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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Under the test conditions, the NOAEL of test item was 1000 mg/kg bw/day for reproductive toxicity in rats (OECD 422).

Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-07-31 to 2013-01-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age and body weight at study initiation: Study Day 0: 10-11 weeks (323-419 g for males; 202-287 g for females); Gestation day 0: 12-13 weeks (216-336 g for females)
- Fasting period before study: No
- Housing: Following receipt and until pairing, all F0 animals were housed individually (except during the mating period for 12 rats/sex/group) in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. The 5 rats/sex in the control and high-dosage groups assigned to the post-treatment period were not paired and remained in clean, stainless steel wire-mesh cages until euthanasia. The breeding phase rats (12/sex/group) were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding. The dams and their litters were housed in these cages until euthanasia on lactation day 4. Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until post-cohabitation or post-mating day 25.
- Diet: Basal diet, ad libitum
- Water: Reverse osmosis-purified (on-site) drinking water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 70.5-71.0 °F (21.4-21.7 °C)
- Humidity: 47.9-54.3%
- Air changes: 10 fresh air changes per hour
- Photoperiod: 12 h dark / 12 h light
Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated, protected from light. The test item formulations were stirred for at least 30 minutes prior to daily dispensation and continuously throughout the preparation, sampling, and dose administration procedures.

VEHICLE
- Concentration in vehicle: 0, 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day
- Lot/batch no. (if required): ZV0181, 1BG0536, and 2AI0653, exp. dates: 23 September 2012, 6 July 2013, and 16 September 2013, respectively from Spectrum Chemical Manufacturing Corporation.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Presence of a vaginal copulatory plug or the presence of sperm in vaginal lavage referred to as day 0 of pregnancy.
- 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Prior to the initiation of dose administration, samples for homogeneity determination were collected from the top, middle, and bottom strata of the 50 and 200 mg/mL dosing formulations. In addition, samples for resuspension homogeneity determinations were collected from the top and bottom strata of these same dosing suspensions following refrigerated storage for 9 days and a period of remixing for a minimum of 30 minutes.
- Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared for the first and last weeks of dose administration (corresponding to all groups on study being dosed). One set of samples from each collection was subjected to the appropriate analyses.
- Results: The analyzed dosing formulations were within the range for suspensions (85-115 %) and were homogeneous. The test item was not detected in the vehicle formulation that was administered to the control group (Group 1).
Analytical method (Coffee ST 2013, WIL Report No.: WIL-168204)
HPLC Method:
Instrument: Dionex UltiMate™ 3000 high performance liquid
chromatograph equipped with a charged aerosol detector,
autosampler, and Dionex Chromeleon® software version
6.8, or equivalent system
Column: Phenomenex Phenogel® 50Å, 300 mm × 7.80 mm,
5-μm particle-size
Column Temperature: 40°C
Mobile Phase: Chloroform
Flow Rate: 0.7 mL/minute
Injection Volume: 20 μL
Detector: Charged aerosol detector
Retention Time: Approximately 11 minutes
Run Time: 15 minutes
Formulation preparation: Suspension formulations prepared at 20, 50 and 200 mg/L. The appropriate amount of the test substance for each formulation was weighed in a tared, calibrated glass container. Approximately 70% of the vehicle was added to each container. The formulations were mixed as necessary until uniform. The formulations were then brought to the calibration target with vehicle. Formulations were moved to a heated water bath set at between 45°C to 50°C (Group Low and High) or 55°C to 60°C (Group 2 and 4) while stirring and heated until uniform. The test substance formulations were stirred continuously throughout the preparation and sampling procedures.
Test item stability:
Processed samples were stored at room temperature for a minimum of 3 days before being re-analyzed to assess test item stability. The mean 3-day post-storage values ranged from 102% to 124% of the pre-storage values.
Formulations prepared at target concentrations of 20 and 200 mg/mL were analyzed on the day of preparation. Aliquots of the
formulations were stored refrigerated for at least 4 days or 10 days and analyzed to assess test item stability. The mean post-storage concentration ranged from 95.4% to 116% of pre-storage values.
Formulations prepared at target test item concentrations of 50 and 200 mg/mL met the protocol-specified acceptance criteria for stability
following 5 days of frozen (approximately -20°C) storage.
Results of the stability tests can be found in the attached document.
Duration of treatment / exposure:
Males: 12 males/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses.

Females: 12 females/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 39-53 doses; females that failed to deliver or had no evidence of mating were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 24) for a total of 39-52 doses.

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 39 doses for the females, these animals were assigned to the post-treatment period and remained on study for a 14-day non-dosing period.
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
250, 500 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
- Vehicle control and high dose group: 17 rats/sex/dose
- Low and mid-dose group: 12 rats/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected based on the results of a previous oral (gavage) 14-day range finding study (Toot, Draft, WIL-168201) with test material and an oral (gavage) 1-generation reproduction study in the rat with a chemically similar borate ester. In both the studies, no toxicity was observed at the maximum dose level of 1000 mg/kg bw/day.
- Rationale for animal assignment: Animals were assigned into different groups by computerized randomization procedure based on body weight stratification in a block design.

Positive control:
Not applicable
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were conducted weekly (prior to test item administration during the treatment period). Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. Females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual male body weights were recorded weekly, beginning 1 week prior to test item administration, on the first day of dosing, and weekly thereafter until the scheduled euthanasia. Individual female body weights were recorded weekly until evidence of copulation and GD 0, 4, 7, 11, 14, 17 and 20 and on lactation days 0, 1 and 4.

FOOD CONSUMPTION:
- Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the mating period for animals selected for pairing, while food consumption continued to be recorded until euthanasia for males and females not selected for pairing. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Following mating, food consumption for females with no evidence of mating was measured on a weekly basis until the scheduled euthanasia.
- Food consumption was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.

WATER CONSUMPTION: No
Sperm parameters (parental animals):
Parameters examined in P male parental generations: Testis weight, epididymides weight, seminal vesicle weight, sperm count in testes and epididymides, sperm motility and sperm morphology.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in offspring: Litter size, viability, number and sex of pups, bodyweights of pups and clinical observations of pups.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals following completion of the mating period.
- Maternal animals: All surviving animals on lactation day 4 (females that delivered) or on post-mating day 25 (females that failed to deliver).

GROSS PATHOLOGY: Yes
Necropsy included examination of the external surface, external surfaces of the brain, all orifices and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Moreover the number and location of corpora lutea and implantation sites were examined and uteri were investigated for early implantation loss.
At the time of necropsy, the following tissues and organs were placed in 10% neutral-buffered formalin:
Adrenal glands (2), Aorta, Bone with marrow (sternebrae), Brain, Coagulating glands, Eyes with optic nerve (2)*, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Liver (sections of 2 lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph node (axillary [2], mesenteric, and mandibular [2]), Ovaries** and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary gland, Prostate gland, Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary gland***, Spinal cord (cervical), Spleen, Testes with epididymides (2)$ and vas deferens, Thymus gland, Thyroids [with parathyroids, if present (2)], Trachea, Urinary bladder, Uterus# with cervix and vagina, All gross lesions (when possible)

Organ weights: Adrenal glands, Brain, Epididymides (total and cauda), Heart, Kidneys, liver, Ovaries with oviducts, Prostate gland, Seminal vesicle (with coagulating gland and fluid), Spleen, Testes, Thymus gland and Thyroids with parathyroids.

HISTOPATHOLOGY: Yes
Microscopic examination was performed on all tissues listed previously from all treatment phase males and females in the control and 1000 mg/kg bw/day groups and one female in the 500 mg/kg bw/day group that was found dead. In addition, the mesenteric lymph node, colon, and ileum were examined as possible target organs in both males and females in the low- and mid-dosage groups at the treatment period necropsy and all animals at the post-treatment necropsy. Microscopic evaluations were also performed on gross lesions from all animals and correlated to macroscopic findings if possible.

* = Fixed in Davidson’s solution
** = Ovaries were fixed in 10% neutral-buffered formalin for approximately 48 hours, following which all ovaries were transferred to 70% ethanol.
*** = For females; a corresponding section of skin was taken from the same anatomic area for males.
$ = Testis and epididymis (right only) were fixed in modified Davidson’s solution. Right and left testes and epididymides from males in the post-treatment phase were fixed in modified Davidson’s solution (care was taken to ensure separation between left/right organs).
# = Any uterus that was placed in 10% ammonium sulfide solution for detection of implantation sites was discarded and not preserved in 10% neutral-buffered formalin.
Postmortem examinations (offspring):
On PND 4, surviving F1 rats were euthanized via an intraperitoneal injection of sodium pentobarbital. The carcasses were then discarded.
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. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
Reproductive indices:
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/Total No. of Females Used for Mating) x 100

Female Conception Index (%) = (No. of Females with Confirmed Pregnancy/ No. of Females with Evidence of Mating (or Confirmed Pregnancy)) x 100
Offspring viability indices:
Litter parameters were defined as follows:

Mean Live Litter Size = Total No. of Viable Pups on PND 0/ No. of Litters with Viable Pups PND 0

Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-selection) (% Per Litter) = (Sum of (Viable Pups Per Litter on PND 0 or PND 4 [Pre-selection]/No. of Pups Born Per Litter)/No. of Litters Per Group) x 100

Postnatal Survival for All Other Intervals (% Per Litter) = (Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)/No. of Litters Per Group) x 100

