Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction, OECD 422 Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Rats, with Recovery, Result: The no-observed-adverse-effect level (NOAEL) for male and female reproductive toxicity was considered to be 125 mg/kg/day, NOAEL for systemic toxicity was considered to be 125 mg/kg/day, NOAEL for neonatal toxicity was 50 mg/kg/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
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 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
Vehicle:
arachis oil
Details on 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
Details on mating procedure:
BREEDING PROCEDURES
The 10 rats/sex/group selected for evaluation of reproductive toxicity were paired on a 1:1 basis within each treatment group following 14 days of treatment for the males and females. A breeding record containing the male and female identification numbers and the start date of cohabitation was maintained. Each female was cohabitated with 1 male in a solid-bottom cage containing bedding material. Positive evidence of mating was confirmed by the presence of a vaginal copulatory plug or the presence of sperm following a vaginal lavage and verified by a second biologist. Each mating pair was examined daily. The day when evidence of mating was identified was termed gestation day 0. If evidence of copulation was not detected after 14 days of pairing, any females that had not shown evidence of mating were placed in solid-bottom cages.
For the purpose of calculating pre-coital intervals, rats paired over a 12-hour dark cycle were considered to have been paired for 1 day.

Mating, fertility, and copulation/conception indices were calculated as follows:
- Male (Female) Mating Index (%) = No. of Males (Females) with Evidence of Mating (or Confirmed Pregnant) / Total No. of Males (Females) Used for Mating x 100
- Male Fertility Index (%) = No. of Males Siring a Litter / Total No. of Males Used for Mating x 100
- Male Copulation Index (%) = No. of Males Siring a Litter / No. of Males with Evidence of Mating (or Females with Confirmed Pregnancy) x 100
- Female Fertility Index (%) = No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or confirmed Pregnancy) x 100
- Female Conception Index (%) = No. of Females with Confirmed Pregnancy / No. of Females with Evidence of Mating (or Confirmed Pregnancy) x 100

PARTURITION
All females selected for pairing were allowed to deliver naturally and rear their young to PND 13. During the period of expected parturition, the females were observed twice daily for initiation and completion of parturition and for signs of dystocia. On the day parturition was initiated (PND 0), pups were sexed and examined for gross malformations, and the numbers of stillborn and live pups were recorded. Individual gestation length was calculated using the date delivery started.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
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 (previous 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:
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
Parental animals: Observations and examinations:
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)

Oestrous cyclicity (parental animals):
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. The average cycle length was calculated and reported for complete oestrous cycles (i.e., the total number of returns to metoestrus [M] or dioestrus [D] from oestrus [E] or prooestrus [P] until the detection of evidence of mating), beginning with the first day of dose administration. Oestrous cycle length was determined by counting the number of days from the first M or D in a cycle to the first M or D in a subsequent cycle. For breeding phase females, the cycle during which evidence of mating was observed for a given animal was not included in the mean individual oestrous cycle length calculation.
Sperm parameters (parental animals):
Histology and Microscopic Examinations: Target tissues of testis, epididymis and sternal bone marrow (males only) were also examined from all F0 animals in all groups.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- To reduce variability among the litters, 8 pups per litter, 4 per sex when possible, were randomly selected on PND 4. Standardization of litter size was not performed on litters with fewer than 8 pups. All selections were performed by computerized randomization. Blood samples for possible future thyroid hormone analysis were collected from 2 culled pups/litter (pooled by litter) on PND 4; pup were euthanized by an intraperitoneal injection of sodium pentobarbital following blood collection and discarded. Remaining culled pups (not used for blood collection) were weighed, euthanized by an intraperitoneal injection of sodium pentobarbital on PND 4, and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, Thyroid Hormone Analysis

