p-tert-butylstyrene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

In-life phase: 07 June 2017 to 01 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Lot/Batch no.: 122016TBSSV
Purity: 95.9%

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Age at first dose: 11-12 weeks
Weight at first dose: Males-390-465 g and Females-239.1-292.6 g
Animals were acclimated to laboratory conditions for at least five days prior to the first dose and released from acclimation by a staff veterinarian. During that time, animals were identified by a temp
orary number that was recorded on each cage label.
Feed: Certified Global Teklad Laboratory Diet 2018 (pellets) was provided ad libitum.
Water: Filtered water was provided ad libitum via an automatic watering system supplemented with water bottles as needed.
Housing: Animals were housed in one room in polycarbonate cages suspended on stainless steel racks. Each cage was affixed with a cage card containing pertinent animal and study information.
Temperature: 20 to 26°C
Humidity: 30-70%
Light-dark cycle: 12-hour light/12-hour dark
Air changes: Minimum of 10 air changes per hour

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

The vehicle/control substance was considered 100% pure for formulation purposes. The test substance was used as received and formulated with no adjustments. The vehicle/control substance,
peanut oil, was used as received; no formulations were necessary. Formulations for Groups 2 (5 mg/mL/10 mg/kg bw/day), 3, (15 mg/mL/30 mg/kg bw/day) 4 (30 mg/mL/60 mg/kg bw/day) and 5 (50 mg/mL/100 mg/kg bw/day) were prepared weekly by adding the appropriate amount of the control test substance to the required amount of vehicle/control substance and mixinged until visually uniform. Formulations were stored in a refrigerator (2-8 degree Celsius) until used for dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability analysis of the dose formulations was performed as part of the method validation. Samples were analyzed for test substance concentration using a validated method. Since all dose formulations were solutions; therefore, homogeneity analysis was not required.

Duration of treatment / exposure:

The animals were dosed via oral gavage at a volume of 2 mL/kg bw. Dosing volumes were based on the most recent body weights. F0 males were dosed for at least 28 days. The F0 females were dosed for two weeks prior to cohabitation, during cohabitation, through gestation, and to at least Postnatal Day (PND) 12. The first day of dosing was designated as Day 1 for each animal.

Frequency of treatment:

Once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

for females only

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

for males only

No. of animals per sex per dose:

12 rats per sex per dose

Control animals:

yes, concurrent vehicle

Details on study design:

F0 males were dosed for at least 28 days. The F0 Females were dosed for two weeks prior to cohabitation, during cohabitation, through gestation, and to at least postnatal day (PND) 12. The first
day of dosing was designated as SD1 for each animal.

Observations and examinations performed and frequency:

Post dosing, F0 males and females were examined regularly for mortality, moribundity, general health, body weight, food consumption and signs of toxicity. Physical examinations included evaluations of skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, autonomic, and central nervous systems, and somatomotor and behavior patterns. Prior to scheduled necropsy, routine hematological, clinical chemistry and hormonal analysis were done.

Sacrifice and pathology:

Gross necropsy of F0 animals included examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. For all F0 females, the number of implantation sites was recorded. All reproductive organs (testes, epididymides, prostate, seminal vesicles with coagulating glands, ovaries, uterus with cervix; sex appropriate) and thyroids from all F0 animals were collected and weighed as soon as possible after dissection; paired organs were weighed together.
Histopathological examination of tissues from the selected animals in the control and high dose group revealed test substance-related changes in the bone marrow (femur) of females only, the pancreas, thyroid and thymus in males only, and the kidney and liver in both sexes. Therefore, the following tissues from selected animals in 10 mg/kg bw/day and 30 mg/kg bw/day were processed to slides and examined microscopically: bone marrow (femur, females only, 5/group); pancreas, thyroid, and thymus (males only, 5/group); kidney and liver (both sexes, 5/sex/group).

Statistics:

See under "any other information on materials and methods incl. tables"

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

gross pathology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Key result

Dose descriptor:

LOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

10 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

kidney

liver

seminal vesicle

seminiferous tubules

testes

thyroid gland

Treatment related:

yes

Dose response relationship:

yes

Conclusions:

Under the study conditions, no NOAEL could be determined and the LOAEL for systemic toxicity in parental male rats was determined to be 10 mg/kg bw/day (due to lesions in thyroid at this dose). The NOAEL for systemic toxicity in parental female rats was considered to be 10 mg/kg bw/day due to adverse body weight changes, clinical chemistry, organ weight and microscopic findings in liver, kidney and thyroid.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats according to OECD Guideline 422, in compliance with GLP. Male rats were administered the test substance at doses of 0, 10, 30 or 100 mg/kg bw/day for 14 d prior to cohabitation and during cohabitation for at least a total of 28 d. Female rats were dosed with the test substance at doses of 0, 10, 30 or 60 mg/kg bw/day prior to cohabitation, during cohabitation, through pregnancy, and throughout lactation to Postnatal Day 13 (PND 13). Treatment-related effects in male rats included a significant effect on the body weight, body weight changes, organ weights, and microscopic findings. In female rats, repeat dose toxicity was observed at doses of 30 and 60 mg/kg bw/day primarily due to reduced body weight and microscopic findings in liver, kidney and thyroid. Important microscopic findings consisted of cytoplasmic alteration in hepatocytes at doses greater than 30 mg/kg bw/day in both sexes, thyroid follicular cell hypertrophy in males from 10 mg/kg bw/day and vacuolation of distal tubules in the kidney in males and females at the respective high doses. The test substance induced reproductive toxicity at doses of 30, 60 (females only) and 100 (males only) mg/kg bw/day. Treatment-related degeneration/atrophy of seminiferous tubules in all high dose males resulted in complete loss of fertility at this dose. Although degeneration of spermatids was only identified in one animal administered 30 mg/kg bw/day, this change was associated with decreased fertility and fecundity; therefore, testicular changes were considered adverse at or above 30 mg/kg bw/day. Decreases in testes and epididymal weight correlated with these changes in the high dose males. Under the study conditions, no NOAEL could be determined and the LOAEL for systemic toxicity in parental male rats was determined to be 10 mg/kg bw/day (due to lesions in thyroid at this dose). The NOAEL for systemic toxicity in parental female rats was considered to be 10 mg/kg bw/day due to adverse body weight changes, clinical chemistry, organ weight and microscopic findings in liver, kidney and thyroid (Murphy, 2019b).

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 January 2018 - 11 July 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:

The study director said that, the deviations did not impact the study, nor did they affect the quality or integrity of the study or the interpretation of the results in the report.