Where N= PND 0-1 and 1-4
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Description (incidence and severity):
Not relevant
Mortality:
mortality observed, non-treatment-related
Description (incidence):
See details on results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
See details on results
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
See details on results
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- One female in the 500 mg/kg bw/day group was found dead on study day 13 prior to pairing, but this was not considered test item-related.
- Test item-related clinical findings were primarily noted at the time of dose administration or approximately 1 hour following dose administration and included clear and red material around the nose and mouth and salivation. These findings were observed in the 500 and 1000 mg/kg bw/day group males and females generally throughout the treatment period. Limited occurrences of red and/or clear material around the nose and mouth or salivation were also observed in the 250 mg/kg bw/day group males and females. The aforementioned clinical findings were considered test item-related but not adverse.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Test item-related, lower mean body weight gains were noted for the 1000 mg/kg bw/day group males generally throughout the treatment period, resulting in lower mean body weight gains when the entire pre-mating period (study days 0-13) and entire treatment period (study days 0-27) were evaluated. These effects corresponded to decrements in mean food consumption for the 1000 mg/kg bw/day group males during the pre-mating period. As a result, mean body weights for the 1000 mg/kg bw/day group males were lower than the control group during study days 13-28. During the post-treatment period, mean body weight gains and food consumption for the 1000 mg/kg bw/day group males were similar to the control group, but mean body weights continued to be lower than the control group throughout the post-treatment period. Mean body weights, body weight gains, and food consumption for the 250 and 500 mg/kg bw/day group males were similar to the control group.
- Mean body weights, body weight changes, and food consumption for the 250, 500, and 1000 mg/kg bw/day group females were unaffected by test item administration during the pre-mating period and remainder of the treatment period for females that were not selected for breeding. Mean body weight gain and food consumption for the 1000 mg/kg bw/day group during the overall post-treatment period (study days 39-52) was similar to the control group, but mean body weights in the 1000 mg/kg bw/day group females remained lower than the control group throughout the post-treatment period.
- During lactation, a test item-related, mean body weight loss with corresponding lower mean food consumption was noted for the 1000 mg/kg bw/day group during lactation days 1-4 (compared to a mean body weight gain in the control group) and resulted in a mean body weight for the 1000 mg/kg bw/day group females that was 5.7% lower than the control group on lactation day 4. No test item-related effects on mean body weights, body weight gains, and food consumption were noted in the 250, 500, and 1000 mg/kg bw/day groups during gestation and the 250 and 500 mg/kg bw/day groups during lactation days 1-4.

HAEMATOLOGY AND CLINICAL CHEMISTRY
- Test item-related higher mean reticulocyte count and lower mean corpuscular volume and mean corpuscular hemoglobin values were observed for males in the 1000 mg/kg bw/day group following the 14-day post-treatment period. In addition, test item-related higher mean red blood cell count, hemoglobin value, and hematocrit percentage were noted for females in the 1000 mg/kg bw/day group following the 14-day post-treatment period. These changes were considered adaptive responses and were not adverse.
- At the end of the treatment period, test item-related, non-adverse, higher mean alanine aminotransferase and urea nitrogen values were noted for males in the 1000 mg/kg bw/day group, and higher mean potassium levels were noted for females in the 1000 mg/kg bw/day group. Following a 14-day post-treatment period, lower mean glucose values were noted in the 1000 mg/kg bw/day group males and females. For the males, all individual glucose values and the group mean value were within the historical control database range. However, the glucose values for 4 of 5 females in the 1000 mg/kg bw/day group and the group mean value were lower than the minimum mean value in the historical control database. The lower mean glucose values for the 1000 mg/kg bw/day group males and females were not considered adverse. There were no test item-related effects on hematology or serum chemistry parameters in the 250 and 500 mg/kg bw/day group male and females.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
- No test item related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, and morphology) in males at any dosage level.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- F0 male and female mating and fertility, male copulation, and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- There was no test item-related internal findings or effects on organ weights observed at any dosage level at the scheduled necropsy.
- The differences were slight and not statistically significant, with the following exceptions. Mean adrenal gland weight relative to final body weight for the 1000 mg/kg bw/day group females on lactation day 4 was significantly (p<0.05) higher than the control group, but was attributed to the lower mean final body weight for the 1000 mg/kg bw/day group females and was not considered test item-related. In addition, the mean thymus weight relative to final body weight for the 1000 mg/kg bw/day group females at the end of the post-treatment period was significantly (p<0.05) higher than the control group and was attributed to the lower mean final body weight for the 1000 mg/kg bw/day group females.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- No test item-related internal findings were observed at any dosage level in females that failed to deliver or males and females at the scheduled necropsy. Macroscopic findings observed in the test item-treated groups occurred infrequently and/or in a manner that was not dose-related.
- The mean numbers of unaccounted-for sites, implantation sites, and corpora lutea in the 250, 500 and 1000 mg/kg bw/day groups were similar to the control group values.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- At the end of the treatment period, an increased incidence of granulomatous inflammation of the mesenteric lymph nodes and lymphoid hyperplasia of the colon and ileum were noted in the 1000 mg/kg bw/day group males and/or females. The lymphoid hyperplasia in the ileum and colon had resolved after the 14-day post-treatment period and was most likely an adaptive physiologic response to immune stimulation.
- Granulomatous inflammation of the mesenteric lymph nodes was still evident in all of the 1000 mg/kg bw/day group females and 4 of 5 of the males after the 14-day post-treatment period. Although not completely resolved, the presence of this finding was less severe. Thus, granulomatous inflammation of the mesenteric lymph nodes was considered test item-related and adverse, but it is noted that the inflammation was partially resolved after the 14-day post-treatment period.
Dose descriptor:
NOAEL
Remarks:
Systemic toxicity
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lower mean food consumption and granulomatous inflammation of the mesenteric lymph node were observed in both males and females at 1000 mg/kg bw/day.
Dose descriptor:
NOAEL
Remarks:
Reproductive performance
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
no
Clinical signs:
not examined
Description (incidence and severity):
Not relevant
Mortality:
not examined
Body weight and weight changes:
not examined
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Clinical signs:
no effects observed
Description (incidence and severity):
See details on results
Description (incidence and severity):
Not relevant
Mortality / viability:
no mortality observed
Description (incidence and severity):
See details on results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See details on results
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:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
See details on results
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
VIABILITY (OFFSPRING)
- Mean number of pups born, live litter size, and the percentage of males at birth in the 250, 500, and 1000 mg/kg bw/day groups were similar to the control group values. In addition, postnatal survival through PND 4 in the 250, 500 and 1000 mg/kg bw/day groups was unaffected by maternal test item administration.

CLINICAL SIGNS (OFFSPRING)
- General physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by maternal test item administration.

BODY WEIGHT (OFFSPRING)
- Mean F1 birth weights (PND 1) for the 1000 mg/kg bw/day group were 9.9% (males) and 7.6% (females) lower than the control group. During PND 1-4, mean pup body weight gains were lower than the control group and resulted in mean pup body weights in the 1000 mg/kg bw/day group that were 17.8% (males) and 18.6% (females) lower than the control group on PND 4. Mean pup body weights and body weight changes in the 250 and 500 mg/kg bw/day groups were not remarkably different than the control group.

GROSS PATHOLOGY (OFFSPRING)
- There were no remarkable internal findings observed at the necropsy of pups that were found dead during PND 0-4.
Dose descriptor:
NOAEL
Remarks:
Neonatal toxicity
Generation:
F1
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Lower mean F1 birth weights (PND 1), pup body weight gains during PND 1-4, and pup body weights on PND 4 were noted for the 1000 mg/kg bw/day group males and females.
Critical effects observed:
no
Clinical signs:
not examined
Description (incidence and severity):
Not relevant
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Reproductive effects observed:
no

See attached Document for Tables of Results

Conclusions:
Under the test conditions, the NOAEL of test item was 1000 mg/kg bw/day for reproductive toxicity in rats.
Executive summary:

Test Guidance

OECD 422: A Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Rats, with Recovery.

 

Materials and methods

The test item in the vehicle (peanut oil) was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats. The low- and mid-dose groups each consisted of 12 rats/sex and the high-dose group consisted of 17 rats/sex. Dosage levels were 250, 500, and 1000 mg/kg bw/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 17 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 10 or 11 weeks of age at the beginning of test item administration. Twelve males/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses. Twelve females/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 39-53 doses; females that failed to deliver or had no evidence of mating were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 24) for a total of 39-52 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 28 doses for males and 39 doses for the females, these animals were assigned to the post-treatment period and remained on study for a 14-day non-dosing period.

 

All animals were observed twice daily for mortality and moribundity. Detailed physical examinations, body weights, and food consumption were recorded at appropriate intervals. FOB and locomotor activity data were recorded for 6 treatment phase males/group following approximately 28 days of dose administration and for 6 treatment phase females/group on lactation day 4. All F0 females were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Pups were euthanized and discarded on PND 4. F0 males were euthanized following completion of the mating period or following the 14-day recovery period. F0 females were euthanized on lactation day 4 for females that delivered, on post-mating day 25 for females that failed to deliver, on post-cohabitation day 25 for females that had no evidence of mating, or following the 14-day recovery period. Clinical pathology evaluations (hematology and serum chemistry) were performed on 6 treatment phase F0 animals/sex/group and 5 recovery phase F0 animals/sex in the control and high-dose groups at the respective necropsies. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Spermatogenic endpoints (sperm motility, morphology, and numbers) were recorded for all treatment phase males. Selected tissues were examined microscopically from all F0 animals in the control and high-dosage groups. In addition, the colon, ileum, and mesenteric lymph nodes from all F0 animals in the low- and mid-dosage groups and all animals in the post-treatment phase were examined microscopically.

