were individually housed in solid-bottom cages
after mating: females were individually housed in solid-bottom cages with bedding material.
Control group: remained housed in groups of 2-3 in clean solid-bottom cages
- Diet (e.g. ad libitum): ad libitum (exception: All F0 males and females (including animals not selected for clinical pathology evaluation) were fasted overnight prior to clinical pathology blood collection when food was withheld.)
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY: 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. Municipal water supplying the facility was sampled for contaminants according to Charles River SOPs. No contaminants were present in animal feed or water at concentrations sufficient to interfere with the objectives of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72.2°F to 72.8°F (22.3°C to 22.7°C)
- Humidity (%): 40.6% to 58.0%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12-hour light (0600 hours to 1800 hours)/12-hour dark photoperiod.
Route of administration:
oral: gavage
Details on route of administration:
The vehicle and test substance formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula once daily.
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The control and test substance formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature, protected from light. On each day of dosing, aliquots of the control and test substance formulations were heated and stirred in a water bath set at approximately 50°C for a minimum of 30 minutes prior to dispensing and continuously throughout dosing. The control and 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.

VEHICLE
- Concentration in vehicle: 0, 1.5, 5, 12.5, 30 mg/mL
- Amount of vehicle (if gavage): 0, 15, 50, 125, 300 mg/kg/day
- Dose volume: 10 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Homogeneity and stability of the test substance in the vehicle following 7 days of room temperature storage at concentrations ranging from 10.0 to 100 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 and 7th test substance dosing formulations and from the middle stratum of the first and 7th control group dosing formulations. 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: 28 days (28 doses)
females: 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 to 54 doses.
Frequency of treatment:
once daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
vehicle control arachis (peanut) oil
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
As a result, a high dosage level of 300 mg/kg/day was chosen for the current study and was expected to produce signs of toxicity without causing mortality in these animals (prevoius 13-day range-finding study; Herberth, 2016 ).
No. of animals per sex per dose:
15 [Groups 1 and 5] or 10 rats/sex/group [Groups 2 to 4]
Control animals:
yes, concurrent vehicle
yes, historical
Details on study design:
- Dose selection rationale:
Dosage levels were selected based on the results of a previous 13-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. All animals in the 1000 mg/kg/day group were euthanized during the first week of the study due to significant body weight losses. In addition, 1 of 5 males in the 500 mg/kg/day group was also euthanized due to significant body weight losses. All animals in the 100 and 300 mg/kg/day groups survived to the scheduled necropsy. Lower overall mean body weight gains were observed in males and females at 300 mg/kg/day when compared to the control group. As a result, a high dosage level of 300 mg/kg/day was chosen for the current study and was expected to produce signs of toxicity without causing mortality in these animals.
Lower dosage levels were selected to assess the dose response over a wide range of dosage levels and to establish a no-observed-adverse-effect level (NOAEL).
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 this nature.
Positive control:
Yes: A POSITIVE CONTROL VALIDATION STUDY OF LOCOMOTOR ACTIVITY IN RATS, 2007, Beck, J. M.
Haloperidol at dosage levels of 0.05, 0.1, and 0.5 mg/kg
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily (once in the morning, once in the afternoon)

BODY WEIGHT: Yes
- Time schedule for examinations: weekly
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, 1, 4, 7, 10, 13, and 14 (fasted).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes (per cage basis)
- 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; food consumption was reported as g/animal/day during gestation and lactation.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected from 5 animals/sex/group at the scheduled/primary necropsies (Study Week 3 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 14- or 15-day recovery period; Study Day 42 for males and Study Day 62 for females).
- Anaesthetic used for blood collection: Yes (identity)
- Animals fasted: Yes

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected from 5 animals/sex/group at the scheduled/primary necropsies (Study Week 3 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 14- or 15-day recovery period; Study Day 42 for males and Study Day 62 for females).
- Animals fasted: Yes

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Motor activity was assessed 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).
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: Yes (Thyroid Hormone Analysis)
- Blood (at least 1 mL) was collected via the jugular vein from all F0 males and females on the day of scheduled euthanasia (Study Day 28 for males and Lactation Day 14 for females)

OTHER:
Oestrous Cycles:
- Vaginal lavages were performed daily and the slides were evaluated microscopically to determine the stage of the oestrous cycle of each female for 10 days prior to test substance administration and continuing until evidence of copulation was observed (females selected for the breeding phase) or until termination of the mating period (females with no evidence of mating and recovery phase females) and for all females on the day of the scheduled necropsy
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see Table 1)
HISTOPATHOLOGY: Yes (see Table 1)
Other examinations:
Breeding Procedures
Parturition
FOB Assessments
Statistics:
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Statistical analyses were not conducted if the number of animals was 2 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. Statistical analyses were not conducted on F0 weekly female body weight data after 1 or more animals had entered the gestation phase. 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.

Analyses Conducted by BioSTAT Consultants, Inc.:
All analyses were conducted using SAS version 9.2,20 or higher, software.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F0:
Female no. 6325 in the control group was found dead on Study Day 17. No remarkable clinical observations were noted for this female prior to death. The cause of death was attributed to acute pulmonary inflammation and additional pulmonary findings consisted of oedema, haemorrhage, vascular inflammation, and thrombosis. The inciting cause of pulmonary changes was undetermined, but gavage error could not be excluded. All remaining F0 males and females survived to the scheduled necropsies.
Test substance-related clinical observations of clear and/or red material around the mouth and red material around the nose were noted for F0 males and females in the 50, 125, and 300 mg/kg/day groups approximately 1 hour following dose administration generally throughout the dosing period. Although these observations were considered test substance-related, they generally did not persist to the daily examinations or detailed physical examinations and were not considered adverse. Other clinical observations noted in the 15, 50, 125, and 300 mg/kg/day groups at the daily examinations, weekly detailed physical examinations, and 1 hour following dose administration, including hair loss on various body surfaces, cool body, and cool extremities, were noted infrequently and/or in a manner that was not dose-related.
Mortality:
no mortality observed
Description (incidence):
One F0 female in the control group was found dead on Study Day 17; the cause of death was attributed to acute pulmonary inflammation and additional pulmonary findings consisted of oedema, haemorrhage, vascular inflammation, and thrombosis. The inciting cause of the pulmonary changes was undetermined, but a gavage error could not be excluded. All other F0 and F1 males and females survived to the scheduled necropsies.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F0_Males:
Test substance-related lower mean body weight gains were noted for F0 males in the 300 mg/kg/day group throughout the entire study (Study Days 0-28); the
differences were generally significant (p<0.01) compared to the control group. Consequently, mean body weights in this group were significantly (p<0.01) lower
(7.4% to 13.7%) than the control group from Study Days 13 through 27. Despite mean body weight gains in the 300 mg/kg/day group that were generally significantly (p<0.05 or p<0.01) higher than the control group during the recovery period (Study Days 28 to 41), mean body weights in this group remained 9.7% to 15.5%
lower than the control group.
Mean body weights and body weight gains in the 15, 50, and 125 mg/kg/day group F0 males were unaffected by test substance administration throughout the
study. None of the differences from the control group were statistically significant

