2-tetradecyloxirane, reaction products with boric acid

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

2-tetradecyloxirane, reaction products with boric acid

EC Number:

939-580-3

Molecular formula:

Complex UVCB

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
- 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

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral 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 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

Doses / concentrations

Remarks:

Doses / Concentrations:
250, 500 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

5 rats/sex in the control and high-dosage groups assigned to the post-treatment period
Breeding phase rats:
- 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 the 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.
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

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: Twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Individual detailed physical examinations were recorded 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 was observed for females selected for pairing. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17 and 20 and on lactation days 0 (when possible), 1 and 4. Weekly body weights continued to be recorded for those females that were not selected for pairing.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- No. of animals and time schedule for collection of blood: 6 animals/sex/group at the scheduled necropsies (study day 28 for males and lactation day 4 for females that were selected for pairing) and from 5 animals/sex in the control and high-dose groups following a 14-day non-dosing post-treatment period (study day 42 for males and study day 53 for females).
- Anaesthetic used for blood collection: Yes (isoflurane anesthesia)
- Animals fasted: Yes (overnight fasting)
- Parameters checked:
Haematology: Total leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLATELET), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count (Percent and Absolute), Mean Platelet Volume (MPV), Red cell distribution width (RDW), Hemoglobin Distribution Width (HDW), Differential leukocyte count - Percent and absolute -Neutrophil (NEU), Lymphocyte (LYMPH), Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC), Platelet estimate and Red cell morphology.
Clinical chemistry: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio), [by calculation], Total bilirubin (Total Bili), Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides, Bile acids, Hemolysis, Lipemia and Icterus.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Functional observation battery (FOB) assessments were recorded for 6 animals/sex/group prior to dose administration on study day 27 (males selected for pairing) and on lactation day 4 (females).
- Dose groups that were examined: All treated and control groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: Home cage, handling and open field observations

Sacrifice and pathology:

All animals that were found dead or euthanized (by carbon dioxide inhalation) were subjected to a gross necropsy. F0 males were euthanized following the completion of the mating period or following the 14-day post-treatment period. F0 females that delivered were euthanized on lactation day 4. Females that failed to deliver were euthanized on post-mating day 25 (females with evidence of mating) or post-cohabitation day 25 (females with no evidence of mating). Females not selected for pairing were euthanized following the 14-day non-dosing period.

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.
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.
# = 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.

Other examinations:

None

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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See details on results

Mortality:

mortality observed, 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

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

See details on results

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

See details on results

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

See details on results

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

See details on results

Gross pathological findings:

no effects observed

Description (incidence and severity):

See details on results

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See details on results

Details on results:

CLINICAL SIGNS AND MORTALITY
- 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, WEIGHT GAIN AND FOOD CONSUMPTION
- 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
- 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.
- There were no test item-related effects on haematology parameters in the 250 and 500 mg/kg bw/day group male and females.

CLINICAL CHEMISTRY
- 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 serum chemistry parameters in the 250 and 500 mg/kg bw/day group male and females.

NEUROBEHAVIOUR
- No test item-related effects were noted during the FOB or locomotor activity evaluations for F0 males and females at any dosage level.

ORGAN WEIGHTS
- 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
- 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
- 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

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.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

See attached Documents 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 based on the lower mean food consumption and granulomatous inflammation of the mesenteric lymph node in the males and females at 1000 mg/kg bw/day 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. 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. 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. 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 based on the lower mean food consumption and granulomatous inflammation of the mesenteric lymph node in the males and females at 1000 mg/kg bw/day in rats.