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: Males were euthanized following completion of the mating period or following the 15-day recovery period
- Maternal animals: Females that delivered were euthanized on Lactation Day 14. Females that failed to deliver were euthanized on post-mating day 25 (females with evidence of mating). Females with total litter loss were euthanized within 24 hours of litter loss.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 and Table 2 were prepared for microscopic examination and weighed, respectively.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring On PND 13, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
- Blood samples were collected for thyroid hormone analysis immediately prior to euthanasia from 1 pup/sex/litter, a gross necropsy was performed, and the thyroids (with parathyroids, if present) were weighed (following fixation) and placed in 10% neutral-buffered formalin for possible histopathological examination. Remaining pups (not used for blood collection) were discarded without examination.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
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.
Reproductive indices:
Not performed on F1 litter.
Offspring viability indices:
Each litter was examined daily for survival, and all deaths were recorded. All pups were individually identified by application of tattoo markings on the digits following completion of parturition. A daily record of litter size was maintained. Intact offspring that were found dead from PND 0 to 4 were necropsied using a fresh dissection technique, which included examination of the heart and major vessels. For any pups in which a skeletal abnormality was suspected, the pups were eviscerated, cleared, stained with Alzarin Red S, and examined. A detailed gross necropsy was performed on any pup found dead after PND 4. Tissues were preserved in 10% neutral-buffered formalin for possible future histopathologic examination only as deemed necessary by the gross findings. The carcass of each pup was then discarded.
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 edema, hemorrhage, 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

Gestation:
Test substance-related lower mean body weight gains continued to be observed in the 300 mg/kg/day group F0 females during gestation. The differences from the control group achieved significance (p<0.01) during gestation days 7 to 11, 14 to 17, 17 to 20, and when the overall gestation period (days 0 to 20) was evaluated. In addition, mean body weights in the 300 mg/kg/day group were 4.0% to 21.7% lower than the control group during gestation; the differences were significant (p<0.05 or p<0.01) on gestation days 14, 17, and 20. Mean F0 female body weights and body weight gains in the 15, 50, and 125 mg/kg/day groups were unaffected by test substance administration during gestation. None of the differences from the control group were statistically significant.