F0_Females:
Weekly:
Test substance-related lower mean body weight gains were noted for F0 females in the 300 mg/kg/day during the pre-mating period; only the difference for the overall pre-mating period (Study Days 0 to 13) was significant (p<0.05) compared to the control group. Mean F0 female body weights in the 300 mg/kg/day group were similar to the control group during the pre-mating period. Mean body weight losses or lower mean body weight gains were noted for F0 females in the 300 mg/kg/day group that were not paired for mating throughout the remainder of the treatment period (Study Days 13 to 48) and when the entire treatment period (Study Days 0 to 48) was evaluated; the difference for the entire treatment period was significant (p<0.05). During Study Days 49 to 56, a significantly (p<0.05) higher mean body weight gain was noted for the 300 mg/kg/day group F0 females assigned to the recovery period when compared to the control group. During Study Days 56 to 61, a slight mean body weight loss was noted in this group compared to a lack of body weight gain in the control group. As a result of the higher mean body weight gain during the first week of the recovery period, a significantly (p<0.05) higher mean body weight gain was noted for the 300 mg/kg/day group F0 females compared to the control group when the overall recovery period (Study Days 49 to 61) was evaluated. Mean body weights in the 300 mg/kg/day group F0 females not paired for mating were up to 13.3% lower than the control group during Study Days 21 to 49, but similar to the control group by Study Day 61; only the difference on Study Day 42 was significant (p<0.05). Mean body weights and body weight gains in the 15, 50, and 125 mg/kg/day group F0 females were unaffected by test substance administration during the pre-mating period. 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):
F0_Males:
Test substance-related lower mean food consumption, evaluated as g/animal/day, was noted for the 300 mg/kg/day group F0 males throughout the pre-mating period (Study Days 0 to 13). The differences were significant (p<0.01) compared to the control group and corresponded to lower mean body weight gains noted for this group during this period. During the recovery period, mean food consumption in the 300 mg/kg/day group F0 males was similar to the control group.
Mean food consumption in the 15, 50, and 125 mg/kg/day group F0 males was similar to that in the control group throughout the study. No statistically significant differences were observed.

F0_Females:
Weekly:
Test substance-related lower mean food consumption, evaluated as g/animal/day, was noted for the 300 mg/kg/day group F0 females during the pre-mating period (Study Days 0-13). Differences from the control group were significant (p<0.01) during Study Days 7-13 and corresponded to the lower mean body weight gains observed in this group. Mean food consumption in the 300 mg/kg/day group F0 females not paired for mating was similar to the control group during Study Days 13 to 48; differences were slight and not statistically significant. During the recovery period (Study Days 49-61), mean food consumption in the 300 mg/kg/day group females was similar or slightly higher than the control group. Test substance-related, significantly (p<0.05) lower mean food consumption was noted for the 125 mg/kg/day group during Study Days 7-13. However, in the absence of corresponding effects on mean body weights during this interval this difference was not considered adverse. Mean food consumption in the 15 and 50 mg/kg/day group females was unaffected by test substance administration during the pre-mating period.

Gestation:
Mean F0 maternal food consumption, evaluated as g/animal/day, in the 15, 50, 125, and 300 mg/kg/day groups was unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.

