2-tetradecyloxirane, reaction products with boric acid

Endpoint summary

Description of key information

28-day repeated dose oral toxicity

The NOAEL (oral, 28-day, rat) 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.

90-day repeated dose oral toxicity

Following once daily oral administration of test item to Sprague Dawley rats at 0, 250, 500 or 1000 mg/kg/day for 13 weeks, no adverse effects were noted on any parameter examined. Therefore, the no-observed-adverse-effect level (NOAEL) was 1000 mg/kg/day, the highest dose level tested.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other 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 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

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

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.

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.

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.

Critical effects observed:

not specified

See attached Documents for Tables of Results

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June 2018 to 27 September 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

various deviations with no impact on results or integrity of the study (see below)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

yes

Remarks:

various deviations with no impact on results or integrity of the study (see below)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

CD(Crl:CD[SD])

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMAL ACQUISITION AND ACCLIMATION
- During the 11-day acclimation period, the animals were observed daily with respect to general health and any signs of disease. All animals were given a detailed clinical examination prior to selection for study. These examinations were not reported but were maintained in the study file.
- All animals were acclimated to the oral gavage dosing procedure on two occasions prior to vehicle or test article administration via a sham dose of tap water at the same dose volume intended for use on study.

RANSOMISATION, ASSIGNMENT TO STUDY AND MAINTENANCE
- Using a standard, by weight, randomisation procedure, 40 male and 40 female animals (weighing 255 to 309 g and 186 to 218 g, respectively, at randomisation) were assigned to the control and treatment groups identified in Table 3 (below).
- Animals assigned to study had body weights within ± 20% of the mean body weight for each sex. Extra animals obtained for the study, but not placed on study, were transferred to the stock colony.
- Each animal was assigned an animal number to be used in the Provantis data collection system.
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 pair-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:

super refined

Details on oral exposure:

VEHICLE AND TEST ARTICLE PREPARATION
- Fresh vehicle, Super Refined Peanut Oil, NF NP-LQ-(MH), was dispensed for use on study weekly and was stored at controlled room temperature in amber glass bottles.
- 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 on a hot plate set to 60 ± 5°C for at least 15 minutes, followed by an additional mixing at ambient temperature for at least 15 minutes to achieve nominal concentrations of 25, 50, and 100 mg/mL.
- Formulations were prepared weekly and were stored at controlled room temperature in amber glass vials.

ADMINISTRATION
- The vehicle and test article were administered once daily for 90 days during the study via oral gavage.
- The dose levels were 0 (vehicle), 250, 500, and 1000 mg/kg/day and administered at a dose volume of 10 mL/kg.
- The vehicle and test article formulations were placed in heated water baths set to 36 ± 3 °C and continually stirred prior to and throughout dose administration.
- Individual doses were based on the most recent body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSES
- All analytical work was conducted by MPI Research, using an analytical method developed and validated under MPI Research Study No. 1928-022. No deviations from the analytical method occurred during the course of the study.

ANALYSIS OF DOSING FORMULATIONS
- Dosing formulations prepared for the study were evaluated for homogeneity and concentration.
Appropriate samples (see Table 2, below) were collected using a positive displacement pipette, while stirring, and placed into amber glass vials. Upon receipt of authorization to finalize the study report, the backup samples were discarded.
- Stability of the dosing formulations at the concentrations and storage conditions used on study
was previously established for 10 days under refrigerated (2°C to 8°C) and at ambient
temperature under MPI Research Study No. 1928-022.

RESERVE SAMPLE AND TEST ARTICLE DISPOSITION
- A reserve sample from the batch of test article used in this study was collected and stored in a secure location at MPI Research. Any remaining test article will be shipped to a Sponsor-designated location after completion of the study.

Duration of treatment / exposure:

At least 90 days

Frequency of treatment:

Once Daily

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.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

- Control (0 mg/kg bw/day): 10 males and 10 females
- Test item (250 mg/kg bw/day): 10 males and 10 females
- Test item (500 mg/kg bw/day): 10 males and 10 females
- Test item (1000 mg/kg bw/day): 10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

JUSTIFICATION OF DOSE LEVELS
- The dose levels were selected by the Sponsor on the basis of available data from previous studies. In a previous toxicity study (WIL-168202, OECD Guideline 422), the NOAEL was determined to be 1000 mg/kg/day. Therefore, the high dose in this study was 1000 mg/kg/day (the limit dose). The low and mid dose levels provided a graded dose response.