 

Results

One female in the 500 mg/kg bw/day group was found dead on study day 13 prior to pairing and it was not considered test item-related. Test item-related clinical findings were primarily noted at the time of dose administration or approximately 1 hour following dose administration and included clear and red material around the nose and mouth and salivation. These findings were observed in the 500 and 1000 mg/kg bw/day group males and females generally throughout the treatment period. Limited occurrences of red and/or clear material around the nose and mouth or salivation were also observed in the 250 mg/kg bw/day group males and females. The aforementioned clinical findings were considered test item-related but not adverse. Test item-related, lower mean body weight gains were noted for the 1000 mg/kg bw/day group males generally throughout the treatment period, resulting in lower mean body weight gains when the entire pre-mating period (study days 0-13) and entire treatment period (study days 0-27) were evaluated. These effects corresponded to decrements in mean food consumption for the 1000 mg/kg bw/day group males during the pre-mating period. As a result, mean body weights for the 1000 mg/kg bw/day group males were lower than the control group during study days 13-28. During the post-treatment period, mean body weight gains and food consumption for the 1000 mg/kg bw/day group males were similar to the control group, but mean body weights continued to be lower than the control group throughout the post-treatment period. Mean body weights, body weight gains, and food consumption for the 250 and 500 mg/kg bw/day group males were similar to the control group. Mean body weights, body weight changes, and food consumption for the 250, 500, and 1000 mg/kg bw/day group females were unaffected by test item administration during the pre-mating period and remainder of the treatment period for females that were not selected for breeding. Mean body weight gain and food consumption for the 1000 mg/kg bw/day group during the overall post-treatment period (study days 39-52) was similar to the control group, but mean body weights in the 1000 mg/kg bw/day group females remained lower than the control group throughout the post-treatment period. During lactation, a test item-related, mean body weight loss with corresponding lower mean food consumption was noted for the 1000 mg/kg bw/day group during lactation days 1-4 and resulted in a mean body weight for the 1000 mg/kg bw/day group females that was 5.7% lower than the control group on lactation day 4. No test item-related effects on mean body weights, body weight gains, and food consumption were noted in the 250, 500, and 1000 mg/kg bw/day groups during gestation and the 250 and 500 mg/kg bw/day groups during lactation days 1-4. Test item-related higher mean reticulocyte count and lower mean corpuscular volume and mean corpuscular hemoglobin values were observed for males in the 1000 mg/kg bw/day group following the 14-day post-treatment period. In addition, test item-related higher mean red blood cell count, hemoglobin value, and hematocrit percentage were noted for females in the 1000 mg/kg bw/day group following the 14-day post-treatment period. These changes were considered adaptive responses and were not adverse. At the end of the treatment period, test item-related, non-adverse, higher mean alanine aminotransferase and urea nitrogen values were noted for males in the 1000 mg/kg bw/day group, and higher mean potassium levels were noted for females in the 1000 mg/kg bw/day group. Following a 14-day post-treatment period, lower mean glucose values were noted in the 1000 mg/kg bw/day group males and females; but the values were within the historical control database range for males, whereas in females the observed values were less than the historical control database range. The lower mean glucose values for the 1000 mg/kg bw/day group males and females were not considered adverse. There were no test item-related effects on hematology or serum chemistry parameters in the 250 and 500 mg/kg bw/day group male and females.

 

F0 male and female mating and fertility, male copulation, and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels. No test item related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, and morphology) in males at any dosage level. Mean numbers of implantation sites, corpora lutea, unaccounted-for sites, mean number of F1 pups born, live litter size, the percentage of F1 males at birth, and F1 postnatal survival in the 250, 500, and 1000 mg/kg bw/day groups were similar to the control group values. Mean F1 birth weights (PND 1) for the 1000 mg/kg bw/day group were 9.9% (males) and 7.6% (females) lower than the control group. During PND 1-4, mean pup body weight gains were lower than the control group and resulted in mean pup body weights in the 1000 mg/kg bw/day group that were 17.8% (males) and 18.6% (females) lower than the control group on PND 4. Mean pup body weights and body weight changes in the 250 and 500 mg/kg bw/day groups were not remarkably different than the control group. There were no remarkable internal findings observed at the necropsy of pups that were found dead during PND 0-4. There were no test item-related internal findings or effects on organ weights observed at any dosage level at the scheduled necropsy. At the end of the treatment period, an increased incidence of granulomatous inflammation of the mesenteric lymph nodes and lymphoid hyperplasia of the colon and ileum were noted in the 1000 mg/kg bw/day group males and/or females. The lymphoid hyperplasia in the ileum and colon had resolved after the 14-day post-treatment period and was most likely an adaptive physiologic response to immune stimulation. Granulomatous inflammation of the mesenteric lymph nodes was still evident in all of the 1000 mg/kg bw/day group females and 4 of 5 of the males after the 14-day post-treatment period. Although not completely resolved, the presence of this finding was less severe. Thus, granulomatous inflammation of the mesenteric lymph nodes was considered test item-related and adverse, but it is noted that the inflammation was partially resolved after the 14-day post-treatment period.

Conclusions

Under the test conditions, the NOAEL of test item was 1000 mg/kg bw/day for reproductive toxicity in rats.

Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity
Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Effects on fertility

In an OECD 422: A Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Rats, with Recovery, the test item, in the vehicle (peanut oil) was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats. The low- and mid-dose groups each consisted of 12 rats/sex and the high-dose group consisted of 17 rats/sex. Dosage levels were 250, 500, and 1000 mg/kg bw/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 17 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 10 or 11 weeks of age at the beginning of test item administration. Twelve males/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses. Twelve females/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 39-53 doses; females that failed to deliver or had no evidence of mating were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 24) for a total of 39-52 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 28 doses for males and 39 doses for the females, these animals were assigned to the post-treatment period and remained on study for a 14-day non-dosing period.

 

One female in the 500 mg/kg bw/day group was found dead on study day 13 prior to pairing and it was not considered test item-related. Test item-related clinical findings were primarily noted at the time of dose administration or approximately 1 hour following dose administration and included clear and red material around the nose and mouth and salivation. These findings were observed in the 500 and 1000 mg/kg bw/day group males and females generally throughout the treatment period. Limited occurrences of red and/or clear material around the nose and mouth or salivation were also observed in the 250 mg/kg bw/day group males and females. The aforementioned clinical findings were considered test item-related but not adverse. No test item-related effects were noted during the FOB or locomotor activity evaluations for F0 males and females at any dosage level. Test item-related, lower mean body weight gains were noted for the 1000 mg/kg bw/day group males generally throughout the treatment period, resulting in lower mean body weight gains when the entire pre-mating period (study days 0-13) and entire treatment period (study days 0-27) were evaluated. These effects corresponded to decrements in mean food consumption for the 1000 mg/kg bw/day group males during the pre-mating period. Mean body weights, body weight gains, and food consumption for the 250 and 500 mg/kg bw/day group males were similar to the control group. Mean body weights, body weight changes, and food consumption for the 250, 500, and 1000 mg/kg bw/day group females were unaffected by test item administration during the pre-mating period and remainder of the treatment period for females that were not selected for breeding. No test item-related effects on mean body weights, body weight gains, and food consumption were noted in the 250, 500, and 1000 mg/kg bw/day groups during gestation and the 250 and 500 mg/kg bw/day groups during lactation days 1-4.

 

F0 male and female mating and fertility, male copulation, and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels. No test item related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, and morphology) in males at any dosage level. Mean numbers of implantation sites, corpora lutea, unaccounted-for sites, mean number of F1 pups born, live litter size, the percentage of F1 males at birth, and F1 postnatal survival in the 250, 500, and 1000 mg/kg bw/day groups were similar to the control group values. Mean F1 birth weights (PND 1) for the 1000 mg/kg bw/day group were 9.9% (males) and 7.6% (females) lower than the control group. During PND 1-4, mean pup body weight gains were lower than the control group and resulted in mean pup body weights in the 1000 mg/kg bw/day group that were 17.8% (males) and 18.6% (females) lower than the control group on PND 4. Mean pup body weights and body weight changes in the 250 and 500 mg/kg bw/day groups were not remarkably different than the control group. There were no remarkable internal findings observed at the necropsy of pups that were found dead during PND 0-4. There were no test item-related internal findings or effects on organ weights observed at any dosage level at the scheduled necropsy.

 

Under the test conditions, the NOAEL of test item was 1000 mg/kg bw/day for reproductive toxicity in rats.

Extended one-generation reproductive toxicity study

The extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that suggest adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity.


Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity screening

Under the test conditions, the NOAEL of test item was 500 mg/kg bw/day for neonatal toxicity, based on the lower mean F1 birth weights, pup body weight gains during PND 1-4 and pup body weights on PND 4 at 1000 mg/kg bw/day in male and female rats (OECD 422).