Lactation:
Evaluation of mean food consumption in the 300 mg/kg/day group during lactation was precluded by euthanasia of females that delivered by Lactation Day 2.
Mean maternal food consumption, evaluated as g/animal/day, in the 15, 50, and 125 mg/kg/day groups was unaffected by test substance administration during lactation. None of the differences from the control group were statistically significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item-related lower leukocyte and reticulocyte values were noted in the 300 mg/kg/day group males, and lower activated partial thromboplastin time (APTT) values were noted in the 125 and 300 mg/kg/day groups at the primary necropsy. Lower mean leukocyte, neutrophil, lymphocyte, and eosinophil (statistically significant) values were noted in the 300 mg/kg/day group males. Leukocyte, lymphocyte, and eosinophil values were additionally lower than the historical control database range. The finding correlated with decreased cellularity of the sternal bone marrow. Lower leukocyte values were attributed to test item-related stress-response, and were not considered to be a direct toxicologic effect.
Lower absolute and percentage reticulocyte values were noted in the 300 mg/kg/day group males. Values were not statistically significantly different from the control group and were within the historical control database range. Lower values were attributed to test item-related stress-response and associated reduced feed intake. Statistically significantly lower mean APTT values were noted in the 300 mg/kg/day group males and the 125 mg/kg/day group females. Mean values were within the historical control database range in the males, but were lower in the females.
There were no other test item-related effects on haematology or coagulation parameters at the primary necropsy. However, statistically significantly higher mean percentage reticulocyte value was noted in the 125 mg/kg/day group females when compared with the control group. Other erythrocyte parameters were similar to the control group, there were no microscopic correlates, and the difference was attributed to biological variability. Statistically significant findings that involved percentage leukocyte differential counts were not itemized above, and were not considered toxicologically important because absolute cell counts are more relevant for interpretative purposes.
At recovery, leukocyte parameters in the 300 mg/kg/day group males were mildly increased when compared with the dosing period. In contrast to the dosing period, mean absolute and percentage reticulocyte values in the 300 mg/kg/day group males were statistically significantly higher than the control group; the mean red cell distribution width (RDW) value was additionally higher. Mean APTT values in the 300 mg/kg/day group males and 300 mg/kg/day group females were similar to the control groups.
Other statistically significant differences were observed when the control and 300 mg/kg/day groups were compared at the recovery necropsy, including lower mean RBC (males and females), HGB (females), MCHC (females), absolute and percentage reticulocytes (females), higher RDW (females), and lower absolute basophil (males) values. While statistically significantly different from the control groups, values were similar to the dosing period and were thus not considered to be test item-related. Lower absolute neutrophil values were noted in the 300 mg/kg/day group females; values in both the control and 300 mg/kg/day groups were similarly decreased when compared to the primary necropsy, and the difference was not considered to be test item-related.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Test item-related, statistically significantly lower mean serum protein and lipid values and higher mean serum total bilirubin values were noted in the 300 mg/kg/day group males, and higher mean serum phosphorus and bile acid values were noted in the 50 (bile acid) and 125 mg/kg/day group females at the primary necropsy. Higher mean serum bilirubin value noted in the 300 mg/kg/day group males was due to 1 higher individual value (No. 6207), with a microscopic correlate of hepatocellular vacuolation. Higher mean and individual bile acid values were noted in the 50 and 125 mg/kg/day group females.
Group mean and several individual values were additionally higher than the historical control database range. Other serum liver-specific parameters were similar to the control groups. Lower mean serum total protein, albumin, globulin, albumin/globulin (A/G) ratio, and cholesterol values noted in the 300 mg/kg/day group males were attributed to test item-related stress-response and associated lower feed intake/weight loss, and not considered to be a direct toxicologic effect.
Higher mean and individual serum phosphorus values were noted in the 125 mg/kg/day group females. Serum calcium and electrolyte (sodium, potassium, chloride) values were similar to the control group.
There were no test item-related effects on serum chemistry parameters at the recovery necropsy. Test item-related differences noted at the primary necropsy were similar to the control groups at recovery. However, some statistically significant differences were noted when the 300 mg/kg/day group males and 300 mg/kg/day group females were compared to the control groups. Higher mean serum A/G ratio values were noted in the males and females; measured protein values were similar to the control groups and the magnitude difference was minimal. Lower mean serum glucose values (males) and lower mean serum alanine aminotransferase (ALT) values (females) were noted. The findings were not noted at the primary necropsy, were of minimal magnitude difference, and in the case of lower ALT values, were in a direction of no known toxicologic importance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Home cage 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), with the following exceptions. A significantly (p<0.05) lower number of males sitting or standing normally and corresponding higher mean number of males alert and oriented toward the observer were noted for in the 300 mg/kg/day group males during Study Week 3. In the absence of a dose response these differences were not attributed to test substance administration.

Handling parameters, Open field parameters and 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).
Immunological findings:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis (F0 Males):
No test item-related differences in mean serum total thyroxine (T4) values were noted at the primary necropsy.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item-related lower mean final body weight was noted in the 300 mg/kg/day group males at the primary necropsy. Test item-related, statistically significantly lower left and right testes (absolute and relative to brain weights) and epididymides weights (absolute and relative to final body and brain weights) were noted in the 300 mg/kg/day group males. Lower weights correlated microscopically with tubular degeneration and reduced luminal sperm, respectively, and were considered to be a direct toxicologic effect.
Higher adrenal gland weights (relative to final body weight) and lower spleen weights (absolute and relative to brain weight) were noted in the 300 mg/kg/day group males, and lower thymus weights (absolute and relative to final body and brain weights) were noted in the 125 and 300 mg/kg/day group males and females. Lower thymus weights (absolute and relative to brain weight) were also noted in the 15 mg/kg/day group females. Higher adrenal gland and lower spleen and thymus weights were attributed to the test item-related stress-response. Some organ weight differences in the 300 mg/kg/day group males were statistically significant when compared to the control group but were considered to be a result of a test item-related effect on final body weight (lower heart and pituitary [absolute] and higher kidney [relative to final body weight] weights).
Other statistically significant differences were observed at the primary necropsy when the control and test substance-treated groups were compared, but were attributed to biological variability because of lack of dose-response relationship and minimal magnitude difference (lower kidney weights relative to final body weight in the 15 mg/kg/day group males and relative to brain weight in the 50 mg/kg/day group females; higher brain weights [absolute and relative to final body weight] in the 125 mg/kg/day group females; lower liver weights [relative to brain weights] in the 15 and 125 mg/kg/day group females).
At recovery, lower final body weight persisted in the 300 mg/kg/day group males. Test item-related lower epididymides (absolute and relative to brain weights) and testes (absolute and relative to final body and brain weights) weights were noted in the 300 mg/kg/day group males. Some other statistically significant differences in organ weights noted at the recovery necropsy were attributed to test item-related effect on final body weight (higher brain and pituitary [relative to final body weight] and lower kidney and thyroid gland [absolute] weights in the 300 mg/kg/day group males). Other statistically significant differences were not considered to be related to administration of the test item because findings were not noted at the primary necropsy, were of minimal magnitude difference, and/or were attributed to changes in the control group (lower pituitary [absolute and relative to brain weight] and higher thyroid/parathyroid gland [absolute and relative to final body and brain weights] weights in the 300 mg/kg/day group females).
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related unscheduled deaths. One control group female (No.6325) assigned to the recovery phase was found dead on Study Day 17. The cause of death was attributed to acute pulmonary inflammation. Additional pulmonary findings consisted of oedema, haemorrhage, vascular inflammation, and thrombosis. No macroscopic observations were noted. The inciting cause of pulmonary changes was undetermined, but gavage error could not be excluded. Thrombosis was characterized by laminated fibrin expanding a large diameter vessel. Similar pulmonary thrombi were noted in one 15 mg/kg/day and three 300 mg/kg/day group males and one 15 mg/kg/day, two 50 mg/kg/day, and three 125 mg/kg/day group females at the primary necropsy. Other microscopic findings noted in the unscheduled death control group female consisted of moderate hepatocellular necrosis consistent with hypoxic change and mild splenic lymphoid depletion.
Test item-related macroscopic findings at the primary necropsy were noted in the reproductive organs of the 300 mg/kg/day group males, thymus of the 300 mg/kg/day group males and 15, 50 and 300 mg/kg/day group females, and liver of the 50, 125 and 300 mg/kg/day group males and the 300 mg/kg/day group females at the primary necropsy. Small left and right epididymides and soft and small left and right testes noted in the 300 mg/kg/day group males correlated microscopically with reduced luminal sperm (epididymides), and tubular degeneration (testes). Small thymus correlated microscopically with reduced cortical lymphocytes.
Pale liver correlated microscopically with hepatocellular vacuolation. There were no other macroscopic observations considered to be related to administration of the test item at the primary necropsy. However, some macroscopic observations deserve mention. Dark red discoloration and/or areas within the lungs were noted in the 15 and 300 mg/kg/day group males and 50 and 125 mg/kg/day group females at the primary necropsy. Macroscopic observations correlated with large vessel thrombosis. The relationship to administration of the test item is uncertain. Small prostate gland and seminal vesicles were noted in a single 300 mg/kg/day group male (No. 6207); there were no microscopic correlates and the finding was attributed to biological variability. A nodule was noted in the left epididymis of a single 15 mg/kg/day group male, which correlated with sperm granuloma; there was no dose-response relationship and the finding was attributed to spontaneous change. Ovarian cysts were noted in three 300 mg/kg/day group females; the observation correlated microscopically with follicular cyst in 1 female, and was considered to represent a spontaneous finding due to low incidence.
At recovery, test item-related small left and right testes were observed in the 300 mg/kg/day group males, which correlated with degeneration/atrophy of seminiferous tubules. Ovarian considered to be test item-related.
A significant (p<0.01) greater number of mean number of unaccounted-for sites was noted in the 300 mg/kg/day group F0 females (8.0 per dam) compared to the concurrent control group (0.4 per dam). This was consistent with lower mean numbers of pups born and live litter size (see Section 6.3.1.) in this group. The mean number of implantation sites in the 300 mg/kg/day group was similar to the control group value.
Mean numbers of implantation sites and unaccounted-for sites in the 15, 50, and 125 mg/kg/day groups were similar to the control group. Differences were slight and not statistically significant.
Neuropathological findings:
no effects observed
Description (incidence and severity):
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), with the following exception. A significantly (p<0.01) lower mean forelimb grip strength was noted for males in the 300 mg/kg/day group during Week 3. However, the value in the 300 mg/kg/day group was within the range of the Charles River Ashland historical control data (v3.1) and in the absence of any other signs of neurobehavioral toxicity at this dosage level, the difference in forelimb grip strength was not considered to be test substance-related.