Observations and examinations performed and frequency:

IN-LIFE EXAMINATIONS
- Cageside observations: All animals were observed for morbidity, mortality, injury, and the availability of food and water twice daily.
- Cageside clinical observations: Each animal was observed daily (except on days of detailed clinical examinations) while still in the cage for clinical signs of disease, toxicity, and injury.
- Detailed clinical observations: A detailed clinical examination of each animal was performed weekly during the study. On occasion, clinical observations were recorded at unscheduled intervals. The observations included, but were not limited to, changes in the skin, fur, eyes, mucous membranes, and occurrence of secretions and excretions.
- Neurobehavioral observations: A detailed neurobehavioral evaluation of each animal was performed weekly during the study outside of the home cage, with the exception of activity level and posture measurements, which were performed inside the home cage. The observations included, but were not limited to changes to autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, and reactivity to handling, as well as the presence of clonic or tonic movements, stereotypy (e.g., excessive grooming, repetitive circling), or bizarre behavior (e.g., self-mutilation, walking backwards) were also recorded.
- Veterinary observations: On occasion, veterinary observations were conducted during the course of the study. All treatments and observations were recorded. The veterinary treatments and observations performed prior to randomization were not reported but were maintained in the study file.
- Functional observational battery: Functional Observational Battery (FOB) evaluations were conducted without knowledge on the part of the testers of the treatment groups on all designated animals during Week 13 (Day 87). FOB evaluations included those conducted in the home-cage, during handling, in the open-field, and others. During open-field evaluations, each animal was placed in a black plexiglass box and observed for a minimum of 3 minutes. The parameters evaluated in the FOB were based on those outlined in Moser, et al. The observations included, but were not limited to, evaluation of activity and arousal, posture, rearing, bizarre behavior, clonic and tonic movements, gait, mobility, stereotypy, righting reflex, response to stimulus (approach, click, tail pinch, and touch), palpebral closure, pupil response, piloerection, exophthalmus, lacrimation, salivation, and respiration. Qualitative and/or quantitative measures of defecation and urination were also recorded. Forelimb and hindlimb grip strength was measured using the procedure described by Meyer, et al., and hindlimb splay was quantitatively measured as described by Edwards and Parker. Pain perception was assessed by measuring the latency of response to a nociceptive (thermal) stimulus when each animal was placed on a hot plate apparatus set to 52 °C (± 1 °C) as described by Ankier. Body weight and temperature were also measured. A glossary of FOB terms used in the study is attached.
- Motor activity: Motor activity evaluations were conducted on all designated animals at approximately 1 hour postdose during Week 13 (Day 88). Each animal was placed into the assigned Hamilton-Kinder enclosure for monitoring. The duration of monitoring was 30 minutes with the data summarized into 10 minute segments. A range of different activities were assessed in a three dimensional array and were recorded. Only basic movement, fine movement, rearing, and distance (cm) were used in comparisons between treated and control animals as the most representative activity parameters.
- Body weights: Body weights for all animals were measured and recorded 3 days following receipt, prior to randomization on Day-1, and weekly during the study. Body weight change was calculated for individual animals relative to the previous body weight. The body weights recorded 3 days following receipt are not reported but are maintained in the study file.
- Food consumption: Food consumption was measured and recorded weekly during the study.
- Opthalmoscopic examinations: Ophthalmoscopic examinations were conducted on all animals by a veterinary ophthalmologist pretest and prior to the terminal necropsy.
- Clinical pathology: Clinical pathology evaluations were conducted on all animals prior to the terminal necropsy. Bone marrow smears were collected and preserved.

Sacrifice and pathology:

- Postmortem study evaluations were performed on all animals euthanized at the scheduled necropsy.