Prenatal developmental toxicity

The No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity was considered to be 750 mg/kg/day, the highest dose level evaluated (OECD 414).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-07-31 to 2013-01-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 422
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age and body weight at study initiation: Study Day 0: 10-11 weeks (323-419 g for males; 202-287 g for females); Gestation day 0: 12-13 weeks (216-336 g for females)
- Fasting period before study: No
- Housing: Following receipt and until pairing, all F0 animals were housed individually (except during the mating period for 12 rats/sex/group) in clean, stainless steel wire-mesh cages suspended above cage-board. The cage-board was changed at least 3 times per week. The 5 rats/sex in the control and high-dosage groups assigned to the post-treatment period were not paired and remained in clean, stainless steel wire-mesh cages until euthanasia. The breeding phase rats (12/sex/group) were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding. The dams and their litters were housed in these cages until euthanasia on lactation day 4. Females with no evidence of mating or that failed to deliver were housed in plastic maternity cages until post-cohabitation or post-mating day 25.- Diet: Basal diet, ad libitum- Water: Reverse osmosis-purified (on-site) drinking water, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 70.5-71.0 °F (21.4-21.7 °C) - Humidity: 47.9-54.3%
- Air changes: 10 fresh air changes per hour
- Photoperiod: 12 h dark / 12 h light
Route of administration:
oral: gavage
Vehicle:
peanut oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated, protected from light. The test item formulations were stirred for at least 30 minutes prior to daily dispensation and continuously throughout the preparation, sampling, and dose administration procedures.
VEHICLE- Concentration in vehicle: 0, 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw/day- Lot/batch no. (if required): ZV0181, 1BG0536, and 2AI0653, exp. dates: 23 September 2012, 6 July 2013, and 16 September 2013, respectively from Spectrum Chemical Manufacturing Corporation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Prior to the initiation of dose administration, samples for homogeneity determination were collected from the top, middle, and bottom strata of the 50 and 200 mg/mL dosing formulations. In addition, samples for resuspension homogeneity determinations were collected from the top and bottom strata of these same dosing suspensions following refrigerated storage for 9 days and a period of remixing for a minimum of 30 minutes.
- Samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared for the first and last weeks of dose administration (corresponding to all groups on study being dosed). One set of samples from each collection was subjected to the appropriate analyses
- Results: The analyzed dosing formulations were within the range for suspensions (85-115 %) and were homogeneous. The test item was not detected in the vehicle formulation that was administered to the control group (Group 1). Analytical method (Coffee ST 2013, WIL Report No.: WIL-168204)
HPLC Method:Instrument: Dionex UltiMate™ 3000 high performance liquidchromatograph equipped with a charged aerosol detector,autosampler, and Dionex Chromeleon® software version6.8, or equivalent system
Column: Phenomenex Phenogel® 50Å, 300 mm × 7.80 mm,5-μm particle-size
Column Temperature: 40°C
Mobile Phase: Chloroform
Flow Rate: 0.7 mL/minuteInjection
Volume: 20 μLDetector: Charged aerosol detectorRetention
Time: Approximately 11 minutes
Run Time: 15 minutes
Formulation preparation: Suspension formulations prepared at 20, 50 and 200 mg/L.
The appropriate amount of the test substance for each formulation was weighed in a tared, calibrated glass container. Approximately 70% of the vehicle was added to each container. The formulations were mixed as necessary until uniform. The formulations were then brought to the calibration target with vehicle. Formulations were moved to a heated water bath set at between 45°C to 50°C (Group Low and High) or 55°C to 60°C (Group 2 and 4) while stirring and heated until uniform. The test substance formulations were stirred continuously throughout the preparation and sampling procedures.
Test item stability: Processed samples were stored at room temperature for a minimum of 3 days before being re-analyzed to assess test item stability.
The mean 3-day post-storage values ranged from 102% to 124% of the pre-storage values.Formulations prepared at target concentrations of 20 and 200 mg/mL were analyzed on the day of preparation.
Aliquots of theformulations were stored refrigerated for at least 4 days or 10 days and analyzed to assess test item stability. The mean post-storage concentration ranged from 95.4% to 116% of pre-storage values.
Formulations prepared at target test item concentrations of 50 and 200 mg/mL met the protocol-specified acceptance criteria for stabilityfollowing 5 days of frozen (approximately -20°C) storage.
Results of the stability tests can be found in the attached document.
Details on mating procedure:
- Impregnation procedure: Cohoused- If cohoused:- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: Presence of a vaginal copulatory plug or the presence of sperm in vaginal lavage referred to as day 0 of pregnancy.
- 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
Duration of treatment / exposure:
Males: 12 males/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses. Females: 12 females/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 39-53 doses; females that failed to deliver or had no evidence of mating were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 24) for a total of 39-52 doses.
Frequency of treatment:
Once daily
Duration of test:
Until lactation day 4
No. of animals per sex per dose:
- Vehicle control and high dose group: 17 rats/sex/dose- Low and mid-dose group: 12 rats/sex/dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were selected based on the results of a previous oral (gavage) 14-day range finding study (Toot, Draft, WIL-168201) with test material and an oral (gavage) 1-generation reproduction study in the rat with a chemically similar borate ester. In both the studies, no toxicity was observed at the maximum dose level of 1000 mg/kg bw/day

- Rationale for animal assignment: Animals were assigned into different groups by computerized randomization procedure based on body weight stratification in a block design.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were conducted weekly (prior to test item administration during the treatment period).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual female body weights were recorded weekly until evidence of copulation and GD 0, 4, 7, 11, 14, 17 and 20 and on lactation days 0, 1 and 4.

FOOD CONSUMPTION:- Individual food consumption was recorded on the corresponding weekly body weight days until pairing. Food intake was not recorded during the mating period for animals selected for pairing, while food consumption continued to be recorded until euthanasia for animals not selected for pairing. Once evidence of mating was observed, female food consumption was recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4. Following mating, food consumption for females with no evidence of mating was measured on a weekly basis until the scheduled euthanasia. - Food consumption was reported as g/animal/day and g/kg/day for the corresponding body weight change intervals.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on lactation day 4
- Organs examined: according to guidelines
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: YesExaminations included:- Gravid uterus weight: No- Number of corpora lutea: Yes - Number of implantations: Yes- Number of early resorptions: No (dams delivered)- Number of late resorptions: No (dams delivered)
Fetal examinations:
- External examinations: Yes, F1 rats delivered and the dams and her litter were euthanized on LD/PND 4. All pups were examined.
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. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
Indices:
Litter parameters were defined as follows:Mean Live Litter Size = Total No. of Viable Pups on PND 0/ No. of Litters with Viable Pups PND 0 Postnatal Survival Between Birth and PND 0 or PND 4 (Pre-selection) (% Per Litter) = (Sum of (Viable Pups Per Litter on PND 0 or PND 4 [Pre-selection]/No. of Pups Born Per Litter)/No. of Litters Per Group) x 100Postnatal Survival for All Other Intervals (% Per Litter) = (Sum of (Viable Pups Per Litter at End of Interval N/Viable Pups Per Litter at Start of Interval N)/No. of Litters Per Group) x 100Where N= PND 0-1 and 1-4
Historical control data:
Results were compared with historical control data.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
See details on results
Description (incidence and severity):
Not relevant
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
See details on results
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
See details on results
Gross pathological findings:
no effects observed
Description (incidence and severity):
See details on results
Neuropathological findings:
not examined
Description (incidence and severity):
See details on results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
See details on results
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
not examined
Details on maternal toxic effects:
Maternal toxic effects:yesDetails on maternal toxic effects:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)- One female in the 500 mg/kg bw/day group was found dead on study day 13 prior to pairing, but this was not considered test item-related.- Test item-related clinical findings were primarily noted at the time of dose administration or approximately 1 hour following dose administration and included clear and red material around the nose and mouth and salivation. These findings were observed in the 500 and 1000 mg/kg bw/day group males and females generally throughout the treatment period. Limited occurrences of red and/or clear material around the nose and mouth or salivation were also observed in the 250 mg/kg bw/day group males and females. The aforementioned clinical findings were considered test item-related but not adverse.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Test item-related, lower mean body weight gains were noted for the 1000 mg/kg bw/day group males generally throughout the treatment period, resulting in lower mean body weight gains when the entire pre-mating period (study days 0-13) and entire treatment period (study days 0-27) were evaluated. These effects corresponded to decrements in mean food consumption for the 1000 mg/kg bw/day group males during the pre-mating period. As a result, mean body weights for the 1000 mg/kg bw/day group males were lower than the control group during study days 13-28. During the post-treatment period, mean body weight gains and food consumption for the 1000 mg/kg bw/day group males were similar to the control group, but mean body weights continued to be lower than the control group throughout the post-treatment period. Mean body weights, body weight gains, and food consumption for the 250 and 500 mg/kg bw/day group males were similar to the control group.- Mean body weights, body weight changes, and food consumption for the 250, 500, and 1000 mg/kg bw/day group females were unaffected by test item administration during the pre-mating period and remainder of the treatment period for females that were not selected for breeding. Mean body weight gain and food consumption for the 1000 mg/kg bw/day group during the overall post-treatment period (study days 39-52) was similar to the control group, but mean body weights in the 1000 mg/kg bw/day group females remained lower than the control group throughout the post-treatment period.- During lactation, a test item-related, mean body weight loss with corresponding lower mean food consumption was noted for the 1000 mg/kg bw/day group during lactation days 1-4 (compared to a mean body weight gain in the control group) and resulted in a mean body weight for the 1000 mg/kg bw/day group females that was 5.7% lower than the control group on lactation day 4. No test item-related effects on mean body weights, body weight gains, and food consumption were noted in the 250, 500, and 1000 mg/kg bw/day groups during gestation and the 250 and 500 mg/kg bw/day groups during lactation days 1-4.

HAEMATOLOGY AND CLINICAL CHEMISTRY
- Test item-related higher mean reticulocyte count and lower mean corpuscular volume and mean corpuscular hemoglobin values were observed for males in the 1000 mg/kg bw/day group following the 14-day post-treatment period. In addition, test item-related higher mean red blood cell count, hemoglobin value, and hematocrit percentage were noted for females in the 1000 mg/kg bw/day group following the 14-day post-treatment period. These changes were considered adaptive responses and were not adverse.
- At the end of the treatment period, test item-related, non-adverse, higher mean alanine aminotransferase and urea nitrogen values were noted for males in the 1000 mg/kg bw/day group, and higher mean potassium levels were noted for females in the 1000 mg/kg bw/day group. Following a 14-day post-treatment period, lower mean glucose values were noted in the 1000 mg/kg bw/day group males and females. For the males, all individual glucose values and the group mean value were within the historical control database range. However, the glucose values for 4 of 5 females in the 1000 mg/kg bw/day group and the group mean value were lower than the minimum mean value in the historical control database. The lower mean glucose values for the 1000 mg/kg bw/day group males and females were not considered adverse. There were no test item-related effects on hematology or serum chemistry parameters in the 250 and 500 mg/kg bw/day group male and females.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)- No test item related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, and morphology) in males at any dosage level.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- F0 male and female mating and fertility, male copulation, and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- There was no test item-related internal findings or effects on organ weights observed at any dosage level at the scheduled necropsy.- The differences were slight and not statistically significant, with the following exceptions. Mean adrenal gland weight relative to final body weight for the 1000 mg/kg bw/day group females on lactation day 4 was significantly (p<0.05) higher than the control group, but was attributed to the lower mean final body weight for the 1000 mg/kg bw/day group females and was not considered test item-related. In addition, the mean thymus weight relative to final body weight for the 1000 mg/kg bw/day group females at the end of the post-treatment period was significantly (p<0.05) higher than the control group and was attributed to the lower mean final body weight for the 1000 mg/kg bw/day group females.