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

CAS No.: 1746-23-2
Identification: para-tertiary-butyl styrene; TBS
Storage: 2 to 8°C in a refrigerator

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Species and Strain: Sprague Dawley Rat
Supplier: Envigo, Frederick, MD
Method of Identification: Cage card/Microchip
Number of Animals Received: 55 (male) and 55 (female)
Number Used on Study: 50 (male) and 50 (female)
Age at First Dose: 9-10 weeks
Weight Range at First Dose: 299.5 to 378 g (male) and 207.1 to 246 g (female)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were acclimated to laboratory conditions for at least five days prior to the first dose and released from acclimation by a staff veterinarian. During that time, animals were identified by a temporary number that was recorded on each cage label.

Feed: Certified Global Teklad Laboratory Diet 2018 (pellets) was provided ad libitum, except when fasted overnight for sample collection and/or prior to necropsy on the following days: SD 92 and 120.
Water: Filtered water was provided ad libitum via an automatic watering system supplemented with water bottles as needed
Bedding: Certified Sani Chips® hardwood bedding
Housing: Animals were housed in one room, two or three per cage, in polycarbonate cages suspended on stainless steel racks. Each cage was affixed with a cage card containing pertinent animal and study information.
Temperature Range: 20 to 26°C
Humidity Range: 30 to 70%
Light Cycle: 12-hour light/12-hour dark, interrupted as necessary for study-related events
Air Changes Minimum of 10 air changes per hour

The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The water is routinely analyzed for contaminants and specific microbes. The bedding was analyzed by the manufacturer for acceptable levels of heavy metals, aflatoxins, bacteria, yeasts, molds, and organophosphates prior to certification. No contaminants were known to be present in the feed, water, or bedding at levels that might have interfered with achieving the objectives of the study. Environmental controls were set to maintain animal room conditions. Actual temperature and relative humidity in the animal room were monitored continuously by a computerized system. All environmental parameters were maintained within the protocol requirements. In addition to standard husbandry procedures, environmental enrichment was provided according to company SOPs.

Route of administration:

oral: gavage

Vehicle:

peanut oil

Details on oral exposure:

The dose formulations were placed at room temperature prior to dosing. The animals were dosed for 91 days via oral gavage at a volume of 2.0 mL/kg. Dosing volumes were based on the animals’ most recent body weights. The first day of dosing was designated as SD 1 for each animal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose Formulations
The vehicle/control substance was considered 100% pure for formulation purposes, and was used as received. The test substance was used as received and formulated ‘as is’ with no adjustments for purity. The test substance density of 0.8840 (provided by the Sponsor, calculated displacement factor of 1.131) and vehicle density of 0.9154 g.cm-3were applied for dose formulation preparation. The dose formulations are stable for at least 24 hours and 7 days after formulation when stored at room temperature and 5 ± 3°C, respectively, based the stability analysis of the dose formulations that was performed under Smithers Avanza Study No. 2388-13941 as part of the method validation. The dose formulations were prepared every two to seven days. Formulations for Group 4 (25 mg/mL) were prepared by mixing the appropriate amounts of test substance and vehicle/control substance and then mixed until visually clear or inverted approximately 30 times. Formulations for Group 3 (5 mg/mL) were prepared by diluting the formulation for Group 4 with the appropriate amount of vehicle/control substance, and then inverting approximately 30 times or mixed until visually clear. The formulations for Group 2 (1.5 mg/mL) were prepared by diluting the formulation for Group 3 with the appropriate amount of vehicle/control substance, and then inverted approximately 30 times or mixed until visually clear. Formulations were stored at room temperature until used for dosing on the day of formulation. Otherwise, formulations were stored in a refrigerator (5 ± 3°C) until used for dosing.

Dosage Sampling
Samples (2-mL, in duplicate) were collected from each test substance dose formulation and the vehicle/control substance. All samples were stored under conditions set to maintain 5 ± 3°C and analyzed. All remaining samples not used for analysis weredisposed of in accordance with company SOPs, appropriate regulatory requirements, and/or information contained in the Material Safety Data Sheetsafter authorization from the Study Director prior to finalization of the report.

Dosage Analysis
Stability analysis of the dose formulations was performed under Smithers Avanza Study No. 2388-13941 as part of the method validation. Test substance formulations were solutions; therefore, homogeneity sampling and analysis was not required. Samples from dose formulations prepared for Weeks 1, 4, 8, 9, and 12 were analyzed for test substance concentration verification by Smithers Avanza using a validated method.

Dose concentration analysis of samples from dose formulations prepared for or Weeks 1, 4, 8, 9, and 12 showed that the test substance was properly formulated (mean % target results ranging from 91.3% to 110.1% of the target concentrations) with the exception of Group 3 and 4 Week 8 formulations which were above the acceptance criteria (mean % target results being 112.8% and 115.8% of the target concentrations, respectively). No test substance was detected in the control formulation.

Duration of treatment / exposure:

91 days

Frequency of treatment:

once a day

Remarks:

0, 3, 10, 50 mg/kg bw/day

No. of animals per sex per dose:

10 animals/sex/group for the main phase and 5 animal/sex/group in Groups 1 and 4 for the recovery phase

Control animals:

yes

Details on study design:

Animals were initially accepted into the randomization pool based upon prestudy body weights, physical examinations, and ophthalmologic examinations. They were assigned to study groups using computer-generated random numbers such that the mean body weight for each group was not statistically different (p ≤ 0.05) from the control mean. Males and females were randomized separately. Following randomization, each study animal was assigned a unique number. The animals were assigned to groups (ten animals/sex/group for the main phase and five animal/sex/group in Groups 1 and 4 for the recovery phase).

Observations and examinations performed and frequency:

(a) Physical Examinations: At least once prior to randomization into study groups, Study Day 1 (prior to initiation of dosing), Weekly thereafter, Prior to necropsy, Unscheduled observations were recorded. Physical examinations included evaluation of skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, autonomic, and central nervous systems, and somatomotor and behavior patterns.
(b) Cageside Observations: >=2 times daily. Cageside observations included observation for mortality, moribundity, general health, and signs of toxicity.
(c) Body Weights: At least once prior to randomization into study groups, SD1 (prior to initiation of dosing), Weekly thereafter, At time of unscheduled termination, Day prior to necropsy (unfasted), Prior to necropsy (fasted)
(d) Food Consumption: Weekly
(e) Ophthalmologic Examinations: All animals prior to randomization into study groups, All surviving animals prior to scheduled necropsies (within 2 days). Ophthalmologic observations were conducted using indirect ophthalmoscopy and slit-lamp biomicroscopy (as needed) following administration of 1% Tropicamide® mydriatic solution.
(f) Functional Observation Battery (FOB): SD 90. For neurotoxicity observations (FOB, grip strength, and locomotor activity), animals were transported to the testing room and acclimated to white noise for at least 10 minutes prior to testing. Animals were assessed and scored FOB.
(g) Locomotor Activity: SD 85. For locomotor activity, animals were placed into an activity chamber for 30 minutes. The Kinder Scientific Motor Monitor II recorded the total number of occurrences of basic movement, fine movement, and rearing during the 30-minute testing timeframe. Data was tabulated in 10-minute intervals.