Statistics:

- See attached document for description of statistical methods.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- No test item-related clinical or veterinary observations were noted in either sex. All clinical observations noted were sporadic and lacked a dose-response relationship or were typical findings for this species and were unrelated to the test article.
- All veterinary observations resulted from events that were unrelated to the test article. Aside from sporadic food and environmental enrichment, only one animal required drug treatment; a control male (animal number 1008) that was submitted for severe swelling and erythema of the left hind food that caused limb impairment. This animal was treated with meloxicam from Days 35 to 39. This systemic treatment did not impact the study due to its short duration and it was necessary to maintain animal welfare.

Mortality:

no mortality observed

Description (incidence):

- No mortalities occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- There was no test item-related effect on body weight in either sex at < 500 mg/kg/day.
- Decrements in body weight change were noted by Week 4 and occurred sporadically over the duration of treatment in both sexes at 1000 mg/kg/day. This resulted in lower overall (through 13 weeks of dosing) body weight change of -16 and -10% in males and females, respectively. Although test item-related, this was not considered adverse based on the magnitude of the changes.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- There were no test item-related effects on food consumption.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

- There were no test item-related ophthalmoscopic findings.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

HEMATOLOGY
- Test item-related minimal decreases in hemoglobin concentration and hematocrit were present in both sexes at 1000 mg/kg/day, and minimal to mild decreases in mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) were present in males at ≥500 mg/kg/day and females at 1000 mg/kg/day. These changes collectively reflected a decrease in erythrocyte size and hemoglobin content and were considered not adverse due to the overall minimal to mild severity.
- Test item-related mild decreases in lymphocyte count were present in both sexes at 1000 mg/kg/day that lacked microscopic correlates and were considered not adverse. Effects on hematology endpoints related to the test item are further detailed in Text Table 7 (attached).
- All other fluctuations among individual and mean values, regardless of statistical significance, were considered sporadic, consistent with biologic variation and/or negligible in magnitude, and not related to test article administration.

COAGULATION
- Test item-related minimal to mild prolongations in activated partial thromboplastin time (APTT) were present in males at ≥500 mg/kg/day, and a test article-related minimal prolongation in prothrombin time was present in males at 1000 mg/kg/day that overall reflected decreased blood coagulability. These changes were considered not adverse due to the overall minimal tomild severity. Effects on coagulation endpoints related to the test item are further detailed in Table 8 (attached).
- Statistical differences in fibrinogen concentration were present in males at 500 and 1000 mg/kg/day that were not considered test article-related or biologically meaningful due to the small magnitude of the difference and considerable overlap of individual values with the range of control values.
- All other fluctuations among individual and mean values were considered sporadic, consistent with biologic variation and/or negligible in magnitude, and not related to test article administration.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Test item-related mild decreases in albumin and globulin concentration were present in males at 1000 mg/kg/day.
- Test item-related mild to moderate decreases in triglyceride concentration were present in males at 1000 mg/kg/day and females at ≥250 mg/kg/day. Test article-related mild decreases in cholesterol concentration were also present in both sexes at ≥500 mg/kg/day. These effects reflected altered lipid metabolism.
- Effects on clinical chemistry endpoints related to the test item are further detailed in Table 9 (attached)
- One female at 250 mg/kg/day (Animal No. 2504) had a moderate increase in aspartate aminotransferase (AST) activity and a mild increase in alanine aminotransferase (ALT) activity that lacked corresponding findings in the tissues examined microscopically and were considered incidental due to the sporadic nature of the changes and lack of similar findings at higher dose levels.
- All other fluctuations among individual and mean values, regardless of statistical significance, were considered sporadic, consistent with biologic variation and/or negligible in magnitude, and not related to test article administration.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related effects on any FOB parameters.
- Statistically significantly lower rearing counts were noted in males at 250 mg/kg/day; however, due to a lack of a dose responsive effect, these changes were considered spurious.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related changes for females and no clear test article-related organ weight changes for males.
- For males, mean liver weight and liver-to-brain weight ratios were decreased at 1000 mg/kg/day and liver-to-body weight ratios were decreased at all doses. However, there was no microscopic correlate for these reductions in weight parameters and no differences for liver weights and ratios for females at any dose. The reductions in liver weight parameters for males was not considered biologically meaningful based on the small magnitude, the direction of change and the absence of microscopic correlate.
- Spleen and epididymis of males at 1000 mg/kg/day had reductions in mean weights and organto-brain weight ratios but no differences in mean organ-to-body weight ratios. There were no microscopic correlates for the weight differences for epididymis or testis and no corresponding reduction in testes weight parameters. For spleen, there were no microscopic correlates for the weight parameter differences for males and there were no reductions in weight parameters for females at any dose. These differences in weight parameters for males were not considered biologically meaningful based on an absence of microscopic correlates or correlative findings.
- All other organ weight differences were within the range seen in species of this strain/breed, age, and sex at this facility or were considered incidental and not test article-related. These differences lacked a dose response relationship, had no correlative microscopic findings, and/or lacked similar differences in the other sex.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- There were no test item-related macroscopic findings.
- The only macroscopic observation in rats of this study occurred in a single control female and was considered incidental.