Lactation:
Evaluation of mean body weights and body weight gains in the 300 mg/kg/day group during lactation was precluded by euthanasia of females that delivered by Lactation Day 2. Mean body weights and body weight gains in the 15, 50, and 125 mg/kg/day groups were unaffected by test substance administration during lactation. Differences from the control group were transient, not statistically significant, and/or had no effect on the overall lactation interval.
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):
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.
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):
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.
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 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 item-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 elongatingspermatids; 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.
Leukemia 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:
not examined
Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
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.
Reproductive function: sperm measures:
effects observed, treatment-related
Description (incidence and severity):
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 substance, and were considered to be adverse in the 300 mg/kg/day group males.
Reproductive performance:
no effects observed
Description (incidence and severity):
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.
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:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
testes
seminiferous tubules
Sertoli cells
cauda epididymis
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
female reproductive system
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
PND 0 Litter Data and Postnatal Survival
A lower number of pups born, corresponding higher number of unaccounted-for sites, and consequently mean live litter size on PND 0 were observed in the 300 mg/kg/day group (6.0 and 2.8 pups, respectively) when compared to the control group (13.6 pups for both); the differences were significant (p<0.01). Significantly (p<0.05) lower postnatal survival was noted in this group from PND 0 to 1. Further evaluation of postnatal survival in the 300 mg/kg/day group was precluded by the death/euthanasia of all pups in this group by PND 2.
Postnatal survival in the 125 mg/kg/day group was slightly lower than the control group during PND 0 to 1, 1 to 4, and birth to PND 4. Although the values were within the Charles River Ashland historical control data and the differences from the control group were not statistically significant, the slightly lower postnatal survival observed in the 125 mg/kg/day group was considered test substance-related due to the increase in the number of pups found dead and missing which was consistent with the test substance-related effects observed at 300 mg/kg/day and considered to be a dose-response effect. Postnatal survival in the 15 and 50 mg/kg/day groups were unaffected by test substance administration. The mean number of pups born, live litter size and the percentage of males at birth in the 15, 50, and 125 mg/kg/day groups were similar to the control group values.
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
A test substance-related increase in the number of pups found dead and missing (presumed cannibalized) was noted in the 125 and 300 mg/kg/day groups compared to the control group. The pups in the 125 and 300 mg/kg/day groups were found dead or missing during PND 0 to 7 and PND 0 to 2, respectively. The number of pups found dead and/or missing in the 15 and 50 mg/kg/day groups was unaffected by maternal test substance administration.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Evaluation of offspring body weights in the 300 mg/kg/day group was precluded by the death or euthanasia of the majority of pups on PND 0 or 1. The pup body weight of the single pup in the 300 mg/kg/day group that was alive on PND 1 was 29.4% lower than the control group. Mean pup birth weights (PND 1) in the 125 mg/kg/day group were similar to the control group. However, test substance-related lower mean F1 male and female pup body weight gains were noted in the 125 mg/kg/day group compared to the control group during PND 1 to 4 and 4 to 7; the differences were generally significant (p<0.05). Mean body weight gains in this group were similar to the control group during PND 7 to 10 and 10 to 13. As a result of the lower body weight gains during the first week of the postnatal period, mean F1 male and female pup body weights in the 125 mg/kg/day group were lower (up to 15.0% and 13.5% for males and females, respectively) than the control group during PND 4 to 13; the greatest deficit was observed on PND 7. In the 50 mg/kg/day group, mean F1 male and female pup body weight gains were similar to the control group during PND 1 to 4 followed by test substance-related lower mean body weight gains during PND 4 to 7; the differences were significant (p<0.05) compared to the control group during PND 4 to 7. The lower mean body weight gains observed at 50 mg/kg/day were not considered adverse because there were no test substance-related effects on postnatal survival noted at this dosage level. Mean body weight gains in this group were similar to the control group during PND 7 to 10 and 10 to 13. Mean F1 male and female pup body weights in the 50 mg/kg/day group were up to 13.2% and 10.9% lower for males and females, respectively, than the control during PND 4 to 13; the greatest deficit was observed on PND 7. Mean F1 male and female pup body weights and body weight changes during PND 1 to 13 in the 15 mg/kg/day group were unaffected by parental administration of the test substance. No statistically significant differences from the control group were noted.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Evaluation of organ weights in the 300 mg/kg/day group was precluded by the death/euthanasia of all pups in this group by PND 2. There were no test substance-related effects on thyroid weights in the F1 males and females at any dosage level on PND 13. Differences from the control group were considered to be the result of normal biological variation and were not considered to be of toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsies of Pups Found Dead
The numbers of pups (litters) found dead during PND 0-13 numbered 1(1), 0(0), 1(1), 6(4), and 34(5) in the control, 15, 50, 125, and 300 mg/kg/day groups, respectively. Aside from the presence or absence of milk in the stomach, the following foetal malformations and developmental variations were noted in the 300 mg/kg/day group. Pup nos. 6279-03 and 6279-07 had anasarca, Pup No. 6279-04 had a cleft palate (palatine plates not joined, entire length), Pup Nos. 6279-07 and 6315-03 had hydrocephaly (increased cavitation of the lateral, bilateral, and third ventricles), Pup Nos. 6282-05, 6282-09, and 6315-03 had microphthalmia (orbit smaller than normal), Pup No. 6282-09 had only 12 pairs of ribs present, Pup No. 6315-04 had a right-sided aortic arch (the aortic arch and descending aorta coursed to the right of the vertebral column, right carotid and subclavian arteries arose independently from the aortic arch [no brachiocephalic arch]; left carotid and subclavian arteries arose from a common vessel from the aortic arch), and Pup No. 6315-03 had sternoschisis (sternal band nos. 2 through 4 not joined). In addition, fetal developmental variations observed in fetuses in the 300 mg/kg/day group included renal papilla(e) not fully developed and/or distended ureter(s) for Pup Nos. 6315-01 and 6315-03 and a major blood vessel variation (right carotid and subclavian arteries arose independently from the aortic arch [no brachiocephalic trunk]) for Pup No. 6315-02. No foetal malformations or developmental variations were noted for pups that were found dead in the 15, 50, or 125 mg/kg/day groups.