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).

Motor Activity:
Motor activity patterns (total activity as well as ambulatory activity counts) in F0 animals were unaffected by test substance administration at all concentrations when evaluated on Study Week 3 (males) and 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 analyzed 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 item-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):
Direct test item-related microscopic findings at the primary necropsy were noted in the epididymides and testes of the 300 mg/kg/day group males and in the liver of the 50, 125, and 300 mg/kg/day group males and a single 300 mg/kg/day group female at the primary necropsy.
Epididymal findings were bilateral with similar magnitude severity grades, and consisted of reduced luminal sperm admixed with cellular debris, compatible with sloughed, degenerate germ cells. Findings were most pronounced in the caput and corpus, with milder changes within the cauda.
Testicular tubular degeneration was similar in severity grades in left and right testes, and was characterized by variable degeneration/loss of germ cells and multinucleated germ cell formation. Germ cell degeneration was characterized by shrunken, hypereosinophilic germ cells. Minimal severity grade was characterized by individual germ cell degeneration and segmental depletion primarily affecting spermatocytes and round spermatids; reduced elongating spermatids; and retained step 19 spermatids in late stage tubules. Mild severity grade was characterized by more pronounced germ cell depletion accompanied by variable germ cell disorganization and exfoliation. Moderate severity grade was characterized by widespread, moderate depletion of spermatocytes and round and elongating spermatids, and moderate germ cell disorganization and exfoliation. Marked severity grade was characterized by generalized depletion of germ cells with scattered Sertoli cell-only tubules. Spermatogonia were present in all severity grades.
Hepatocellular vacuolation was characterized by discrete, variably-sized, vacuoles with well-defined margins in a centrilobular to diffuse distribution. The finding correlated with pale liver macroscopically. Higher total serum bilirubin was noted in a single 300 mg/kg/day group male (No.6207) with mild hepatocellular vacuolation. Microscopic findings considered to be secondary to test item-related stress consisted of reduced thymic cortical lymphocytes in the 15, 50, 125, and 300 mg/kg/day group males and females, and decreased sternal bone marrow cellularity in the 300 mg/kg/day group males. The thymic change was characterized by thinning of the cortex accompanied in some animals by increased lymphocyte apoptosis. Reduced marrow cellularity was characterized by less densely organized progenitor cells and increased prominence of adipocytes. There were no other histologic changes considered to be related to administration of the test item at the primary necropsy. However, several findings deserve mention. Thrombosis of large caliber pulmonary vessels with congestion of surrounding parenchyma was noted in the single unscheduled death control group female and in the 15 and 300 mg/kg/day group males and 15, 50, and 125 mg/kg/day group females. Thrombosis was accompanied by congestion and occasionally, vascular inflammation. Associated pulmonary congestion correlated macroscopically with dark red discoloration/areas in the lungs. The relationship between pulmonary thrombosis and administration of the test item was considered uncertain because of absence of the finding in the 300 mg/kg/day group females, presence of the finding in the single unscheduled death control female, absence of cardiovascular changes in any other tissues evaluated, and the findings were incompatible with the mode of action of the test item.
Unilateral testicular tubular atrophy was noted in a single 125 mg/kg/day group male; the finding was consistent with spontaneous change and not considered to be test item-related. Sperm granuloma was noted in two 15 mg/kg/day group males; there was no dose-response relationship, and the finding was attributed to spontaneous change. Ovarian follicular cysts, characterized by mildly ectatic follicles lined by flattened follicular cells, were noted in a single 300 mg/kg/day group female. The finding was attributed to spontaneous change and not considered to be test item-related.
Leukaemia was noted in the liver of one 50 mg/kg/day group female (No. 6283) at the primary necropsy, characterized by infiltrates of round cells with round to indented nuclei that effaced normal marrow architecture and infiltrated hepatic parenchyma. There were no macroscopic or clinical pathology correlates, and the finding was attributed to spontaneous neoplasia. 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. At recovery, epididymal and testicular findings were more pronounced in the 300 mg/kg/day group males when compared with males at the primary necropsy. Marked to severe tubular degeneration/atrophy, characterized by a preponderance of tubules lined almost exclusively by Sertoli cells with variable numbers of sloughed, degenerate germ cells, was noted in all 300 mg/kg/day group males. No other microscopic findings considered to be test item-related were noted.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Reproductive Performance:
No test substance-related effects on reproductive performance were observed at any dosage level. No statistically significant differences were noted between the control and test substance-treated groups. All females in all groups were gravid. The mean numbers of days between pairing and coitus in the test substance-treated groups were similar to the control group value. The mean lengths of oestrous cycles in these groups were also similar to the control group value. None of these differences were statistically significant.