Neuropathological findings:

no effects observed

Description (incidence and severity):

- There were no test item-related observations noted during the weekly neurobehavioral evaluations.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Test item-related, non-adverse microscopic findings occurred in the mesenteric lymph node and jejunum of rats at all doses (≥250 mg/kg/day) and are summarized in Table 10 (attached).
- Histiocytosis (minimal to moderate) occurred in the mesenteric lymph node of females at all doses (≥250 mg/kg/day). For males, histiocytosis (minimal to mild) occurred at ≥500 mg/kg/day. Incidence and severity were dose related for females at all doses but were comparable at 500 and 1000 mg/kg/day for males. Histiocytosis was characterized as aggregates of pale staining large mononuclear cells with vacuolated cytoplasm within the medulla or medullary cords.
- The jejunum of rats at all doses (≥250 mg/kg/day) had the presence of variably sized vacuoles (vacuolation) in the villi. For males, vacuolation ranged from minimal to mild and incidence was dose related. For females, vacuolation was minimal and noted at similar incidence at all doses. Vacuoles were not present in other sections of small intestine at 1000 mg/kg/day. These vacuoles in affected animals did not appear to be intracellular and were not associated with tissue reactions.
- All other microscopic observations were considered incidental and not test article-related as they were of low incidence and/or severity, had no dose response relationship in incidence or severity, occurred in control rats at similar incidence/severity, and/or were known background findings for the species/strain.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

MOTOR ACTIVITY
- There were no test item-related effects on motor activity endpoints.
- Statistically significant decreases in basic movement, fine movement, and total distance noted among males at 250 mg/kg/day and statistically significant increases in basic movement in females at 250 and 1000 mg/kg/day, and fine movement, and total distance in females at 1000 mg/kg/day relative to controls were not considered test item-related due to the lack of a dose response and difference in the sexes.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of adverse effects for any parameter examined

Critical effects observed:

no

ANALYSIS OF DOSING FORMULATIONS

- The analytical data demonstrated acceptable performance of the method for all reported results and that dose formulations were prepared at the intended target concentration and were homogenous (see Table 6, attached).

Conclusions:

Following once daily oral administration of test item to Sprague Dawley rats at 0, 250, 500 or 1000 mg/kg/day for 13 weeks, no adverse effects were noted on any parameter examined. Therefore, the no-observed-adverse-effect level (NOAEL) was 1000 mg/kg/day, the highest dose level tested.

Executive summary:

GUIDELINE

The objective of this study was to evaluate the potential toxicity of the test article after oral administration for at least 90 days. This study was based on the US EPA Office of Prevention, Pesticides, and Toxic Substances, Guideline 870.3100 and OECD Section 4 (Part 408), and in accordance with the Guide for the Care and Use of Laboratory Animals.

 

METHODS

Test item in the vehicle (super refined peanut oil) was administered orally by gavage once daily for a minimum of 90 consecutive days to three groups of Crl:CD(SD) rats (10 males and 10 females per group). Dosage levels were 250, 500 and 1000 mg/kg bw/day. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 10 mL/kg for all groups. Assessment of toxicity was based on mortality, cageside and detailed clinical observations, neurobehavioral observations, functional observational battery (FOB) assessments, motor activity, body weight, and food consumption; ophthalmoscopic examinations; and clinical and anatomic pathology.

 

RESULTS

No mortalities or clinical/neurobehavioral observations of systemic toxicity were noted. There were no test item-related effects on FOB parameters, motor activity or ophthalmoscopic findings, effects on food consumption or organ weights, and no macroscopic findings.