Sacrifice and pathology:

Blood samples and urine specimens were collected from all surviving animals. Animals were fasted overnight (with water available) prior to sample collection. Blood samples were also taken from moribund animals prior to euthanasia, if possible. Samples/specimens were transported to the Clinical Pathology Laboratory for analysis. All remaining samples were discarded prior to finalization of the report.

Termination
Moribund animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy. On SD 92 for main phase animals and 120 for recovery animals, all surviving animals were euthanized by carbon dioxide inhalation followed by exsanguination prior to necropsy.

Necropsy
Animals were necropsied as soon as possible after the time of death or discovery. Animals were necropsied, bone marrow smears were prepared, required organs were weighed, and protocol-specified tissues were collected and preserved. No organ weights were collected from animals found dead or euthanized prior to scheduled termination and no bone marrow smears were prepared from animals found dead. Gross necropsy included examination of the external surface of the body, all orifices, the cranial, thoracic, and abdominal cavities and their contents. Organ weights were collected from all animals at scheduled necropsy as soon as possible after dissection and paired organs were weighed together. The following organs were weighed as follows: adrenal glands, brain, heart, kidneys, liver, spleen, thymus, testes with epididymides, ovaries, and uterus with cervix. Protocol-specified tissues, including gross lesions, were preserved in 10% neutral buffered formalin (NBF) with the exception of the eyes (and associated ocular tissue) and testes (with epididymides), which were preserved in modified Davidson’s fixative and subsequently transferred to 10% NBF. Tissues from animals found dead were preserved in 10% NBF only. Animal identification from each study animal was also retained with the tissues at the time of necropsy. Two bone marrow smears were prepared from the left femur and the slides were air-dried, fixed in methanol, and stored at room temperature for possible future evaluation. No analysis of the bone marrow smears was deemed necessary; therefore, the unstained slides were discarded prior to report finalization.

Histopathology
Preserved tissues were transferred to VPS where the protocol-required tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by a board-certified veterinary pathologist. The pancreas, thyroid, liver, testis, and epididymis were identified as potential target tissues by the pathologist, therefore tissues from animals in Groups 2 and 3 (main) and Group 1 and 4 (recovery) were processed to slides and microscopically examined.

Other examinations:

Clinical biochemistry parameters analysed
Albumin, Alkaline Phosphatase, Alanine Aminotransferase, Aspartate Aminotransferase, Blood Urea Nitrogen, Calcium, Cholesterol, Creatine Kinase, Chloride, Creatinine, Gamma Glutamyl Transferase, Glucose, Potassium, Sodium, Phosphorus, Total Bilirubin, Total Protein, Triglycerides

Hematology parameters analysed
White Blood Cells, Red Blood Cells, Hemoglobin, Hematocrit, Mean Corpuscular Volume, Mean Corpuscular Hemoglobin, Mean Corpuscular Hemoglobin Concentration, Mean Platelet Volume, Platelets, Red Cell Distribution Width, Leukocyte Differential Count, Absolute Neutrophil, Absolute Lymphocytes, Absolute Monocytes, Absolute Eosinophils, Absolute Basophils, Reticulocyte Count, Absolute Reticulocytes

Coagulation parameters analysed
Prothrombin Time, Activated Partial Thromboplastin Time, Fibrinogen

Urinanalysis parameters analysed
Volume, Color, Clarity/Appearance, Specific Gravity, Glucose, Bilirubin, Ketones, Blood, pH, Protein, Urobilinogen, Nitrites, Leukocytes, White Blood Cells, Red Blood Cells, Epithelial Cells, Bacteria, Mucus, Yeast, Fecal, Sperm, Hyaline Cast, Granular Cast, Amorphous Crystals, Triple Phosphate Crystals, Calcium Oxalate Crystals, Other Observations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment with TBS had no effect on physical examinations. There were observations noted that were associated with accidental dosing gavage errors. Additionally, there were incidental observations that included abrasions (Group 1 – one female; Group 2 – one female; Group 3 – two males; Group 4 – two males, two females), and alopecia (Group 1 – one male, two females; Group 3 – one female; Group 4 – two males, four females) at various sites. Abnormal respiration (soft, loud, or wheezing) was observed in several animals that survived to scheduled termination (Group 1 – two females; Group 3 – one female; Group 4 – one male) and were associated with dosing of a viscous material via oral gavage. Female 25224 (4f) was observed as thin on SD 85 and at termination on SD 92 and correlated with a test substance-related reduction in body weight. Female 25228 (4f) had a small movable tissue mass on the abdomen when observed on SD 57, 64, and 71 that was not considered test substance-related due to the transient finding and no similar findings in any other animal.
Treatment with TBS had no effect on cageside observations. Male 25211 (4m) was observed with rough haircoat on SD 17. Female 25197 (3f) exhibited alopecia of the neck and dorsal thoracic sides (bilateral) from SD 58 to scheduled termination on SD 92. Observations were considered incidental since each observation was only observed in one animal.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Treatment with TBS had no effect on mortality. There were five early deaths; three animals were found dead and two animals were euthanized as moribund. All deaths were considered accidental and gavage related. Animal 25209 (4m; 50 mg/kg/day) was observed with gasping, hunched posture, and rough haircoat on SD 40, and was found dead the same day. Animal 25227 (4f) was found dead on SD 88 with no previous clinical signs of toxicity. Animal 25136 (1m; 0 mg/kg/day) was observed with brown discharge from the nose and swelling of the left axillary on SD 91, and when examined by the veterinarian, was also observed to have left forelimb lameness. The animal was treated with meloxicam on SD 91, and was found dead on SD 92. Animal 25211 (4m) exhibited swelling of the left axillary on SD 17 and 20, and loud respiration on SD 20. This animal was not dosed on SD 18 per veterinarian directive and was treated with a diet gel recovery pack on SD 17 and 19. Animal 25211 (4m) was euthanized as moribund, per the veterinarian, on SD 20. Animal 25173 (2m; 3 mg/kg/day) exhibited soft abnormal respiration and salivation on SD 50. Body weight loss, rough haircoat, hypoactivity, and red/brown nasal discharge were noted by the veterinarian at examination. Per the veterinarian, this animal was euthanized as moribund on SD 50. All other animals survived until the scheduled termination.
Animals 25210 (4m), 25220 (4f), and 25225 (4f) were given nutritional support (diet gel recovery packs) for various durations throughout the study. Animals 25210 (4m) and 25220 (4f) exhibited body weight loss (> 15.0 g) and Animal 25225 (4f) had blood present around the mouth and nose area after dosing on SD 32.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment with TBS impacted body weights and body weight gains/changes. The body weight gains of Group 4 (50 mg/kg/day) males were significantly less than the control in weeks 4, 5 and 12 of the main phase, whcih resulted in significantly lower total body weight gains than the control at the end of both main and recovery phases. Mean body weights of Group 4 males were lower than the control beginning on SD 29 of the main phase through SD 119 of the revcovery phase, and statistically significant lower than the control on SD 36 and from SD 57 through 91 of the main phase (8.4 to 11.2% difference compared to the control). The body weight gains of Group 4 females were statistically significant less than the control in weeks 1, 2, 5 and 12 of the main phase, which resulted in statistically significant lower total body weight gains than the control at the end of both main and recovery phases. The total body weight gain of Group 3 (10 mg/kg/day) females was also statistically significant lower than the control at the end of the main phase. Mean body weights of Group 4 females were statistically significantly lower than the control beginning on SD 8 of the main phase through SD 106 of the recovery phase (5.6 to 21.8% difference compared to the control). Mean body weights of Group 3 females were also lower than the control throughout the main phase beginning on SD 15 and were statistically significantly lower than the control on SD 78 and 85 (7.4% and 8.8% difference compared to the control, respectively). Group 4 females gained significantly more weight than the control during the recovery phase, which resulted in a partial recovery of body weight; however, the mean total body weight gain throughout the entire study (SD 1 to 119) was still statistically significantly less than the control.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Treatment with TBS had no effect on food consumption. During the main phase, mean total food consumption (SD 1 to 91) of Group 3 (10 mg/kg/day) and Group 4 (50 mg/kg/day) males was significantly higher than the control. There were several intervals in which Group 3 and 4 males and Group 4 females had significantly higher or lower mean food consumption than the control. These significant differences did not correlate with the changes in body weight and were considered incidental due to the animals being group housed (2 to 3/cage). During recovery, there were several intervals that mean food consumption of Group 4 males was significantly lower than the control (SD 92 to 99, 99 to 106, 113 to 119), and over the entire recovery phase (SD 92 to 119). No significant differences were noted in the females during the recovery phase or for all recovery animals during the entire study (SD 1 to 119).