Gestation Length and Parturition:
Significantly (p<0.01) longer mean gestation lengths were noted for the 125 and 300 mg/kg/day groups (22.0 and 22.5 days, respectively) compared to the control group (21.3 days). In the 300 mg/kg/day group, 3 of the 6 females that delivered had gestation lengths of 23 days and in conjunction with the test substance-related effects on intrauterine and postnatal survival in this group, the longer gestation length was attributed to the test substance. The value in the 125 mg/kg/day group was within the Charles River Ashland historical control data range (20.9 to 22.1 days) and in the absence of similar effects on pre- and postnatal survival, the effect at 125 mg/kg/day was not considered test substance-related. Mean gestation lengths in the 15 and 50 mg/kg/day groups were similar to those in the control group. No statistically significant differences were noted. No signs of dystocia were noted at any dosage level.
Key result
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
gross pathology
other: gestation length
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
testes
Sertoli cells
germ cells
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
female reproductive system
Organ:
germ cells
ovary
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified
Conclusions:
A dosage level of 125 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for male and female reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 125 mg/kg/day based on lower body weights, body weight gains, and reduced food consumption in the 300 mg/kg/day group males and females. The NOAEL for neonatal toxicity was 50 mg/kg/day based on lower pup body weights and body weight gains at 125 and/or 300 mg/kg/day.
Executive summary:

The objectives of the GLP-Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test were to investigate the potential toxic effects of the test substance, when administered to rats for 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 in accordance with OECD Guideline 422 and separate analytical validation, homogeneity and stability of the test item in gavage formulations containing peanut oil and test substance ranging in concentration from 10.0 to 100 mg/mL was conducted with an HPLC method using CAD for the determination of the test item concentration (Sen, 2016). The assay validation was subsequently extended to include the determination of the test substance concentration in peanut oil formulations at concentrations as low as 1.00 mg/mL. Assay specificity/selectivity, ruggedness, calibration reproducibility, precision, accuracy, and test substance stability in calibration standards and processed QC samples stored at room temperature were assessed. In addition, formulations prepared at target concentrations of 1, 10, and 100 mg test substance/mL were analyzed to assess test substance homogeneity and, following 7 days of room temperature storage, resuspension homogeneity. Finally, test substance stability following 18 hours and following 7 days of room temperature storage was assessed in formulations prepared at target concentrations of 1, 10, and 100 mg test substance/mL. The results of the test substance homogeneity at all tested concentrations and following 7 days of room temperature storage met the protocol-specified acceptance criteria, i.e., the RSD for the mean concentration was ≤10%. Assessment of test substance stability following 18 hours and following 7 days of room temperature storage in formulations prepared at target concentrations of 1, 10, and 100 mg test substance/mL met the previously stated protocol-specified acceptance criteria for stability.

In the combined 28 -day repeated dose oral toxicity study with reproduction/developmental toxicity screening 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 15, 50, 125, and 300 mg/kg/day. A concurrent control group of 15 rats/sex received the control article (arachis oil and mineral oil mixture; the percentage of mineral oil matched that in the high-dosage group) on a comparable regimen. The dose volume was 10 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 to 54 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 to 42 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- or 14-day non-dosing recovery period, respectively. 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.

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 (Lactation Day 14 for females) and recovery necropsies; only primary necropsy male samples were analyzed. F0 males were euthanized following completion of the mating period or 15-day recovery period and F0 females were euthanized on Lactation Day 14 for females that delivered, post-mating day 25 for females that failed to deliver, or following the 14-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals found dead and all animals in the control and high-dose groups at the primary necropsy. In addition, the testes, epididymides, ovary, uterus, vagina, thymus, liver, lungs, and sternal bone marrow (males) were identified as potential target tissues and were examined from all F0 animals.

There was no test substance-related mortality noted at any dosage level (except one F0 female in the control group was found dead on day 17 caused by acute pulmonary inflammation).

Test substance-related lower mean body weight gains and corresponding decreased food consumption were noted for F0 males in the 300 mg/kg/day group throughout the treatment period (Study Days 0-28) resulting in mean body weights that were up to 13.7% lower than the control group. Mean body weights in the 300 mg/kg/day group F0 males remained lower than the control group during the recovery period (Study Days 28 to 41) despite mean body weight gains that were higher than the control group. Test substance-related lower mean body weight gains were noted for F0 females in the 300 mg/kg/day group compared to the control group during the pre-mating period and gestation. Lower mean food consumption was noted for F0 females in the 300 mg/kg/day group during the pre-mating period; during gestation, mean food consumption in this group was similar to the control group. When the entire treatment period (Study Days 13 to 48) was evaluated, a lower mean body weight gain was noted for F0 females in the 300 mg/kg/day group that were not paired for mating due to the mean body weight losses and lower mean body weight gains observed in this group following the pre-mating period which resulted in mean body weights that were up to 13.3% lower than the control group during Study Days 13 to 48.