 

Mean body weight change over the duration of treatment was slightly lower (up to -16%) in both sexes at 1000 mg/kg/day when compared to control values. This was not considered adverse based on the magnitude of the changes.

 

Test item-related effects on clinical pathology endpoints were noted and included decreases in hemoglobin concentration and hematocrit in both sexes at 1000 mg/kg/day; decreases in mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) in males at ≥ 500 mg/kg/day and females at 1000 mg/kg/day; prolongations in activated partial thromboplastin time (APTT) in males at ≥ 500 mg/kg/day and prolongation in prothrombin time in males at 1000 mg/kg/day; decreases in lymphocyte count in both sexes at 1000 mg/kg/day; decreases in triglyceride concentration in males at 1000 mg/kg/day and females at ≥ 250 mg/kg/day, and mild decreases in cholesterol concentration in both sexes at ≥ 500 mg/kg/day; decreases in albumin and globulin concentration in males at 1000 mg/kg/day. None of these changes were considered adverse based on the magnitude and/or lack of microscopic correlates.

 

Test item-related non-adverse microscopic findings were noted in the mesenteric lymph node and jejunum of rats at all dose levels. Histiocytosis occurred in the mesenteric lymph node of females at ≥ 250 mg/kg/day and in males at ≥ 500 mg/kg/day. Vacuolation of the jejunum was also observed in rats at ≥ 250 mg/kg/day in a dose-related incidence. There were higher occurrences noted in the males compared to females. These findings were not considered adverse in nature.

 

CONCLUSION

Following once daily oral administration of test item to Sprague Dawley rats at 0, 250, 500 or 1000 mg/kg/day for 13 weeks, no adverse effects were noted on any parameter examined. Therefore, the no-observed-adverse-effect level (NOAEL) was 1000 mg/kg/day, the highest dose level tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

28-day repeated dose oral toxicity

A key study was performed in accordance with 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.

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 followingapproximately 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.

 

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.

 

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 hyperplasiaof 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.

 

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.

 

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.

 

90-day repeated dose oral toxicity

The objective of this study was to evaluate the potential toxicity of the test article after oral administration for at least 90 days. This study was based on the US EPA Office of Prevention, Pesticides, and Toxic Substances, Guideline 870.3100 and OECD Section 4 (Part 408), and in accordance with the Guide for the Care and Use of Laboratory Animals.

 

Test item in the vehicle (super refined peanut oil) was administered orally by gavage once daily for a minimum of 90 consecutive days to three groups of Crl:CD(SD) rats (10 males and 10 females per group). Dosage levels were 250, 500 and 1000 mg/kg bw/day. A concurrent control group (Group 1) received the vehicle on a comparable regimen. The dose volume was 10 mL/kg for all groups. Assessment of toxicity was based on mortality, cageside and detailed clinical observations, neurobehavioral observations, functional observational battery (FOB) assessments, motor activity, body weight, and food consumption; ophthalmoscopic examinations; and clinical and anatomic pathology.

 

No mortalities or clinical/neurobehavioral observations of systemic toxicity were noted. There were no test item-related effects on FOB parameters, motor activity or ophthalmoscopic findings, effects on food consumption or organ weights, and no macroscopic findings.

 

Mean body weight change over the duration of treatment was slightly lower (up to -16%) in both sexes at 1000 mg/kg/day when compared to control values. This was not considered adverse based on the magnitude of the changes.

 

Test item-related effects on clinical pathology endpoints were noted and included decreases in hemoglobin concentration and hematocrit in both sexes at 1000 mg/kg/day; decreases in mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) in males at ≥ 500 mg/kg/day and females at 1000 mg/kg/day; prolongations in activated partial thromboplastin time (APTT) in males at ≥ 500 mg/kg/day and prolongation in prothrombin time in males at 1000 mg/kg/day; decreases in lymphocyte count in both sexes at 1000 mg/kg/day; decreases in triglyceride concentration in males at 1000 mg/kg/day and females at ≥ 250 mg/kg/day, and mild decreases in cholesterol concentration in both sexes at ≥ 500 mg/kg/day; decreases in albumin and globulin concentration in males at 1000 mg/kg/day. None of these changes were considered adverse based on the magnitude and/or lack of microscopic correlates.