GROSS PATHOLOGY (PARENTAL ANIMALS)- No test item-related internal findings were observed at any dosage level in females that failed to deliver or males and females at the scheduled necropsy. Macroscopic findings observed in the test item-treated groups occurred infrequently and/or in a manner that was not dose-related.- The mean numbers of unaccounted-for sites, implantation sites, and corpora lutea in the 250, 500 and 1000 mg/kg bw/day groups were similar to the control group values.

HISTOPATHOLOGY (PARENTAL ANIMALS)- At the end of the treatment period, an increased incidence of granulomatous inflammation of the mesenteric lymph nodes and lymphoid hyperplasia of the colon and ileum were noted in the 1000 mg/kg bw/day group males and/or females. The lymphoid hyperplasia in the ileum and colon had resolved after the 14-day post-treatment period and was most likely an adaptive physiologic response to immune stimulation.- Granulomatous inflammation of the mesenteric lymph nodes was still evident in all of the 1000 mg/kg bw/day group females and 4 of 5 of the males after the 14-day post-treatment period. Although not completely resolved, the presence of this finding was less severe. Thus, granulomatous inflammation of the mesenteric lymph nodes was considered test item-related and adverse, but it is noted that the inflammation was partially resolved after the 14-day post-treatment period.
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
effects observed, treatment-related
Fetal body weight changes:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): See details on results
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
See details on results
Changes in sex ratio:
no effects observed
Description (incidence and severity):
See details on results
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
See details on results
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
See details on results
External malformations:
no effects observed
Description (incidence and severity):
See details on results
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
not examined
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes
Details on embryotoxic / teratogenic effects:VIABILITY (OFFSPRING)
- Mean number of pups born, live litter size, and the percentage of males at birth in the 250, 500, and 1000 mg/kg bw/day groups were similar to the control group values. In addition, postnatal survival through PND 4 in the 250, 500 and 1000 mg/kg bw/day groups was unaffected by maternal test item administration.

CLINICAL SIGNS (OFFSPRING)
- General physical condition (defined as the occurrence and severity of clinical findings) of all F1 pups in this study was unaffected by maternal test item administration.

BODY WEIGHT (OFFSPRING)
- Mean F1 birth weights (PND 1) for the 1000 mg/kg bw/day group were 9.9% (males) and 7.6% (females) lower than the control group. During PND 1-4, mean pup body weight gains were lower than the control group and resulted in mean pup body weights in the 1000 mg/kg bw/day group that were 17.8% (males) and 18.6% (females) lower than the control group on PND 4. Mean pup body weights and body weight changes in the 250 and 500 mg/kg bw/day groups were not remarkably different than the control group.

GROSS PATHOLOGY (OFFSPRING)
- There were no remarkable internal findings observed at the necropsy of pups that were found dead during PND 0-4.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
maternal dose
Sex:
male/female
Basis for effect level:
fetal/pup body weight changes
Abnormalities:
effects observed, treatment-related
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects

See attached document for Tables of Results

Conclusions:
Under the test conditions, the NOAEL of test item was 500 mg/kg bw/day for neonatal toxicity, based on the lower mean F1 birth weights, pup body weight gains during PND 1-4 and pup body weights on PND 4 at 1000 mg/kg bw/day in male and female rats.
Executive summary:

Test Guidance

OECD 422: A Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Rats, with Recovery.

 

Materials and methods

The test item in the vehicle (peanut oil) was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats. The low- and mid-dose groups each consisted of 12 rats/sex and the high-dose group consisted of 17 rats/sex. Dosage levels were 250, 500, and 1000 mg/kg bw/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 17 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 10 or 11 weeks of age at the beginning of test item administration. Twelve males/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses. Twelve females/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 39-53 doses; females that failed to deliver or had no evidence of mating were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 24) for a total of 39-52 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 28 doses for males and 39 doses for the females, these animals were assigned to the post-treatment period and remained on study for a 14-day non-dosing period.

 

All animals were observed twice daily for mortality and moribundity. Detailed physical examinations, body weights, and food consumption were recorded at appropriate intervals. FOB and locomotor activity data were recorded for 6 treatment phase males/group following approximately 28 days of dose administration and for 6 treatment phase females/group on lactation day 4. All F0 females were allowed to deliver and rear their pups until lactation day 4. F1 clinical observations and body weights were recorded on PND 1 and 4. Pups were euthanized and discarded on PND 4. F0 males were euthanized following completion of the mating period or following the 14-day recovery period. F0 females were euthanized on lactation day 4 for females that delivered, on post-mating day 25 for females that failed to deliver, on post-cohabitation day 25 for females that had no evidence of mating, or following the 14-day recovery period. Clinical pathology evaluations (hematology and serum chemistry) were performed on 6 treatment phase F0 animals/sex/group and 5 recovery phase F0 animals/sex in the control and high-dose groups at the respective necropsies. Complete necropsies were conducted on all F0 animals, and selected organs were weighed. Spermatogenic endpoints (sperm motility, morphology, and numbers) were recorded for all treatment phase males. Selected tissues were examined microscopically from all F0 animals in the control and high-dosage groups. In addition, the colon, ileum, and mesenteric lymph nodes from all F0 animals in the low- and mid-dosage groups and all animals in the post-treatment phase were examined microscopically.

 

Results

One female in the 500 mg/kg bw/day group was found dead on study day 13 prior to pairing and it was not considered test item-related. Test item-related clinical findings were primarily noted at the time of dose administration or approximately 1 hour following dose administration and included clear and red material around the nose and mouth and salivation. These findings were observed in the 500 and 1000 mg/kg bw/day group males and females generally throughout the treatment period. Limited occurrences of red and/or clear material around the nose and mouth or salivation were also observed in the 250 mg/kg bw/day group males and females. The aforementioned clinical findings were considered test item-related but not adverse. Test item-related, lower mean body weight gains were noted for the 1000 mg/kg bw/day group males generally throughout the treatment period, resulting in lower mean body weight gains when the entire pre-mating period (study days 0-13) and entire treatment period (study days 0-27) were evaluated. These effects corresponded to decrements in mean food consumption for the 1000 mg/kg bw/day group males during the pre-mating period. As a result, mean body weights for the 1000 mg/kg bw/day group males were lower than the control group during study days 13-28. During the post-treatment period, mean body weight gains and food consumption for the 1000 mg/kg bw/day group males were similar to the control group, but mean body weights continued to be lower than the control group throughout the post-treatment period. Mean body weights, body weight gains, and food consumption for the 250 and 500 mg/kg bw/day group males were similar to the control group. Mean body weights, body weight changes, and food consumption for the 250, 500, and 1000 mg/kg bw/day group females were unaffected by test item administration during the pre-mating period and remainder of the treatment period for females that were not selected for breeding. Mean body weight gain and food consumption for the 1000 mg/kg bw/day group during the overall post-treatment period (study days 39-52) was similar to the control group, but mean body weights in the 1000 mg/kg bw/day group females remained lower than the control group throughout the post-treatment period. During lactation, a test item-related, mean body weight loss with corresponding lower mean food consumption was noted for the 1000 mg/kg bw/day group during lactation days 1-4 (compared to a mean body weight gain in the control group) and resulted in a mean body weight for the 1000 mg/kg bw/day group females that was 5.7% lower than the control group on lactation day 4. No test item-related effects on mean body weights, body weight gains, and food consumption were noted in the 250, 500, and 1000 mg/kg bw/day groups during gestation and the 250 and 500 mg/kg bw/day groups during lactation days 1-4. Test item-related higher mean reticulocyte count and lower mean corpuscular volume and mean corpuscular hemoglobin values were observed for males in the 1000 mg/kg bw/day group following the 14-day post-treatment period. In addition, test item-related higher mean red blood cell count, hemoglobin value, and hematocrit percentage were noted for females in the 1000 mg/kg bw/day group following the 14-day post-treatment period. These changes were considered adaptive responses and were not adverse. At the end of the treatment period, test item-related, non-adverse, higher mean alanine aminotransferase and urea nitrogen values were noted for males in the 1000 mg/kg bw/day group, and higher mean potassium levels were noted for females in the 1000 mg/kg bw/day group. Following a 14-day post-treatment period, lower mean glucose values were noted in the 1000 mg/kg bw/day group males and females. For the males, all individual glucose values and the group mean value were within the historical control database range. However, the glucose values for 4 of 5 females in the 1000 mg/kg bw/day group and the group mean value were lower than the minimum mean value in the historical control database. The lower mean glucose values for the 1000 mg/kg bw/day group males and females were not considered adverse. There were no test item-related effects on hematology or serum chemistry parameters in the 250 and 500 mg/kg bw/day group male and females.