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment with TBS had no effect on ophthalmology; no ocular lesions were seen in any treated animals. Persistent hyaloid ghost vessel was observed in the left eye of Animal 25156 (1f) on SD 91 and focal retinal hemorrhage around the optic nerve was observed in the left eye of Animal 25163 (1f) on SD 91 and was observed as resolving on SD 119. These findings were considered incidental due to the findings being unilateral in nature and occurring in control group animals.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology
Study Day 92 – Terminal Necropsy
Mild (51-59%) decreases in absolute eosinophil counts in females administered 50 mg/kg/day and males administered ≥ 10 mg/kg/day compared with concurrent controls were potentially related to the test substance; however, due to the lack of correlating changes in the bone marrow or lymphoid tissues, the relationship to the test substance was uncertain. Decreases in eosinophil counts were not considered toxicologically or biologically important.

Study Day 120 – Recovery Necropsy
No test substance-related changes in hematology parameters were observed at recovery necropsy.

Coagulation
Study Day 92 – Terminal Necropsy
Test substance-related mild decreases in fibrinogen concentrations in males (38%) and females (42%) administered 50 mg/kg/day compared with concurrent controls were not biologically or toxicologically significant but correlated with increased liver weight parameters and microscopic hepatocellular cytoplasmic alteration.

Study Day 120 – Recovery Necropsy
No test substance-related changes in coagulation parameters were observed at recovery necropsy.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Study Day 92 – Terminal Necropsy
Test substance-related changes at a dose level of 50 mg/kg/day occurred in alkaline phosphatase (ALPi) and/or gamma-glutamyl transferase activities, and albumin, globulin, cholesterol and triglyceride concentrations in both sexes; and albumin and globulin concentrations in females at doses ≥ 10 mg/kg/day and cholesterol concentrations in males at doses ≥ 10 mg/kg/day. All changes were assessed relative to concurrent controls. Minimal (~ 2-fold) increases in ALPi activities were observed in animals administered 50 mg/kg/day and minimal (< 2-fold) increases in gamma-glutamyl transferase activities were observed in females administered 50 mg/kg/day. Dose-related minimal decreases in albumin (6%) and globulin (11%) concentrations occurred in males administered 50 mg/kg/day and in females (albumin 13% and globulin 10%) administered 10 mg/kg/day, with mild decreases in albumin (24%) and globulin (20%) in females administered 50 mg/kg/day. Increases in ALPi and gamma-glutamyl transferase activities, and decreases in albumin, globulin and fibrinogen concentrations correlated with increases in liver weight parameters and microscopic hepatocellular cytoplasmic alteration. Dose-related minimal (29%) or moderate (66%) decreases in cholesterol occurred in males administered ≥ 10mg/kg/day and females (52%) administered 50 mg/kg/day; and slight (42-48%) decreases in triglyceride concentrations in animals administered 50 mg/kg/day.
Test substance-related changes in clinical chemistry consisted of minimal (35-53%) increases in blood urea nitrogen (BUN) in males and females administered 50 mg/kg/day and concurrent minimal (28%) increases in creatinine concentrations in males administered 50 mg/kg/day. Dose-related minimal (2-4%) increases in chloride concentrations occurred in females at ≥ 3 mg/kg/day and males at ≥ 10 mg/kg/day. None of these clinical chemistry changes were considered biologically important at the magnitudes observed in this study; however, increases in ALPi and gamma-glutamyl transferase activities, and decreases in albumin, globulin and fibrinogen concentrations correlated with hepatocellular cytoplasmic alteration and increased liver weight parameters.