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

A test substance-related longer gestation length was observed at 300 mg/kg/day. F0 gestation lengths in the 15, 50, and 125 mg/kg/day groups and oestrous cycle length, reproductive performance (mating, fertility, copulation/conception indices), and the process of parturition in all test substance-treated groups were unaffected by test substance administration.

A test substance-related greater number of unaccounted-for sites was noted for the 300 mg/kg/day group F0 females.

Test item-related microscopic findings were noted in the reproductive organs of the 300 mg/kg/day group males, and in the liver of the 50, 125, and 300 mg/kg/day group males and 300 mg/kg/day group females. Small epididymides noted macroscopically correlated with lower epididymal weights and bilateral reduced luminal sperm, and small, soft testes correlated with lower testicular weights and bilateral tubular degeneration. Testicular and epididymal degenerative changes were more pronounced at recovery. Findings were considered to be a direct toxicologic effect of the test item and were considered to be adverse in the 300 mg/kg/day group males. Hepatocellular vacuolation was characteristic of lipid accumulation and correlated with pale liver noted macroscopically. Hepatocellular vacuolation was considered to be non-adverse because of low grade severity and absence of corresponding serum chemistry alterations, with the exception of higher serum total bilirubin values noted in a single 300 mg/kg/day group male. Higher serum bile acid values were noted in the 50 and 125 mg/kg/day group females, with no microscopic correlates.

Microscopic findings at the primary necropsy attributed to test item-related stress-response consisted of reduced thymic cortical lymphocytes noted in the 15, 50, 125, and 300 mg/kg/day group males and females, which correlated with lower thymus weights and small thymus macroscopically, and decreased cellularity noted in the sternal bone marrow in the 300 mg/kg/day group males. Both thymus and sternal bone marrow were similar to the control groups at the recovery necropsy. Additional findings attributed to stress-response consisted of lower final body weights, lower spleen, and higher adrenal gland weights, lower total white cell, neutrophil, lymphocyte, and eosinophil counts, lower reticulocyte parameters, and lower serum protein and lipid parameters in the 300 mg/kg/day group males. Lower final body weights were noted in the 300 mg/kg/day group males at recovery, but haematology parameters were increased when compared with values at the primary necropsy, indicating partial recovery. Thrombosis of large pulmonary vessels was noted in the 15 and 300 mg/kg/day group males and the 15, 50, and 125 mg/kg/day group females; pulmonary thrombosis was additionally noted in 1 control group female that died. The finding was associated with congestion and in some animals, vascular inflammation, and dark red discoloration/areas were observed macroscopically. The cause of the finding was unknown with uncertain relationship to administration of the test item.

Leukaemia was noted in the liver of one 50 mg/kg/day group female, but was considered to represent a spontaneous finding given occurrence in a lower dosage group animal, absence of the finding at recovery, and known low incidence of spontaneous leukaemia in young rats.

Under the conditions of this screening study, test substance-related effects on F0 male reproductive organs were noted at 300 mg/kg/day. Macroscopic findings of small and/or soft testes and epididymides corresponded with lower mean testes and epididymal weights and microscopic findings of reduced luminal sperm (epididymides) and tubular degeneration (testes) and were of greater magnitude at the recovery necropsy. In addition, a test substance-related longer gestation length was noted at 300 mg/kg/day, therefore, a dosage level of 125 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for male and female reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 125 mg/kg/day based on lower body weights, body weight gains, and reduced food consumption in the 300 mg/kg/day group males and females. The NOAEL for neonatal toxicity was 50 mg/kg/day based on lower pup body weights and body weight gains at 125 and/or 300 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
125 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
good quality due to well documented guideline study.
System:
other: male and female reproductive system

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose - oral toxicity

The objectives of the GLP-Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (Herberth, 2017) were to investigate the potential toxic effects of the test substance, when administered to rats for 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 in accordance with OECD Guideline 422 and separate analytical validation, homogeneity and stability of the test item in gavage formulations containing peanut oil and test substance ranging in concentration from 10.0 to 100 mg/mL was conducted with an HPLC method using CAD for the determination of the test item concentration (Sen, 2016). The assay validation was subsequently extended to include the determination of magnesium metaborate concentration in peanut oil formulations at concentrations as low as 1.00 mg/mL. Assay specificity/selectivity, ruggedness, calibration reproducibility, precision, accuracy, and test substance stability in calibration standards and processed QC samples stored at room temperature were assessed. In addition, formulations prepared at target concentrations of 1, 10, and 100 mg test substance/mL were analyzed to assess test substance homogeneity and, following 7 days of room temperature storage, resuspension homogeneity. Finally, test substance stability following 18 hours and following 7 days of room temperature storage was assessed in formulations prepared at target concentrations of 1, 10, and 100 mg test substance/mL. The results of the test substance homogeneity at all tested concentrations and following 7 days of room temperature storage met the protocol-specified acceptance criteria, i.e., the RSD for the mean concentration was ≤10%. Assessment of test substance stability following 18 hours and following 7 days of room temperature storage in formulations prepared at target concentrations of 1, 10, and 100 mg test substance/mL met the previously stated protocol-specified acceptance criteria for stability.

In the combined 28-day repeated dose oral toxicity study with reproduction/developmental toxicity screening 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 15, 50, 125, and 300 mg/kg/day. A concurrent control group of 15 rats/sex received the control article (arachis oil and mineral oil mixture; the percentage of mineral oil matched that in the high-dosage group) on a comparable regimen. The dose volume was 10 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 to 54 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 to 42 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- or 14-day non-dosing recovery period, respectively. 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.

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 (Lactation Day 14 for females) and recovery necropsies; only primary necropsy male samples were analyzed. F0 males were euthanized following completion of the mating period or 15-day recovery period and F0 females were euthanized on Lactation Day 14 for females that delivered, post-mating day 25 for females that failed to deliver, or following the 14-day recovery period. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Selected tissues were examined microscopically from all F0 animals found dead and all animals in the control and high-dose groups at the primary necropsy. In addition, the testes, epididymides, ovary, uterus, vagina, thymus, liver, lungs, and sternal bone marrow (males) were identified as potential target tissues and were examined from all F0 animals.

There was no test substance-related mortality noted at any dosage level (except one F0 female in the control group was found dead on day 17 caused by acute pulmonary inflammation).