F0 male and female mating and fertility, male copulation, and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels. No test item related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, and morphology) in males at any dosage level. Mean numbers of implantation sites, corpora lutea, unaccounted-for sites, mean number of F1 pups born, live litter size, the percentage of F1 males at birth, and F1 postnatal survival in the 250, 500, and 1000 mg/kg bw/day groups were similar to the control group values. Mean F1 birth weights (PND 1) for the 1000 mg/kg bw/day group were 9.9% (males) and 7.6% (females) lower than the control group. During PND 1-4, mean pup body weight gains were lower than the control group and resulted in mean pup body weights in the 1000 mg/kg bw/day group that were 17.8% (males) and 18.6% (females) lower than the control group on PND 4. Mean pup body weights and body weight changes in the 250 and 500 mg/kg bw/day groups were not remarkably different than the control group. There were no remarkable internal findings observed at the necropsy of pups that were found dead during PND 0-4.There were no test item-related internal findings or effects on organ weights observed at any dosage level at the scheduled necropsy. At the end of the treatment period, an increased incidence of granulomatous inflammation of the mesenteric lymph nodes and lymphoid hyperplasia of the colon and ileum were noted in the 1000 mg/kg bw/day group males and/or females. The lymphoid hyperplasia in the ileum and colon had resolved after the 14-day post-treatment period and was most likely an adaptive physiologic response to immune stimulation. Granulomatous inflammation of the mesenteric lymph nodes was still evident in all of the 1000 mg/kg bw/day group females and 4 of 5 of the males after the 14-day post-treatment period. Although not completely resolved, the presence of this finding was less severe. Thus, granulomatous inflammation of the mesenteric lymph nodes was considered test item-related and adverse, but it is noted that the inflammation was partially resolved after the 14-day post-treatment period.

Conclusions

Under the test conditions, the NOAEL of test item was 500 mg/kg bw/day for neonatal toxicity, based on the lower mean F1 birth weights, pup body weight gains during PND 1-4 and pup body weights on PND 4 at 1000 mg/kg bw/day in male and female rats.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 May 2018 to 22 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
animal room temperature deviation with no impact on results or integrity of the study (see below)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
HSD: Sprague Dawley SD
Details on test animals or test system and environmental conditions:
ANIMAL ACQUISITION AND ACCLIMATION
- All animals were given a detailed clinical examination and body weights were recorded prior to selection on GD 0.
- The findings from the clinical examinations recorded during the acclimation period were not reported but were maintained in the study file.

RANDOMIZATION, ASSIGNMENT TO STUDY AND MAINTENANCE
- Using a standard, by weight, randomization procedure, 80 female animals (weighing 179 to 245 g, at randomization) were assigned to the control and treatment groups as shown in Table C below.
- Animals assigned to study had body weights within ± 20 % of the mean body weight. Extra animals obtained, but not placed on study, were euthanized by carbon dioxide inhalation followed by an MPI Research SOP approved method to ensure death. The carcasses were discarded.
- Each animal was assigned an animal number to be used in the Provantis data collection system and was implanted with a microchip bearing a unique identification number. The individual animal number, implant number, and study number comprised a unique identification for each animal. Each cage was identified by the animal number, study number, group number, and sex.
- The animals were individually housed in solid bottom cages with nonaromatic bedding in an environmentally controlled room. Animal enrichment was provided according to SOP. Fluorescent lighting was provided for approximately 12 hours per day. Temperature and humidity were continuously monitored, recorded, and maintained to the maximum extent possible within the ranges of 68 to 79 °F and 30 to 70 %, respectively. The actual temperature and humidity data are not reported but are maintained in the study file.


Route of administration:
oral: gavage
Vehicle:
peanut oil
Remarks:
Spectrum Chemical Manufacturing Corporation New Brunswick, NJ (Lot number 2GK0218) or Spectrum Chemical Manufacturing Corporation Gardena, CA (Lot number 1GL0495); Expiry date 30 Nov 2018; Storage conditions controlled room temperature under nitrogen.
Details on exposure:
VEHICLE AND TEST ARTICLE PREPARATION
- Fresh vehicle, peanut oil, was dispensed for use on study weekly and was stored at controlled room temperature and protected from light.
- The test article, was used as received from the Sponsor. No adjustment was made for purity when preparing the test article formulations. Formulations of the test article were prepared by mixing the appropriate amount of vehicle with the appropriate amount of test article to achieve nominal concentrations of 50, 100, and 150 mg/mL. The formulations were mixed on a hot plate or in a water bath set at 60 ± 2 °C until uniform in appearance. - Formulations were prepared weekly and were stored at controlled room temperature and protected from light.

ADMINISTRATION
- The vehicle and test article were administered once daily from GD 0 to 19 at approximately the same time each day (± 2 hours from the GD 0 dose) via oral gavage.
- The dose levels for the treated groups were 250, 500, and 750 mg/kg/day at a dose volume of 5 mL/kg.
- The vehicle and test article formulations were continually stirred prior to and throughout dose administration.
- Individual doses were based on the most recent body weights.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
ANALYSIS OF DOSING FORMULATIONS
- Dosing formulations prepared for the study were evaluated for homogeneity and concentration.
- Appropriate samples (see Table B, attached) were collected using a positive displacement pipette, while stirring and placed into amber glass scintillation vials.
- Upon receipt of the authorization to finalize the main study report, backup samples were discarded.
- The stability of the formulations at 50 and 200 mg/mL was previously established for 10 days of refrigerated storage (WIL-168204).

ANALYSES
- All analytical work was conducted by MPI Research, using an analytical method developed and validated under MPI Research Study Number 1928-022. No deviations from the analytical method occurred during the course of this study.
Details on mating procedure:
A total of 88 time-mated female HSD: Sprague Dawley SD rats (approximately 9.5 to 10 weeks of age) were received from Envigo RMS, Inc., from May 1 to May 2, 2018.
Duration of treatment / exposure:
Gestation Day 0 through 19
Frequency of treatment:
Once daily
Duration of test:
Gestation Day 0 to Gestation Day 20 when each surviving female was euthanized by carbon dioxide inhalation.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 time-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
JUSTIFICATION FOR ROUTE OF EXPOSURE
- The oral route is a potential route of accidental exposure of this test article in humans.

JUSTIFICATION OF DOSE LEVELS
- The dose levels were selected by the Sponsor, or in consultation with the Sponsor, on the basis of available data from a previous range-finding developmental toxicity study in rats (MPI Research Study Number 1928-025), in which no effects were noted at dose levels up to 750 mg/kg/day.
Maternal examinations:
CAGESIDE OBSERVATIONS
- All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily.

DETAILED CLINICAL OBSERVATIONS
- Daily from GD 0 through 20 (75 minutes ± 15 minutes postdose on dosing days), each animal was removed from the cage and given a detailed clinical examination.
- On occasion, clinical observations were recorded at unscheduled intervals.
- The observations included, but were not limited to, evaluation of the skin, fur, eyes, ears, nose, oral cavity, thorax, abdomen, external genitalia, limbs and feet, as well as evaluation of respiration.

VETERINARY OBSERVATIONS
- On occasion, veterinary observations were conducted during the course of the study. All treatments and observations were recorded.
- The medical treatments and observations are not reported but are maintained in the study file.

BODY WEIGHT AND BODY WEIGHT CHANGES
- Body weights for all animals were measured and recorded on GD 0, 3, 6, 9, 12, 15, 18, and 20.
- Individual body weight change was calculated for the following GD intervals: 0-3, 3-6, 6-9, 9-12, 12-15, 15-18, 18 20 and 0-20.
- Adjusted body weight (GD 20 body weight minus gravid uterine weight) and adjusted body weight change (GD 0 to 20) were also calculated.

FOOD CONSUMPTION
- Food consumption was measured and recorded on the corresponding body weight days and calculated for the same intervals.