Study Day 120 – Recovery Necropsy
No test substance-related changes in clinical chemistry parameters were observed at recovery necropsy.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No test substance-related changes in urinalysis parameters were observed at terminal or recovery necropsy.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Treatment with TBS did not affect the functional observational battery or grip strength. A score of zero was considered normal. Several animals in all Groups exhibited abnormalities when assessed. Stained fur, low activity and loud respiration/vocalization were the primary findings and were not considered test substance-related due to a similar incidence of the findings in the control and dosed groups. Means of rearings, grooms, grip strength (forelimb and hindlimb), fecal boli, urine pool counts, and hindlimb splay were comparable across groups.
Treatment with TBS did not affect locomotor activity. Mean number of basic movements, fine movements, and rearings decreased over the three ten-minute intervals, showing normal habituation in all groups. Additionally, females had lower overall motor activity compared to males, as expected.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Terminal Necropsy (SD 92)
Test substance-related changes in organ weight parameters at terminal necropsy consisted of decreases in absolute testes weight, and testes weight relative to body and brain weight in males administered 50 mg/kg/day and increases in absolute liver weight, and liver weight relative to body and brain weight of females administered ≥ 10 mg/kg/day and males administered 50 mg/kg/day. Increases in liver weight parameters, increases in ALPi and gamma-glutamyl transferase activities, and decreases in albumin, globulin and fibrinogen concentrations correlated with hepatocellular cytoplasmic alteration. Decreases in testes weight parameters correlated with macroscopic observations of small testes and microscopic degeneration/atrophy of seminiferous tubules.

Recovery Necropsy (SD 120)
Test substance-related changes in organ weight parameters at recovery necropsy consisted of decreases in absolute testes weight, and testes weight relative to body and brain weight in males administered 50 mg/kg/day. Decreases in testes weight parameters correlated with macroscopic observations of small testes and microscopic degeneration/atrophy of seminiferous tubules. Other differences in organ weights were not attributed to test substance administration due to the lack of dose response, small magnitude and/or lack of correlative morphologic changes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Early deaths
Cause of death was considered accidental (gavage-related) in all five animals (1m – 0 mg/kg/day – 25136; 2m – 3 mg/kg/day – 25173; 4m – 50 mg/kg/day – 24209, 25211, 4f – 25227) based on macroscopic observations of esophageal perforation, or microscopic inflammation of the epicardium of the heart, pleura of the lung, surface of the diaphragm and/or interstitium surrounding the aorta and/or myofiber degeneration of the esophagus or inflammation of surrounding tissue. Minimal to moderate myeloid hyperplasia in the femoral and/or sternal bone marrow of animals 25136, 25173, 25209 and 25211 was considered secondary to inflammation caused by gavage error. Test substance-related findings in animals 25173 (2m); 25209 and 25211 (4m) consisted of severe degeneration/atrophy of the seminiferous epithelium in the testes with marked hypospermia in the epididymides in addition to minimal cytoplasmic alteration of hepatocytes in the liver in animal 25209. Adrenal gland hypertrophy and lymphoid depletion in the thymus of animals 25209 and 25211 (50 mg/kg/day) were not considered test substance-related, but most likely due to stress induced by gavage error. Testicular and epididymal changes in animals 25173 and 25211 (moribund kill), administered 3 and 50 mg/kg/day were considered background due to the unilateral distribution of changes.

Terminal Necropsy (SD 92)
Test substance-related macroscopic observations at terminal necropsy were limited to bilaterally small testis in 8 of 10 males administered 50 mg/kg/day that correlated with decreased testicular weight parameters and microscopically with degeneration/atrophy of seminiferous tubules.

Recovery Necropsy (SD 120)
Test substance-related macroscopic observations at recovery necropsy were limited to bilaterally small testis in 3 of 3 males administered 50 mg/kg/day that correlated with decreased testicular weight parameters and microscopically with degeneration/atrophy of seminiferous tubules. All other gross alterations seen in the current study were typical of sporadic, naturally occurring background changes commonly observed in rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Terminal Necropsy (SD 92)
At terminal necropsy, test substance-related changes occurred in the liver, testes and epididymides in males at a dose level of 50 mg/kg/day, in the liver and thyroid in females and pancreas of males at doses ≥ 10 mg/kg/day and the pancreas of females administered ≥ 3 mg/kg/day. In the males administered 50 mg/kg/day, test substance-related degeneration/atrophy of seminiferous tubules was characterized by shrunken tubular profiles, complete absence of elongating spermatids and spermatozoa within tubular epithelium, multifocal to segmental loss of round spermatocytes. Additional changes characterizing degeneration/atrophy of seminiferous tubules included scattered multinucleated giant cells, disorganized spermatogenesis, sloughed round/pachytene spermatocytes, degenerate cellular debris within the lumina and occasional Sertoli cell-only tubules. Severely affected animals had ≥ 90% of tubular cross sections affected. Minimally affected animals had < 10% tubules affected. In affected animals, Leydig cells were normal morphologically; however, due to tubular shrinkage appeared artifactually increased in density. These findings correlated with decreased testes weight parameters and macroscopic observations of small testes. The epididymides of eight of ten males administered 50 mg/kg/day had severe hypospermia and ten males had minimal or moderate cellular debris within the lumina. Severe hypospermia was characterized by lack of sperm throughout the epididymis although the tubular tails were completely filled with eosinophilic cellular debris (moderate cellular debris). Tubules in the body of the epididymis were partially collapsed (atrophy) resulting in increased thickness of the epithelium. The absence of sperm in the head and body of the epididymis coupled with the presence of cellular debris in the tail was consistent with testicular changes occurring within the duration of the study. In females administered ≥ 10 mg/kg/day and males administered 50 mg/kg/day, cytoplasmic alteration of hepatocytes was characterized by diffuse eosinophilia with minimal granularity of the cytoplasm in minimally affected animals, in mildly affected animals this change was characterized by cytoplasmic eosinophilia and granulation with minimal cell swelling sometimes with minimal vacuolation of the cytoplasm. Increased liver weight parameters and minimal increases in ALPi and/or gamma-glutamyl transferase activities at a dose level of 50 mg/kg/day and decreases in albumin, globulin and fibrinogen concentrations in males administered 50 mg/kg/day and in females administered ≥ 10 mg/kg/day correlated with hepatocyte cytoplasmic alteration. Dose-related minimal or moderate decreases in cholesterol occurred in males administered ≥ 10 mg/kg/day and females administered 50 mg/kg/day; and slight decreases in triglyceride concentrations in animals administered 50 mg/kg/day may also have been related to these hepatic changes. Thyroid follicular cell hypertrophy in females administered ≥ 10 mg/kg/day was characterized by follicular cell enlargement with a mild decrease in colloid within follicles. In the pancreas of males administered ≥ 10 mg/kg/day and females administered ≥ 3 mg/kg/day, zymogen depletion in acinar cells was noted. Of uncertain relationship to test substance administration was an increased incidence of minimal or mild myocardial degeneration with chronic-active inflammation affecting four males administered 50 mg/kg/day. Also of uncertain relationship to test substance administration were vacuolation of distal tubules in the kidney of one male administered 50 mg/kg/day. Male Animal 25210, administered 50 mg/kg/day, had granulomatous inflammation with foreign material within the interstitium surrounding the esophagus. This finding was consistent with a gavage error.