Test substance-related lower mean body weight gains and corresponding decreased food consumption were noted for F0 males in the 300 mg/kg/day group throughout the treatment period (Study Days 0-28) resulting in mean body weights that were up to 13.7% lower than the control group. Mean body weights in the 300 mg/kg/day group F0 males remained lower than the control group during the recovery period (Study Days 28 to 41) despite mean body weight gains that were higher than the control group. Test substance-related lower mean body weight gains were noted for F0 females in the 300 mg/kg/day group compared to the control group during the pre-mating period and gestation. Lower mean food consumption was noted for F0 females in the 300 mg/kg/day group during the pre-mating period; during gestation, mean food consumption in this group was similar to the control group. When the entire treatment period (Study Days 13 to 48) was evaluated, a lower mean body weight gain was noted for F0 females in the 300 mg/kg/day group that were not paired for mating due to the mean body weight losses and lower mean body weight gains observed in this group following the pre-mating period which resulted in mean body weights that were up to 13.3% lower than the control group during Study Days 13 to 48.

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

A test substance-related longer gestation length was observed at 300 mg/kg/day. F0 gestation lengths in the 15, 50, and 125 mg/kg/day groups and oestrous cycle length, reproductive performance (mating, fertility, copulation/conception indices), and the process of parturition in all test substance-treated groups were unaffected by test substance administration.

A test substance-related greater number of unaccounted-for sites was noted for the 300 mg/kg/day group F0 females.

Test item-related microscopic findings were noted in the reproductive organs of the 300 mg/kg/day group males, and in the liver of the 50, 125, and 300 mg/kg/day group males and 300 mg/kg/day group females. Small epididymides noted macroscopically correlated with lower epididymal weights and bilateral reduced luminal sperm, and small, soft testes correlated with lower testicular weights and bilateral tubular degeneration. Testicular and epididymal degenerative changes were more pronounced at recovery. Findings were considered to be a direct toxicologic effect of the test item and were considered to be adverse in the 300 mg/kg/day group males. Hepatocellular vacuolation was characteristic of lipid accumulation and correlated with pale liver noted macroscopically. Hepatocellular vacuolation was considered to be non-adverse because of low grade severity and absence of corresponding serum chemistry alterations, with the exception of higher serum total bilirubin values noted in a single 300 mg/kg/day group male. Higher serum bile acid values were noted in the 50 and 125 mg/kg/day group females, with no microscopic correlates.

Microscopic findings at the primary necropsy attributed to test item-related stress-response consisted of reduced thymic cortical lymphocytes noted in the 15, 50, 125, and 300 mg/kg/day group males and females, which correlated with lower thymus weights and small thymus macroscopically, and decreased cellularity noted in the sternal bone marrow in the 300 mg/kg/day group males. Both thymus and sternal bone marrow were similar to the control groups at the recovery necropsy. Additional findings attributed to stress-response consisted of lower final body weights, lower spleen, and higher adrenal gland weights, lower total white cell, neutrophil, lymphocyte, and eosinophil counts, lower reticulocyte parameters, and lower serum protein and lipid parameters in the 300 mg/kg/day group males. Lower final body weights were noted in the 300 mg/kg/day group males at recovery, but haematology parameters were increased when compared with values at the primary necropsy, indicating partial recovery. Thrombosis of large pulmonary vessels was noted in the 15 and 300 mg/kg/day group males and the 15, 50, and 125 mg/kg/day group females; pulmonary thrombosis was additionally noted in 1 control group female that died. The finding was associated with congestion and in some animals, vascular inflammation, and dark red discoloration/areas were observed macroscopically. The cause of the finding was unknown with uncertain relationship to administration of the test item.

Leukaemia was noted in the liver of one 50 mg/kg/day group female, but was considered to represent a spontaneous finding given occurrence in a lower dosage group animal, absence of the finding at recovery, and known low incidence of spontaneous leukaemia in young rats.

Under the conditions of this screening study, test substance-related effects on F0 male reproductive organs were noted at 300 mg/kg/day. Macroscopic findings of small and/or soft testes and epididymides corresponded with lower mean testes and epididymal weights and microscopic findings of reduced luminal sperm (epididymides) and tubular degeneration (testes) and were of greater magnitude at the recovery necropsy. In addition, a test substance-related longer gestation length was noted at 300 mg/kg/day, therefore, a dosage level of 125 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for male and female reproductive toxicity of the test item when administered orally by gavage to Crl:CD(SD) rats. Under the conditions of this screening study, the NOAEL for systemic toxicity was considered to be 125 mg/kg/day based on lower body weights, body weight gains, and reduced food consumption in the 300 mg/kg/day group males and females. The NOAEL for neonatal toxicity was 50 mg/kg/day based on lower pup body weights and body weight gains at 125 and/or 300 mg/kg/day.

In the non-GLP supporting range finder study (Herberth, 2016) the dosage levels of magnesium metaborate to be evaluated in a potential combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) in rats.

The test substance in the vehicle (arachis [peanut] oil) was administered orally by gavage to 4 groups Crl:CD(SD) rats, each group consisting of 5 males and 5 females, once daily for 13 consecutive days. Dosage levels were 100, 300, 500, and 1000 mg/kg/day administered at a dose volume of 10 mL/kg. A concurrent control group composed of 5 rats/sex received the control substance (arachis [peanut] oil and mineral oil mixture; the percentage of mineral oil matched that in the high-dosage group) on a comparable regimen. The animals were approximately 11 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 surviving animals on study day 13, and selected organs were weighed.

In the 1000 mg/kg/day group, 2 males were found dead and 3 females were euthanized in extremis on study day 4 or 5. All remaining males and females in this group were euthanized due to excessive toxicity on study day 5. Body weight losses (up to 54 g) and reduced food consumption (≤6 g/day) were noted for males and females in the 1000 mg/kg/day group during study days 0-4. In addition, clinical observations noted at the daily examinations and/or 1 hour following dose administration in this group included unkempt appearance, flushed extremities, thin body, red material around the nose, clear material around the mouth, and yellow material around the urogenital area.