MATERNAL NECROPSY
- A complete necropsy was performed on all euthanized in extremis and surviving dams under procedures approved by a veterinary pathologist. Special emphasis was placed on structural abnormalities or pathologic changes that may have influenced the pregnancy.
- The presence of lesions or other abnormal conditions in the dam were noted and described in the study records.
Ovaries and uterine content:
OVARIAN AND UTERINE EXAMINATION
- On GD 20, each surviving female was euthanized by carbon dioxide inhalation, followed by an SOP approved method to ensure death and immediately subjected to a cesarean section. The skin was reflected from a ventral midline incision to examine mammary tissue and locate any subcutaneous masses. The abdominal cavity was then opened, and the uterus was exposed. The uterus was excised, and the gravid uterine weight was recorded. Beginning at the distal end of the left uterine horn, the location of viable and nonviable fetuses, early and late resorptions for each uterine horn, and the total number of implantations were recorded. The number of corpora lutea on each ovary was also recorded.
- Each implant was categorized according to the following criteria. Viable fetuses responded to touch. Nonviable fetuses did not respond to touch and had no signs of autolysis. Late resorptions were characterized by recognizable fetal form, but undergoing autolysis. Early resorptions were characterized as implantation sites that had no recognizable fetal characteristics. The foetuses were removed by making a dorsal incision longitudinally along both uterine horns. The embryonic membrane of each fetus was gently removed, and each fetus was pulled away from the placenta, fully extending the umbilical cord. The placentae were examined grossly.
- Uteri from females that appeared nongravid were opened and placed in 10 % ammonium sulphide solution for detection of implantation sites. If no foci were detected, the female was considered to be nonpregnant.
- The maternal necropsies and subsequent fetal evaluations were conducted without knowledge of the treatment groups in order to minimize bias.
Fetal examinations:
FETAL EXAMINATIONS
- Each fetus was individually weighed, sexed, tagged, and examined for external malformations and variations. Fetuses were then euthanized by intraperitoneal injection of euthanasia solution.
- Approximately one-half of the fetuses in each litter were placed in Bouin’s solution and the remaining fetuses were fixed in alcohol. All fetuses fixed in Bouin’s solution were examined for soft tissue defects using the Wilson razor-blade sectioning technique. The fetuses fixed in alcohol were macerated in potassium hydroxide, stained with Alizarin Red S, and cleared with glycerin by a method similar to that described by Dawson for subsequent skeletal examination.
- Fetal findings were classified as malformations or developmental variations under procedures approved by a developmental toxicologist. On occasion, additional information to clarify or identify a visceral or skeletal observation was documented. These comments were not reported, but were maintained in the study data. Mechanical artifacts (e.g., tail removed and discarded) occurred during examination and processing of the fetuses. These artifacts were not reported, but were maintained in the study data.
Statistics:
See below
Indices:
Details of calculations are provided in the reproproduction glossary (attached)
Historical control data:
See Appendix M (attached)
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- No adverse effects of the test item were seen on clinical observations at any of the dose levels evaluated.
- At 750 mg/kg/day, incidences of red material around the nose and red discoloration of the hair in the cervical region (8 and 4 animals, respectively, out of 20) were increased compared to concurrent controls (4 and 1 animals, respectively, out of 20).
- These findings are common among animals of this age and strain, did not affect the overall health of the animals, and although test article related, were not considered adverse.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
- No test article-related effects were noted on maternal survival at any of the dose levels evaluated.
- On GD 5, one female at 500 mg/kg/day (animal number 3512) was euthanized moribund due to decreased activity, thin body condition, loss of skin elasticity, pale discoloration of the skin, and irregular breathing.
- On GD 6, one female at 250 mg/kg/day (animal number 2505) was euthanized moribund due to decreased activity, thin body condition, loss of skin elasticity, pale discoloration of the skin, and irregular breathing following a body weight loss of 15 g over GD 3-6.
- Macroscopic observations of fluid and foreign material in the thoracic cavity, red discoloration of the lungs, and white discoloration of the pericardium noted for both animals at necropsy, these deaths were considered to be a result of a gavage-related injury and were not considered to be test article related. All animals survived to scheduled study termination.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- No test article-related effects were noted on mean gestation body weight or body weight change at any of the dose levels evaluated.
- Mean body weight and body weight changes values were similar between treated groups and comparable to those observed in concurrent controls.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- No effect of the test item was observed on mean gestation food consumption at any of the dose levels evaluated.
- Mean food consumption values were similar between treated groups and were comparable to control values.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- No adverse test article-related effect was noted on maternal macroscopic observations at any of the dose levels evaluated.
- A finding of small thymus was noted in 8 of 20 dams at 750 mg/kg/day, which was higher than the incidence observed in concurrent controls (4 of 20).
- Although considered test article related, this finding was not considered adverse in the absence of any other maternal findings.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
- No effect of treatment with the test item was observed from ovarian and uterine implantation data.
- The mean number of corpora lutea, implantation sites, viable fetuses, resorption sites (early, late, and total [early + late]), litter size per dam, and mean pre- and postimplantation loss indices in the treated groups were comparable to controls.
- Likewise, mean gravid uterine weights, adjusted GD 20 body weights, and adjusted GD 0-20 body weight change in the treated groups were comparable to controls, and no test article-related effects were observed from these data.
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
- The pregnancy index was 95 % in the control, 250, and 500 mg/kg/day groups and 100 % in the 750 mg/kg/day group.
- One female each in the control, 250, and 500 mg/kg/day groups was not pregnant, and one female at 250 mg/kg/day had a litter consisting of all resorbed fetuses.
- As a result, there were 19, 18, 19, and 20 GD 20 litters for evaluation in the control, 250, 500, and 750 mg/kg/day groups, respectively.
Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks on result:
other: no adverse effects reported at highest dose evaluated
Abnormalities:
no effects observed
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
- No adverse effect of the test item was noted on fetal body weights at any of the dose levels evaluated. At 750 mg/kg/day, mean fetal body weights (male, female, and combined) were 10 % lower than concurrent controls and outside the range seen historically at the testing facility.
- Although considered test article related, in the absence of any other developmental findings, this decrease in fetal body weights was not considered adverse.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
- No test article-related effect was noted on fetal sex ratios (% of live male fetuses per litter) at any of the dose levels evaluated.
- The fetal sex ratios in the treated groups ranged from 49.2 % to 55.2 % and were comparable to the 52.5 % observed in controls.
External malformations:
no effects observed
Description (incidence and severity):
- No external malformations or developmental variations were observed in any of the foetuses evaluated.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No effect of the test item was noted on fetal skeletal malformations or developmental variations at any of the dose levels evaluated.
- Skeletal malformations were noted at low incidence (1 fetus each in the control and 750 mg/kg/day groups) and were considered spurious and not related to treatment with the test material.
- Litter and fetal incidences of developmental variations observed at 500 and 750 mg/kg/day were lower than that seen in control fetuses and were not considered test article related.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
- No fetal visceral malformations were noted in any of the test item-treated groups.
- Developmental variations observed were of low incidence (1 or 2 fetuses), and were considered spurious and not test article related.
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
maternal dose
Sex:
male/female
Remarks on result:
other: no adverse effects reported at highest dose evaluated
Abnormalities:
no effects observed
Developmental effects observed:
no

ANALYSIS OF DOSING FORMULATIONS

- Homogeneity: The homogeneity of the dose formulations was determined for the 50 and 150 mg/mL groups by assaying samples from the top, middle, and bottom strata.

- The relative standard deviation (RSD) of test item concentrations was within acceptance criteria of ≤ 10 % and ranged from 0.491 % to 2.094 % within each group and confirmed formulations were homogeneous (see Table F, attached).

- Concentration: No peak corresponding to test item was observed in the samples from the control group (0 mg/mL). The results for test item concentration was within the acceptance criteria of ± 15 % and ranged from 87.7 % to 99.3 % of nominal for all treatment groups and confirmed that animals received appropriate doses (see Table G, attached).

Conclusions:
The No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity was considered to be 750 mg/kg/day, the highest dose level evaluated.
Executive summary:

GUIDELINE

The objective of the study was to determine the developmental toxicity, including the teratogenic potential of the test article. This study was based on the Organisation for Economic Cooperation and Development (OECD) 414 (Prenatal Developmental Toxicity Study) Guideline for the Testing of Chemicals, January 2001 and in accordance with the Guide for the Care and Use of Laboratory Animals1 and the Public Health Service Policy on Humane Care and Use of Laboratory Animals.

 

METHODS

The vehicle, peanut oil, or test article, was administered to time-mated female HSD: Sprague Dawley SD rats once daily via oral gavage from Gestation Day (GD) 0 through 19. Animals were assigned to 0 mg/kg/day (Group 1), 250 mg/kg/day (Group 2), 500 mg/kg/day (Group 3) or 750 mg/kg/day (Group 4). Each group consisted of 20 time mated females and a dose volume of 5 mL/kg gave dose concentrations equivalent to 0 mg/mL (Group 1), 50 mg/mL (Group 2), 100 mg/mL (Group 3) and 150 mg/mL (Group 4). Observations of the animals included clinical signs, body weights, food consumption, and anatomical pathology including a uterine examination. All foetuses were given an external and visceral or skeletal examination. The homogeneity and concentration results for test item dose formulations of all groups analyzed during the course of the study met the protocol specified acceptance criteria. The control vehicle samples were devoid of test article. Therefore, all dose formulations used for dosing in this study met the acceptance criteria required by the protocol.

 

RESULTS

No adverse effect of the test item was noted on maternal survival, clinical findings, mean gestation body weight, body weight gain, and food consumption, or gross macroscopic findings at any of the dose levels evaluated. Likewise, no adverse effect of the test item was observed from uterine implantation data, fetal sex ratios, fetal body weights, or fetal external, visceral, or skeletal examinations at any of the dose levels evaluated.

 

CONCLUSION

The No Observed Adverse Effect Level (NOAEL) for maternal and developmental toxicity was considered to be 750 mg/kg/day, the highest dose level evaluated.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Prenatal development toxicity screening

In an OECD 422: A Combined 28-Day Repeated Dose Oral (Gavage) Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in Rats, with Recovery, the test item, in the vehicle (peanut oil) was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats. The low- and mid-dose groups each consisted of 12 rats/sex and the high-dose group consisted of 17 rats/sex. Dosage levels were 250, 500, and 1000 mg/kg bw/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 17 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 10 or 11 weeks of age at the beginning of test item administration. Twelve males/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses. Twelve females/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 39-53 doses; females that failed to deliver or had no evidence of mating were dosed through the day prior to euthanasia (post-mating or post-cohabitation day 24) for a total of 39-52 doses. The extra 5 males and 5 females in the control and high-dose groups that were not used for mating were treated beginning on study day 0; following 28 doses for males and 39 doses for the females, these animals were assigned to the post-treatment period and remained on study for a 14-day non-dosing period.

 

One female in the 500 mg/kg bw/day group was found dead on study day 13 prior to pairing and it was not considered test item-related. Test item-related clinical findings were primarily noted at the time of dose administration or approximately 1 hour following dose administration and included clear and red material around the nose and mouth and salivation. These findings were observed in the 500 and 1000 mg/kg bw/day group males and females generally throughout the treatment period. Limited occurrences of red and/or clear material around the nose and mouth or salivation were also observed in the 250 mg/kg bw/day group males and females. The aforementioned clinical findings were considered test item-related but not adverse. No test item-related effects were noted during the FOB or locomotor activity evaluations for F0 males and females at any dosage level. Test item-related, lower mean body weight gains were noted for the 1000 mg/kg bw/day group males generally throughout the treatment period, resulting in lower mean body weight gains when the entire pre-mating period (study days 0-13) and entire treatment period (study days 0-27) were evaluated. These effects corresponded to decrements in mean food consumption for the 1000 mg/kg bw/day group males during the pre-mating period. Mean body weights, body weight gains, and food consumption for the 250 and 500 mg/kg bw/day group males were similar to the control group. Mean body weights, body weight changes, and food consumption for the 250, 500, and 1000 mg/kg bw/day group females were unaffected by test item administration during the pre-mating period and remainder of the treatment period for females that were not selected for breeding. No test item-related effects on mean body weights, body weight gains, and food consumption were noted in the 250, 500, and 1000 mg/kg bw/day groups during gestation and the 250 and 500 mg/kg bw/day groups during lactation days 1-4.