Recovery Necropsy (SD 120)
At recovery necropsy, test substance-related changes were observed in the testes and epididymides in males, and in the liver, thyroid and pancreas of females at a dose level of 50 mg/kg/day. All three recovery males in the 50 mg/kg/day group had minimal or mild degeneration/atrophy of the seminiferous epithelium in the testes as previously described; however, there was less degeneration and more atrophy present in affected tubules. In the epididymides, moderate cellular debris was present in the tail with moderate to severe hypospermia affecting the remainder of the tubules. In the two male animals (25218 and 25216) with moderate or marked hypospermia, there was a small number of spermatozoa in the proximal tubules indicating that spermatogenesis had begun to recover. Animal 25216 had related mild atrophy of the epididymides. Microscopic degeneration/atrophy of seminiferous tubules correlated with decreases in testes weight parameters and macroscopic observations of small testes. In the liver, one female (25232) had minimal cytoplasmic alteration of hepatocytes as previously described. Notably, this individual had the lowest individual absolute and relative liver weights compared with other females in the group. No increases in group mean liver weights occurred at recovery necropsy. Minimal thyroid follicular cell hypertrophy as previously described was observed in one female (25231) and decreased zymogen was noted in the pancreas of two females (25232 and 25233). All other microscopic alterations seen in the current study were typical of sporadic, naturally occurring background changes commonly observed in rats of this age and strain.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Decrease in testes weight and microscopic findings in testes at 50 mg/kg bw/day dose level

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No treatment related adverse effect observations

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

male reproductive system

Organ:

seminiferous tubules

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Results

(A) Selected Mean Organ Weight Differences at Main Phase Necropsy

Sex	Males				Females
Dose (mg/kg/day)	0	3	10	50	0	3	10	50
Testes
Absolute mean (g)	5.67	5.64	5.49	3.76*	–	–	–	–
(% difference)	–	0	-3	-34	–	–	–	–
Relative to body weight (%)	0.96	0.96	0.92	0.72*	–	–	–	–
(% difference)	–	0	-5	-25	–	–	–	–
Relative to brain weight (%)	2.43	2.51	2.44	1.74	–	–	–	–
(% difference)	–	+3	0	-28	–	–	–	–
Liver
Absolute mean (g)	14.61	15.06	15.11	16.49*	7.56	8.13	8.37	9.43*
(% difference)	–	+3	+4	+13	–	+8	+11	+25
Relative to body weight (%)	2.48	2.56	2.50	3.17*	2.58	2.72	3.05*	3.95*
(% difference)	–	+4	+1	+28	–	+5	+18	+53
Relative to brain weight (%)	6.24	6.70	6.74	7.64*	3.71	3.86	4.12	4.74*
(% difference)	–	+7	+8	+22	–	+4	+11	+28

(% difference) = Percent difference between group means relative to control group;

– = Not applicable; +/- = Increase/Decrease

* Statistically significant, ANOVA and Dunnett, p ≤ 0.05.

Selected Mean Organ Weight Differences at Recovery Phase Necropsy

Sex	Males
Dose (mg/kg/day)	0	50
Testes
Absolute mean (g)	5.70	4.52*
(% difference)	–	-21
Relative to body weight (%)	0.87	0.77
(% difference)	–	-11
Relative to brain weight (%)	2.49	2.06*
(% difference)	–	-17

(% difference) = Percent difference between group means relative to control group

– = Not applicable; +/- = Increase/Decrease

* Statistically significant, ANOVA and Dunnett, p ≤ 0.05.

(B) Incidence of Selected Test Substance-Related Microscopic Findings – Main Phase, Survivors

	Males				Females
Dose Level (mg/kg/day)	0	3	10	50	0	3	10	50
Number Examined	9	9	10	10	-	-	-	-
Testes, seminiferous tubule
Degeneration/atrophy               minimal	0	0	0	1	-	-	-	-
severe	0	0	0	8	-	-	-	-
Number Examined	9	9	10	10	-	-	-	-
Epididymis, lumen
Hypospermia                              severe	0	1^a	1^a	8	-	-	-	-
Cellular debris                           minimal	0	0	0	2	-	-	-	-
moderate	0	1^a	0	8	-	-	-	-
Atrophy                                     minimal	0	0	0	1	-	-	-	-
mild	0	1^a	0	6	-	-	-	-
moderate	0	0	1^a	0	-	-	-	-
Number Examined	9	9	10	10	10	10	10	10
Liver, hepatocyte
Cytoplasmic alteration (diffuse) minimal	0	0	0	4	0	0	9	1
mild	0	0	0	6	0	0	0	9
Number Examined	9	9	10	10	10	10	10	10
Thyroid
Follicular hypertrophy               minimal	0	0	0	0	0	0	2	3
mild	0	0	0	0	0	0	0	3
Number Examined	9	9	10	10	10	10	10	10
Pancreas, acinar
Decreased zymogen                minimal	0	0	1	1	0	2	1	1
mild	0	0	0	9	0	0	1	8

– = Not applicable; a. unilateral and not considered test substance-related.

Incidence of Selected Test Substance-Related Microscopic Findings – Recovery Phase

	Males				Females
Dose Level (mg/kg/day)	0	3	10	50	0	3	10	50
Number Examined	9	9	10	10	-	-	-	-
Testes, seminiferous tubule
Degeneration/atrophy               minimal	0	0	0	1	-	-	-	-
severe	0	0	0	8	-	-	-	-
Number Examined	9	9	10	10	-	-	-	-
Epididymis, lumen
Hypospermia                              severe	0	1^a	1^a	8	-	-	-	-
Cellular debris                           minimal	0	0	0	2	-	-	-	-
moderate	0	1^a	0	8	-	-	-	-
Atrophy                                     minimal	0	0	0	1	-	-	-	-
mild	0	1^a	0	6	-	-	-	-
moderate	0	0	1^a	0	-	-	-	-
Number Examined	9	9	10	10	10	10	10	10
Liver, hepatocyte
Cytoplasmic alteration (diffuse) minimal	0	0	0	4	0	0	9	1
mild	0	0	0	6	0	0	0	9
Number Examined	9	9	10	10	10	10	10	10
Thyroid
Follicular hypertrophy               minimal	0	0	0	0	0	0	2	3
mild	0	0	0	0	0	0	0	3
Number Examined	9	9	10	10	10	10	10	10
Pancreas, acinar
Decreased zymogen                minimal	0	0	1	1	0	2	1	1
mild	0	0	0	9	0	0	1	8

– = Not applicable

(c) Summary of Functional Observational Battery

Group	Sex	Animal	FOB Test	Observation	Domain	Score
1	m	25136	Hand Held	Vocalization During Handling	Activity Arousal	2
		25136	Open Field	Low Activity	Neuromuscular	1
		25140	Hand Held	Loud Respiration (Other)	Physiological	1
4		25207	Open Field	Low Activity	Neuromuscular	1
		25218	Hand Held	Stained Fur	Neuromuscular	1
1	f	25153	Hand Held	Stained Fur	Neuromuscular	1
		25156	Open Field	Low Activity	Neuromuscular	1
		25158	Hand Held	Loud Respiration (Other)	Physiological	1
2		25174	Hand Held	Stained Fur	Neuromuscular	1
3		25200	Hand Held	Loud Respiration (Other)	Physiological	1
4		25222	Hand Held	Stained Fur	Neuromuscular	1
		25228	Hand Held	Stained Fur	Neuromuscular	1

For more detailed results summary tables, kindly refer the attached background material section of the IUCLID.