Scheduled Pup Necropsies (PND 13)
In the 300 mg/kg/day group, PND 13 necropsy evaluations were precluded by the death/euthanasia of all pups in this group by PND 2. No internal findings that could be attributed to parental test substance administration were noted at the necropsy of pups euthanized on PND 13. Pup no. 6340-01 in the 125 mg/kg/day group was missing a tail. No internal findings were noted for pups at any dosage level.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Anogenital Distance
Evaluation of anogenital distance in the 300 mg/kg/day group was precluded by the death or euthanasia of the majority of pups on PND 0 or 1. The anogenital distance (2.51 mm) of the single pup in this group that survived until PND 2 was smaller than the mean control group value (3.68 mm). The anogenital distances (absolute, relative to pup body weight, and relative to the cube root of pup body weight) in the 15, 50, and 125 mg/kg/day groups were similar to the control group values. Differences from the control group were slight and not statistically significant.

Areolae/Nipple Anlagen
Evaluation of areolae/nipple anlagen in the 300 mg/kg/day group was precluded by the death/euthanasia of all pups in this group by PND 2. Areolae/nipple anlagen in the F1 male pups was unaffected by parental administration of the test substance when evaluated on PND 13. The test substance-treated group values were not statistically different from the control group values.

Behaviour (functional findings):
not examined
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
No test item-related differences in mean serum total thyroxine (T4) values were noted at PND 13 in the test item-administered groups.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
body weight and weight gain
Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
Under the conditions of this screening study, test substance-related effects on F0 male reproductive organs were 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 substance when administered orally by gavage to Crl:CD(SD) rats.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-study were to investigate the potential toxic effects of the test substance, magnesium metaborate, 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. The study was conducted according to 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.

There was no test substance-related mortality noted at any dosage level. 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. 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. These clinical observations were generally not observed at the daily examinations on the following day and were not considered adverse.

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. The magnitude of the differences were not of sufficient magnitude to affect mean body weights during the pre-mating period; however, mean body weights in the 300 mg/kg/day group were up to 21.7% lower than the control group during 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. Evaluation of body weight and food consumption parameters in the 300 mg/kg/day group during lactation was precluded by the euthanasia due to 4 females that failed to deliver and euthanasia due to total litter loss of the females that delivered by Lactation Day 2. 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. During the recovery period (Study Days 49 to 61), a higher mean body weight gain was noted in 300 mg/kg/day group F0 females compared to the control group; mean food consumption in this group was similar to the control group. Consequently, the lower mean body weights observed in these animals at the beginning of the recovery period (Study Day 49) were ameliorated by Study Day 61. Mean body weights, body weight gains, and food consumption in the 15, 50, and 125 mg/kg/day group F0 males and females were unaffected by test substance administration throughout the study.