 

F0 male and female mating and fertility, male copulation, and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels. No test item related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, and morphology) in males at any dosage level. Mean numbers of implantation sites, corpora lutea, unaccounted-for sites, mean number of F1 pups born, live litter size, the percentage of F1 males at birth, and F1 postnatal survival in the 250, 500, and 1000 mg/kg bw/day groups were similar to the control group values. Mean F1 birth weights (PND 1) for the 1000 mg/kg bw/day group were 9.9% (males) and 7.6% (females) lower than the control group. During PND 1-4, mean pup body weight gains were lower than the control group and resulted in mean pup body weights in the 1000 mg/kg bw/day group that were 17.8% (males) and 18.6% (females) lower than the control group on PND 4. Mean pup body weights and body weight changes in the 250 and 500 mg/kg bw/day groups were not remarkably different than the control group. There were no remarkable internal findings observed at the necropsy of pups that were found dead during PND 0-4. There were no test item-related internal findings or effects on organ weights observed at any dosage level at the scheduled necropsy.

 

Under the test conditions, the NOAEL of test item was 500 mg/kg bw/day for neonatal toxicity, based on the lower mean F1 birth weights, pup body weight gains during PND 1-4 and pup body weights on PND 4 at 1000 mg/kg bw/day in male and female rats.

Prenatal development toxicity

In an OECD 414: prenatal development toxicity study, the vehicle, peanut oil, or test article, was administered to time-mated female HSD: Sprague Dawley SD rats once daily via oral gavage from Gestation Day (GD) 0 through 19. Animals were assigned to 0 mg/kg/day (Group 1), 250 mg/kg/day (Group 2), 500 mg/kg/day (Group 3) or 750 mg/kg/day (Group 4). Each group consisted of 20 time mated females and a dose volume of 5 mL/kg gave dose concentrations equivalent to 0 mg/mL (Group 1), 50 mg/mL (Group 2), 100 mg/mL (Group 3) and 150 mg/mL (Group 4). Observations of the animals included clinical signs, body weights, food consumption, and anatomical pathology including a uterine examination. All foetuses were given an external and visceral or skeletal examination. The homogeneity and concentration results for test item dose formulations of all groups analyzed during the course of the study met the protocol specified acceptance criteria. The control vehicle samples were devoid of test article. Therefore, all dose formulations used for dosing in this study met the acceptance criteria required by the protocol.

No adverse effect of the test item was noted on maternal survival, clinical findings, mean gestation body weight, body weight gain, and food consumption, or gross macroscopic findings at any of the dose levels evaluated. Likewise, no adverse effect of the test item was observed from uterine implantation data, fetal sex ratios, fetal body weights, or fetal external, visceral, or skeletal examinations at any of the dose levels evaluated.

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of test item for maternal and developmental toxicity was considered to be 750 mg/kg/day, the highest dose level evaluated.

Toxicity to reproduction: other studies

Additional information

Introduction

 

The reproductive and developmental toxicity, and endocrine disruption, potential of the test material were assessed based on the physical chemical properties and toxicity data on the substance and, in the expert statement attached in Section 13, with reference to the residual levels of boric acid (approx. 3 wt%).

 

Metabolism

 

Borate esters are present as components of the UVCB substance. However, the UVCB as a whole is hydrophobic and relatively insoluble (water solubility < 0.17 mg/L at 20 °C and pH 4.4). As a result, borate esters will not be present in solution and hydrolysis and enzymatic metabolism of these components is not expected. This prediction is supported by the absence of reproductive effects characteristic of boric acid exposure in studies with the test material.

 

The absorbable components of this UVCB substance are expected to be metabolised via a number of metabolic pathways although the OECD Toolbox v2.3.0 QSAR system predicts that the metabolism/bioaccumulation will be slow or very slow for the dimer and trimers and moderate for the monomer. Cytochrome P450 (CYP450) enzymes are a superfamily of oxidative catalysts important in the biosynthesis and metabolism of a wide range of endogenous molecules as well as the metabolism of xenobiotics. The four main components of this UVCB substance were subjected to metabolite profiling using the OECD Toolbox v2.3.0 QSAR system and the predicted metabolites were partitioned into chemical categories based on USEPA rules (ECOSAR). The Toolbox predicted a total of 52 potential metabolites from the representative structures; boric acid is not one of the predicted metabolites.

 

Exposure Routes

 

The potential for exposure to this substance is limited by its use and physico-chemical properties. The dermal contact route is considered to be the primary route of occupational exposure. Inhalation exposure is expected to be limited because this substance has a negligible vapour pressure (OECD 2003: negligible <1 Pa; ECHA R15.5: low < 0.1 Pa). Because of the use pattern oral exposure is not an anticipated route of exposure, either to workers or the general public. In the professional work environment dermal exposure will be limited because precautions have been taken because this chemical possesses skin-sensitising properties.

 

Summary of Effects on fertility and developmental toxicity

 

In an OECD 422: combined 28-day repeated dose oral (gavage) toxicity study with reproduction/developmental toxicity screening test in rats, with recovery groups, the test item, in the vehicle (peanut oil) was administered orally by gavage once daily to 3 groups of Crl: CD(SD) rats. The low- and mid-dose groups each consisted of 12 rats/sex and the high-dose group consisted of 17 rats/sex. Dosage levels were 250, 500, and 1000 mg/kg bw/day administered at a dosage volume of 5 mL/kg. A concurrent control group of 17 rats/sex received the vehicle on a comparable regimen. Males and females were approximately 10 or 11 weeks of age at the beginning of test item administration.

 

F0 male and female mating and fertility, male copulation, and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels. No test item related effects were observed on F0 spermatogenesis endpoints (mean testicular and epididymal sperm numbers and sperm production rate, motility, and morphology) in males at any dosage level. Mean numbers of implantation sites, corpora lutea, unaccounted-for sites, mean number of F1 pups born, live litter size, the percentage of F1 males at birth, and F1 postnatal survival in the 250, 500, and 1000 mg/kg bw/day groups were similar to the control group values. Mean F1 birth weights (PND 1) for the 1000 mg/kg bw/day group were 9.9% (males) and 7.6% (females) lower than the control group. During PND 1-4, mean pup body weight gains were lower than the control group and resulted in mean pup body weightsinthe 1000 mg/kg bw/day group that were 17.8% (males) and 18.6% (females) lower than the control group on PND 4. Mean pup body weights and body weight changes in the 250 and 500 mg/kg bw/day groups were not remarkably different than the control group. There were no test item-related internal findings or effects on organ weights observed at any dosage level at the scheduled necropsy.

 

Under the test conditions, the NOAEL of test item was 1000 mg/kg bw/day for reproductive toxicity in rats and the NOAEL of test item was 500 mg/kg bw/day for neonatal toxicity, based on the lower mean F1 birth weights, pup body weight gains during PND 1-4 and pup body weights on PND 4 at 1000 mg/kg bw/day in male and female rats.

A single oral boric acid dose of 3000 mg/kg body weight given to female mice on the day of insemination inhibited embryonal development almost completely. In a study of the influence of boric acid on the development of rat embryos, female Sprague-Dawley (CD) rats were given 0.1 %, 0.2 % or 0.4 % boric acid in the diet from day 0 to day 20 of gestation or 0.8 % from day 6 to day 15.

 

Endocrine disruption potential of boric acid

 

The ECHA Endocrine Disruptor Expert Group focuses on substances with endocrine disrupting properties. The most widely accepted definition for endocrine disruptors is the one by the International Programme for Chemical Safety (IPCS, 2002): "Endocrine disruptors have been defined as exogenous substances that alter function(s) of the endocrine system and consequently cause adverse health effects in an intact organism or its progeny, or (sub)populations".

 

The mechanism of action by which boric acid causes testicular toxicity has been investigated and it has been proposed that decreased testosterone production arises via a CNS mediated mechanism. It is not likely that hormone changes can explain the testicular atrophy observed at high dose levels since it has been shown that spermatogenesis can be maintained in the presence of significantly decreased intra-testicular testosterone levels. The fact that testicular damage was reversible and less extensive in younger sexually immature males than in mature animals also argues against an endocrine disruptor mechanism because younger animals still in development may be expected to be more sensitive to anti-androgenic effects than adults.

 

Inhibition of spermiation has been investigated and the involvement of Sertoli cells is suggested, as effects on these cells can lead to testicular atrophy. The changes in serum hormone levels may reflect an indirect effect on the CNS mediated by paracrine and/or autocrine influences.

 

There is no evidence that boric acid has a mechanism of action relating to endocrine disruption and whilst the exact mechanism has not been fully elucidated there are some proposed mechanisms but these do not involve endocrine disruption.

Justification for classification or non-classification

Classification of the registered substance is not justified in the absence of reproductive/developmental effects at dose levels below those at which maternal systemic toxicity is observed. Classification is also not justified on the basis of the residual levels of boric acid because this is significantly less than the specific concentration limit of 5.5% given by Annex VI of Regulation (EC) No 1272/2008 (CLP) and is expected to remain stable at approximately 3 wt%. There is no evidence that boric acid has a mechanism of action relating to endocrine disruption and residual boric acid in the UVCB test material gives no grounds for concern.

Additional information