Conclusions:

Under the study conditions, NOAEL for male rats was considered to be 10 mg/kg/day due to adverse effects on the testis organ weight and microscopic findings at 50 mg/kg/day, whereas the NOAEL for female rats was considered to be 50 mg/kg/day due to no adverse observations at this dose level.

Executive summary:

A study was conducted to determine the repeated dose 90-d oral toxicity of the test substance in rats according to OECD Guideline 408, in compliance with GLP. One hundred (50/sex) Sprague Dawley rats were randomly assigned to four groups (15 animals/sex in Groups 1 and 4 and 10 animals/sex in Groups 2 and 3). Animals were administered the test substance in control vehicle (peanut oil) at 0, 3, 10 or 50 mg/kg bw/day with a dosing volume of 2 mL/kg once daily via oral gavage for 91 consecutive days. Animals were subjected to a full gross necropsy on Day 92 (terminal; the end of the main phase [Days 1 – 91]) or Day 120 (recovery, 5 animals/sex/group for Groups 1 and 4; the end of the recovery phase [Days 92 – 119]). The parameters evaluated included mortality, physical examinations, cage side observations, body weights, body weight changes, food consumption, ophthalmic examinations, functional observations, locomotor activity, clinical pathology (clinical chemistry, hematology, coagulation, and urinalysis), gross pathology examinations, absolute and relative organ weights and histopathology. Repeated oral administration at doses up to 50 mg/kg bw/day had no effect on mortality, physical examinations, cage side observations, food consumption, ophthalmic examination findings, functional observations and locomotor activity. The body weight gains of males administered 50 mg/kg bw/day and of females administered 10 and 50 mg/kg bw/day were significantly lower than those of the vehicle controls. The mean body weights were statistically significantly lower than vehicle controls in males administered 50 mg/kg bw/day on Days 36 and from Days 57 through 91 of the main phase (8.4 to 11.2% difference compared to the controls) and in females administered 10 mg/kg bw/day at Days 78 and 85 of the main phase (7.4% and 8.8% difference compared to the control, respectively) and 50 mg/kg bw/day from Days 8 of the main phase through Days 106 of the recovery phase (5.6 to 21.8% difference compared to the controls). The effects on body weight were considered test substance-related, but not adverse, since there was no body weight loss and all animals increased body weight during the study. Test substance-related degeneration/atrophy of seminiferous tubules was observed at terminal necropsy in 9 of 10 males at 50 mg/kg bw/day and was considered adverse. Hypospermia, cellular debris and tubule atrophy in the epididymides of affected animals were secondary to testicular changes. Decreases in testes weight and macroscopic observations of small testes correlated with these changes. Although reduced in severity, testicular changes and secondary epidydimal findings were observed at recovery necropsy. This was consistent with a partial reversal of changes during the recovery phase. Important findings in other organs consisted of cytoplasmic alteration in hepatocytes in males administered 50 mg/kg bw/day and females administered ≥10 mg/kg bw/day, and thyroid follicular cell hypertrophy in females administered ≥10 mg/kg bw/day. Increases in liver weight, increases in alkaline phosphatase and/or gamma-glutamyl transferase activities, and decreases in albumin, globulin and fibrinogen concentrations correlated with hepatocellular cytoplasmic alteration. Additionally, decreases in cholesterol in males administered ≥10 mg/kg bw/day and females administered 50 mg/kg bw/day and decreases in triglyceride concentrations in animals administered 50 mg/kg bw/day occurred at terminal necropsy. At recovery necropsy, cytoplasmic alteration of hepatocytes was only observed in one female in the 50 mg/kg bw/day group and there were no correlating changes in clinical pathology or organ weight parameters which was consistent with a complete reversal of changes in males and a partial reversal in females. Thyroid follicular hypertrophy was observed in terminal necropsy females at ≥10 mg/kg bw/day and at recovery necropsy, one female had thyroid follicular hypertrophy consistent with a partial reversal of this finding during the recovery phase. Minimal increases in BUN concentrations in both sexes and creatinine concentrations in males were observed at a dose level of 50 mg/kg bw/day at terminal necropsy. Additionally, minimal increases in chloride concentrations occurred in females at ≥3 mg/kg bw/day and males at ≥10 mg/kg bw/day. These findings were not observed at recovery necropsy which was consistent with a complete reversal of this finding during the recovery phase. Decreased pancreatic acinar cell zymogen was noted at doses ≥10 mg/kg bw/day in males and ≥3 mg/kg bw/day in females. This change was completely reversed in males and partially reversed in females administered 50 mg/kg bw/day during the recovery phase. None of the test substance-related microscopic findings in the liver, thyroid or pancreas, or changes in clinical pathology parameters at terminal or recovery sacrifice were considered adverse due to the small magnitude of the clinical pathology changes, or lack of microscopic degenerative changes. However, increases in ALP and gamma-glutamyl transferase activities correlated with increased liver weight parameters and microscopic hepatocellular cytoplasmic alteration. Under the study conditions, the NOAEL for male rats was considered to be 10 mg/kg bw/day due to adverse effects on the weight and microscopic findings in testes at 50 mg/kg bw/day, whereas the NOAEL for female rats was considered to be 50 mg/kg bw/day due to no adverse observations at this dose level (Murphy, 2019a).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Organ:

seminiferous tubules

testes

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

Sub-chronic toxicity (from 90 day repeated dose toxicity study)