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 estrous 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. This was consistent with the lower number of F1 pups born and live litter size on PND 0 observed in this group. In addition, lower postnatal survival was noted for the 300 mg/kg/day group from PND 0 to 1 due to 6 females with total litter loss by PND 2; the other 4 females in this group failed to deliver and had entirely resorbed litters. Further evaluation of F1 parameters in the 300 mg/kg/day group was precluded by death/euthanasia of all pups by PND 2. Foetal malformations and developmental variations noted for these pups included cleft palate, anasarca, hydrocephaly, microphthalmia, sternoschisis, right-sided aortic arch, 12 pairs of ribs present, 7th cervical ribs, vertebral centra not fully ossified, renal papillae not developed and/or distended ureter, and major blood vessel variation. In the 125 mg/kg/day group, a test substance-related higher number of pups that were found dead and missing (presumed cannibalized) were noted compared to the control group resulting in slightly lower postnatal survival in this group from birth to PND 4; the difference in postnatal survival was not statistically significantly different from the control group and the value was within the Charles River Ashland historical control data v2016.01. There were no test substance-related effects on F1 survival or clinical condition in the 15 and 50 mg/kg/day groups. Although mean F1 pup birth weights (PND 1) in the 125 mg/kg/day group were similar to the control group, test substance-related lower mean body weight gains were noted in this group during PND 1 to 4 and PND 4 to 7. As a result of the lower mean F1 pup body weight gains during the first week of the postnatal period, mean F1 male and female body weights were lower (up to 15.0%) than the control group during PND 4 to 13; the greatest deficit was observed on PND 7. Mean F1 pup body weights in the 125 mg/kg/day group showed slight recovery from PND 7 to 13 and the values were within the Charles River Ashland historical control ranges; however, due to the magnitude of change from control group values, the differences in mean F1 pup body weights at 125 mg/kg/day were considered test substance-related and adverse. In the 50 mg/kg/day group, test substance-related lower mean F1 pup body weight gains were noted during PND 4 to 7 resulting in lower (up to 13.2%) mean F1 male and female body weights compared to the control group. However, in the absence of test substance-related effects on postnatal survival at this dosage level, these deficits were not considered adverse. F1 pup body weights and body weight gains in the 15 mg/kg/day group were unaffected by maternal test substance administration. There were no test substance-related effects noted on F1 anogenital distance or areolae/nipple anlagen (males only) in the 15, 50, and 125 mg/kg/day groups. Necropsy findings for F1 pups that were euthanized on PND 13 were not suggestive of any association with maternal administration of the test substance. There were no test substance-related effects on thyroid/parathyroid weights noted for F1 pups on PND 13. 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.

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 substance 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.

Effect on fertility: via oral route
Endpoint conclusion:
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 codumented guideline study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Toxicity to reproduction - Effects on fertility

The objectives of the GLP compliant key study (Herberth, 2017) were to investigate the potential toxic effects of the test substance magnesium metaborate 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. The study was conducted according to 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.

There was no test substance-related mortality noted at any dosage level. 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. 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. These clinical observations were generally not observed at the daily examinations on the following day and were not considered adverse.

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. The magnitude of the differences were not of sufficient magnitude to affect mean body weights during the pre-mating period; however, mean body weights in the 300 mg/kg/day group were up to 21.7% lower than the control group during 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. Evaluation of body weight and food consumption parameters in the 300 mg/kg/day group during lactation was precluded by the euthanasia due to 4 females that failed to deliver and euthanasia due to total litter loss of the females that delivered by Lactation Day 2. 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. During the recovery period (Study Days 49 to 61), a higher mean body weight gain was noted in 300 mg/kg/day group F0 females compared to the control group; mean food consumption in this group was similar to the control group. Consequently, the lower mean body weights observed in these animals at the beginning of the recovery period (Study Day 49) were ameliorated by Study Day 61. Mean body weights, body weight gains, and food consumption in the 15, 50, and 125 mg/kg/day group F0 males and females were unaffected by test substance administration throughout the study.