One male in the 500 mg/kg/day group was euthanized in extremis on study day 8 due to poor clinical condition including clinical observations of impaired use of hind limbs, a body cool to the touch, and unkempt appearance as well as a marked body weight loss. All other males and females in the control, 100, 300, and 500 mg/kg/day groups survived to the scheduled euthanasia on study day 13. In the 500 mg/kg/day group, clinical observations of red material around the nose and clear material around the mouth were noted at the daily examinations and/or approximately 1 hour following dose administration. No noteworthy clinical observations were noted in the 100 and 300 mg/kg/day groups.

Mean body weight losses and lower mean body weight gains were noted for males and females in the 500 mg/kg/day group generally throughout the treatment period (study days 0-13) resulting in mean body weights that were up to 12.6% and 10.4% lower than the control group for males and females, respectively, during study days 7-13. In the 300 mg/kg/day group, mean body weight gains in males and females were lower than the control group for the overall treatment period (study days 0-13). As a result, mean body weights in this group were 5.6% and 7.7% lower than the control group for males and females, respectively, on study day 13. The body weight effects noted at 300 and 500 mg/kg/day corresponded to lower mean food consumption in these groups compared to the control group throughout the treatment period.

Lower mean food consumption was also noted for the 100 mg/kg/day group males and females throughout the treatment period; however, there were no corresponding effects on body weights noted in this group.

Macroscopic findings noted for males and females in the 500 mg/kg/day group included dark red discoloration of the adrenal glands, small thymus, and pale liver (females only). Dark red discoloration of the adrenal glands was also noted for males in the 300 mg/kg/day group. Lower mean thymus weights (absolute and relative to final body and brain weights) were noted in a dose-responsive pattern for males in the 100, 300, and 500 mg/kg/day groups and females in the 300 and 500 mg/kg/day groups when compared to the control group and corresponded to the macroscopic finding of small thymus in the 500 mg/kg/day group. In addition, mean liver weights (absolute and relative to final body and brain weights) for males in the 500 mg/kg/day group were lower than the control group which corresponded with lower mean body weights in this group.

Excessive toxicity was observed at 1000 mg/kg/day as evidenced by mortality, clinical observations, mean body weight losses, lower mean body weights, and lower mean food consumption which resulted in early group termination of the remaining animals on study day 5. A single male was also found moribund at 500 mg/kg/day. Mean body weight losses and/or lower mean body weight gains, body weights, and food consumption were noted at 300 and 500 mg/kg/day generally throughout the treatment period. Mean food consumption in the 100 mg/kg/day group was also lower than the control group; however, there were no corresponding effects on body weights noted at this dosage level. In the 500 mg/kg/day group, macroscopic findings included dark red discoloration of the adrenal glands and small thymus for males and females and pale liver for females. Dark red discoloration of the adrenal glands was also noted for males at 300 mg/kg/day. In addition, lower mean absolute and relative (to final body and brain weights) thymus weights were noted in a dose-responsive pattern for males in the 100, 300, and 500 mg/kg/day groups and females in the 300 and 500 mg/kg/day groups, which correlated with macroscopic findings of small thymus in the 300 and 500 mg/kg/day groups.

Lower mean liver weights (absolute and relative to final body and brain weights) were noted for males in the 500 mg/kg/day group and corresponded with lower body weight. Based on the results of this study, dosage levels of 15, 50, 125, and 300 mg/kg/day were selected for a combined repeated-dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) of test material administered orally by gavage to Crl:CD(SD) rats. The Lowest Observed Effect Level is determined to be 100 mg/kg/day in this dose finding study.

Repeated dose - dermal toxicity

A short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment. Testing by dermal route is not justified for the test item. The physico-chemical and toxicological properties suggest low potential for significant rate of absorption through the skin (logPow = -3.08, MW of 109.9246 g/mol, low water solubility (< 1x10E-4 g/L Mg or B at 20°C)). Furthermore the results of laboratory animal studies show low acute dermal and oral toxicity. LD50values are reported to be greater than 2000 mg/kg bw for the test item (Sanders, 2017; Sanders, 2016). In the 28-days repeated dose oral toxicity study in rats (Herberth, 2017) lower body weights were reported and a NOAEL of 125 mg/kg bw was therefore derived. The NOAEL for neonatal toxicity was 50 mg/kg/day based on lower pup body weights and body weight gains at 125 and/or 300 mg/kg/day. The test item was neither a skin irritant in an in vitro and in vivo skin irritation study (Warre, 2017, Sanders, 2016b) nor an irritant to the eyes in rabbits (Sanders, 2016c). Based on these results, a dermal repeated dose toxicity study for the test item is considered superfluous. For the hazard assessment purpose (DNEL derivation), a NOAEL from the oral repeated dose 28-days toxicity study in rats (Herberth, 2017), with application of an appropriate assessment factor, allows extrapolation towards repeated dermal route administration.

Repeated dose - inhalative toxicity

A short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment. According to Annex VIII, testing by the inhalation route is justified if exposure of humans via inhalation is likely. In this case testing by the inhalation route is not justified since the liquid test substance has low vapour pressure (0.0365 Pa at 25 °C), so the potential for the generation of inhalable forms is low, also the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be unlikely to occur. For the hazard assessment purpose (DNEL derivation), a NOAEL from the Repeated Dose 28-days Toxicity study combined with reprotoxicity screening in rats (Herberth, 2017), with application of an appropriate assessment factor, allows extrapolation towards repeated inhalation route administration.

Justification for classification or non-classification

No test substance related mortalities or effects during the FOB or motor activity evaluations at any dosage level in the F0 generation in the OECD 422 screening study were found. Test substance-related lower mean body weight gains and decreased food consumption were noted for male and female F0 at 300 mg/kg/day. Also test substance-related effects in the reproductive organs of males and females were noted in the 300 mg/kg/day group.

Concerning possible systemic effects by dermal route of exposure, they could be ruled out because no significant dermal absorption is expected for the test item due to its physico-chemical properties.

The inhalation route is also a not relevant route of exposure for the test item due to its physico-chemical properties.

Based on these data and in accordance with Regulation (EC) No 1272/2008 (CLP Regulation), classification and labelling is not required for systemic organ toxicity after repeated exposure (STOT-RE) for the test item because no other than testis target organs were identified during the study.