 

Test item-related non-adverse microscopic findings were noted in the mesenteric lymph node and jejunum of rats at all dose levels. Histiocytosis occurred in the mesenteric lymph node of females at ≥ 250 mg/kg/day and in males at ≥ 500 mg/kg/day. Vacuolation of the jejunum was also observed in rats at ≥ 250 mg/kg/day in a dose-related incidence. There were higher occurrences noted in the males compared to females. These findings were not considered adverse in nature.

 

Following once daily oral administration of test item to Sprague Dawley rats at 0, 250, 500 or 1000 mg/kg/day for 13 weeks, no adverse effects were noted on any parameter examined. Therefore, the no-observed-adverse-effect level (NOAEL) was 1000 mg/kg/day, the highest dose level tested.

 

28-day repeated dose inhalation toxicity

According to REACH, Annex VIII, Section 8.6.1, short-term repeated dose toxicity should be investigated using one species and the most likely route of human exposure. The substance is a waxy solid with determined vapour pressure of 0.009 Pa at 25 °C and a high onset boiling point of > 400°C at 100.9 kPa. Based on evaluation of the life cycle of the substance, it is therefore expected that inhalation exposure will be low and that the most likely route of exposure for workers and consumers is the dermal route. A short-term repeated dose toxicity study via the inhalation route is, therefore, not appropriate.

90-day repeated dose inhalation toxicity

A 90-day repeat dose study by the inhalation route is scientifically unjustified. The substance is unreactive, insoluble and not inhalable and there is no evidence of dermal absorption. The substance is a UCVB with the majority of components in the molecular weight range of 284 - 853 gm/mol. The substance is slightly soluble in water (0.17 mg/L), with a relatively high octanol/water partition coefficient (log Pow > 9.4) and a low vapour pressure (0.009Pa). The substance is a hydrophobic waxy solid and is only placed on the market diluted in solvent. It is not acutely toxic via the oral route and in a 90-day repeat oral dose study the NOAEL was 1000 mg/kg bw/day because no adverse effects were noted on any parameter examined. If this substance was to reach the respiratory tract, the physical chemical properties and its bioavailability, as inferred from the oral toxicity data, indicates that uptake is likely to occur only by means of micellar solubilization. The potential for inhalation toxicity was not measured. However, the vapour pressure indicates a very low propensity to enter atmospheric air in a respirable form (predicted to be ca. 0.1 ppm under ambient conditions). Thus, respiratory absorption under normal use and handling of this material is expected to be inconsequential.

 

28-day repeated dose inhalation toxicity

According to REACH, Annex VIII, Section 8.6.1, short-term repeated dose toxicity should be investigated using one species and the most likely route of human exposure. However, no evidence of systemic toxicity was demonstrated during an acute study via the dermal route and, in order to minimise the number of vertebrate animals tested, oral dosing by gavage was considered most appropriate for investigation of combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) in rats and 90-day repeated dose toxicity (OECD 408).

90-day repeated dose dermal toxicity

A 90-day repeat dose study by the dermal route is scientifically unjustified. The substance is unreactive, insoluble and a skin sensitiser . The substance is a UCVB with the majority of components in the molecular weight range of 284 - 853 gm/mol. The substance is slightly soluble in water (0.17 mg/L), with a relatively high octanol/water partition coefficient (log Pow > 9.4) and a low vapour pressure (0.009Pa). The substance is a hydrophobic waxy solid and is only placed on the market diluted in solvent. The single-dose dermal application of the test material resulted in no manifestations of systemic toxicity that would suggest systemic absorption through cutaneous barriers. The substance is poorly soluble in water, with the majority of components with MW > 500 and high Log Pow values which indicate a disinclination for absorption. It is not acutely toxic via the oral route and in a 90-day repeat oral dose study the NOAEL was 1000 mg/kg bw/day because no adverse effects were noted on any parameter examined. The substance is a skin sensitiser therefore systemic exposure via the dermal route would be secondary and minimal with respect to local effects. Human health risk mitigation measures will be in place to minimise the sensitisation properties of the substance, resulting in a reduction of potential human exposure.

Justification for classification or non-classification

No adverse effects were reported at the limit of classification in a 90-day repeat oral dose toxicity study in the rat.