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 estrous 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. This was consistent with the lower number of F1 pups born and live litter size on PND 0 observed in this group. In addition, lower postnatal survival was noted for the 300 mg/kg/day group from PND 0 to 1 due to 6 females with total litter loss by PND 2; the other 4 females in this group failed to deliver and had entirely resorbed litters. Further evaluation of F1 parameters in the 300 mg/kg/day group was precluded by death/euthanasia of all pups by PND 2. Foetal malformations and developmental variations noted for these pups included cleft palate, anasarca, hydrocephaly, microphthalmia, sternoschisis, right-sided aortic arch, 12 pairs of ribs present, 7th cervical ribs, vertebral centra not fully ossified, renal papillae not developed and/or distended ureter, and major blood vessel variation. In the 125 mg/kg/day group, a test substance-related higher number of pups that were found dead and missing (presumed cannibalized) were noted compared to the control group resulting in slightly lower postnatal survival in this group from birth to PND 4; the difference in postnatal survival was not statistically significantly different from the control group and the value was within the Charles River Ashland historical control data v2016.01. There were no test substance-related effects on F1 survival or clinical condition in the 15 and 50 mg/kg/day groups. Although mean F1 pup birth weights (PND 1) in the 125 mg/kg/day group were similar to the control group, test substance-related lower mean body weight gains were noted in this group during PND 1 to 4 and PND 4 to 7. As a result of the lower mean F1 pup body weight gains during the first week of the postnatal period, mean F1 male and female body weights were lower (up to 15.0%) than the control group during PND 4 to 13; the greatest deficit was observed on PND 7. Mean F1 pup body weights in the 125 mg/kg/day group showed slight recovery from PND 7 to 13 and the values were within the Charles River Ashland historical control ranges; however, due to the magnitude of change from control group values, the differences in mean F1 pup body weights at 125 mg/kg/day were considered test substance-related and adverse. In the 50 mg/kg/day group, test substance-related lower mean F1 pup body weight gains were noted during PND 4 to 7 resulting in lower (up to 13.2%) mean F1 male and female body weights compared to the control group. However, in the absence of test substance-related effects on postnatal survival at this dosage level, these deficits were not considered adverse. F1 pup body weights and body weight gains in the 15 mg/kg/day group were unaffected by maternal test substance administration. There were no test substance-related effects noted on F1 anogenital distance or areolae/nipple anlagen (males only) in the 15, 50, and 125 mg/kg/day groups. Necropsy findings for F1 pups that were euthanized on PND 13 were not suggestive of any association with maternal administration of the test substance. There were no test substance-related effects on thyroid/parathyroid weights noted for F1 pups on PND 13. 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.

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 substance 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.

Value chosen for the risk assessment:

For the risk assessment the NOAEL for male and female reproductive toxicity of 125 mg/kg/day was chosen, since the exposure duration for the treated test animals of 54 days was the longest in the OECD 422 screening study. Adverse effects were obvious in the F0 male and female reproductive organs in the 300 mg/kg/day group. Developmental effects, e.g. malformations, in the F1 pups found dead in the 300 mg/kg/day group appeared to be more influenced by the toxic effects on the maternal reproductive system, than actual signs of developmental toxicity. The same might be true for the adverse differences in mean body weights of F1 pups at 125 mg/kg/day and recovery can be a expected. Further investigations regarding developmental toxicity (e.g OECD 414) might be necessary.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available (further information necessary)
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

In an OECD 422 screening study reproductive effects in male and female rats at a dose of 300 mg/kg/day were observed. Definite effects on the reproduction organs of the F0 parental animals in males and females were observed. Clear effects on testes, luminal sperm and tubulars are shown in males and effects on estrous cycle, higher number of unaccounted-for sites in females followed by reduced postnatal survival. So it can be concluded that the substance is toxic to reproduction. The F1 pups showed several malformations in the 300 mg/kg/day group. There were no test substance-related effects on F1 survival or clinical condition in the 15 and 50 mg/kg/day groups. The differences in mean F1 pup body weights at 125 mg/kg/day were considered test substance-related and adverse. In expert consultation it is concluded that the malformations of pups found dead in the 300 mg/kg bw group appear to be more influenced by the toxic effects on the maternal reproductive system, than actual signs of developmental toxicity. So the conclusion is that the substance is not a developmental toxicant. Taking these results into account and considering that the OECD 422 study is just a screening study for toxicity to reproduction, a further investigation for developmental toxicity is warranted (OECD 414). The screening study with inconclusive evidence is not sufficient for classification according to Regulation (EC) No 1272/2008 (CLP Regulation) as toxic to reproduction.