2-tetradecyloxirane, reaction products with boric acid

Developmental toxicity / teratogenicity

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

2-tetradecyloxirane, reaction products with boric acid

EC Number:

701-392-2

IUPAC Name:

2-tetradecyloxirane, reaction products with boric acid

Test material form:

other: solid

Details on test material:

- Physical state: Off-white, waxy solid
- Date of receipt: 29 June 2012
- Expiration date of the lot/batch: 29 June 2013
- Storage condition of test material: Room temperature, protected from light
- Stability under test conditions: Considered stable under these conditions

Test animals

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

Administration / exposure

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.

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Abnormalities:

effects observed, treatment-related

Results (fetuses)

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.

Effect levels (fetuses)

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

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Overall developmental toxicity

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

Any other information on results incl. tables

See attached document for Tables of Results

Applicant's summary and conclusion

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.