A study was conducted to determine the repeated dose 90-d oral toxicity of the test substance in rats according to OECD Guideline 408, in compliance with GLP. One hundred (50/sex) Sprague Dawley rats were randomly assigned to four groups (15 animals/sex in Groups 1 and 4 and 10 animals/sex in Groups 2 and 3). Animals were administered the test substance in control vehicle (peanut oil) at 0, 3, 10 or 50 mg/kg bw/day with a dosing volume of 2 mL/kg once daily via oral gavage for 91 consecutive days. Animals were subjected to a full gross necropsy on Day 92 (terminal; the end of the main phase [Days 1 – 91]) or Day 120 (recovery, 5 animals/sex/group for Groups 1 and 4; the end of the recovery phase [Days 92 – 119]). The parameters evaluated included mortality, physical examinations, cage side observations, body weights, body weight changes, food consumption, ophthalmic examinations, functional observations, locomotor activity, clinical pathology (clinical chemistry, hematology, coagulation, and urinalysis), gross pathology examinations, absolute and relative organ weights and histopathology. Repeated oral administration at doses up to 50 mg/kg bw/day had no effect on mortality, physical examinations, cage side observations, food consumption, ophthalmic examination findings, functional observations and locomotor activity. The body weight gains of males administered 50 mg/kg bw/day and of females administered 10 and 50 mg/kg bw/day were significantly lower than those of the vehicle controls. The mean body weights were statistically significantly lower than vehicle controls in males administered 50 mg/kg bw/day on Days 36 and from Days 57 through 91 of the main phase (8.4 to 11.2% difference compared to the controls) and in females administered 10 mg/kg bw/day at Days 78 and 85 of the main phase (7.4% and 8.8% difference compared to the control, respectively) and 50 mg/kg bw/day from Days 8 of the main phase through Days 106 of the recovery phase (5.6 to 21.8% difference compared to the controls). The effects on body weight were considered test substance-related, but not adverse, since there was no body weight loss and all animals increased body weight during the study. Test substance-related degeneration/atrophy of seminiferous tubules was observed at terminal necropsy in 9 of 10 males at 50 mg/kg bw/day and was considered adverse. Hypospermia, cellular debris and tubule atrophy in the epididymides of affected animals were secondary to testicular changes. Decreases in testes weight and macroscopic observations of small testes correlated with these changes. Although reduced in severity, testicular changes and secondary epidydimal findings were observed at recovery necropsy. This was consistent with a partial reversal of changes during the recovery phase. Important findings in other organs consisted of cytoplasmic alteration in hepatocytes in males administered 50 mg/kg bw/day and females administered ≥10 mg/kg bw/day, and thyroid follicular cell hypertrophy in females administered ≥10 mg/kg bw/day. Increases in liver weight, increases in alkaline phosphatase and/or gamma-glutamyl transferase activities, and decreases in albumin, globulin and fibrinogen concentrations correlated with hepatocellular cytoplasmic alteration. Additionally, decreases in cholesterol in males administered ≥10 mg/kg bw/day and females administered 50 mg/kg bw/day and decreases in triglyceride concentrations in animals administered 50 mg/kg bw/day occurred at terminal necropsy. At recovery necropsy, cytoplasmic alteration of hepatocytes was only observed in one female in the 50 mg/kg bw/day group and there were no correlating changes in clinical pathology or organ weight parameters which was consistent with a complete reversal of changes in males and a partial reversal in females. Thyroid follicular hypertrophy was observed in terminal necropsy females at ≥10 mg/kg bw/day and at recovery necropsy, one female had thyroid follicular hypertrophy consistent with a partial reversal of this finding during the recovery phase. Minimal increases in BUN concentrations in both sexes and creatinine concentrations in males were observed at a dose level of 50 mg/kg bw/day at terminal necropsy. Additionally, minimal increases in chloride concentrations occurred in females at ≥3 mg/kg bw/day and males at ≥10 mg/kg bw/day. These findings were not observed at recovery necropsy which was consistent with a complete reversal of this finding during the recovery phase. Decreased pancreatic acinar cell zymogen was noted at doses ≥10 mg/kg bw/day in males and ≥3 mg/kg bw/day in females. This change was completely reversed in males and partially reversed in females administered 50 mg/kg bw/day during the recovery phase. None of the test substance-related microscopic findings in the liver, thyroid or pancreas, or changes in clinical pathology parameters at terminal or recovery sacrifice were considered adverse due to the small magnitude of the clinical pathology changes, or lack of microscopic degenerative changes. However, increases in ALP and gamma-glutamyl transferase activities correlated with increased liver weight parameters and microscopic hepatocellular cytoplasmic alteration. Under the study conditions, the NOAEL for male rats was considered to be 10 mg/kg bw/day due to adverse effects on the weight and microscopic findings in testes at 50 mg/kg bw/day, whereas the NOAEL for female rats was considered to be 50 mg/kg bw/day due to no adverse observations at this dose level (Murphy, 2019a).

Sub-acute toxicity (from combined repeated dose toxicity and reproduction-development screening toxicity study)

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed in rats according to OECD Guideline 422, in compliance with GLP. Male rats were administered the test substance at doses of 0, 10, 30 or 100 mg/kg bw/day for 14 d prior to cohabitation and during cohabitation for at least a total of 28 d. Female rats were dosed with the test substance at doses of 0, 10, 30 or 60 mg/kg bw/day prior to cohabitation, during cohabitation, through pregnancy, and throughout lactation to Postnatal Day 13 (PND 13). Treatment-related effects in male rats included a significant effect on the body weight, body weight changes, organ weights, and microscopic findings. In female rats, repeat dose toxicity was observed at doses of 30 and 60 mg/kg bw/day primarily due to reduced body weight and microscopic findings in liver, kidney and thyroid. Important microscopic findings consisted of cytoplasmic alteration in hepatocytes at doses greater than 30 mg/kg bw/day in both sexes, thyroid follicular cell hypertrophy in males from 10 mg/kg bw/day and vacuolation of distal tubules in the kidney in males and females at the respective high doses. The test substance induced reproductive toxicity at doses of 30, 60 (females only) and 100 (males only) mg/kg bw/day. Treatment-related degeneration/atrophy of seminiferous tubules in all high dose males resulted in complete loss of fertility at this dose. Although degeneration of spermatids was only identified in one animal administered 30 mg/kg bw/day, this change was associated with decreased fertility and fecundity; therefore, testicular changes were considered adverse at or above 30 mg/kg bw/day. Decreases in testes and epididymal weight correlated with these changes in the high dose males. Under the study conditions, no NOAEL could be determined and the LOAEL for systemic toxicity in parental male rats was determined to be 10 mg/kg bw/day (due to lesions in thyroid at this dose). The NOAEL for systemic toxicity in parental female rats was considered to be 10 mg/kg bw/day due to adverse body weight changes, clinical chemistry, organ weight and microscopic findings in liver, kidney and thyroid (Murphy, 2019b).

Justification for classification or non-classification

Based on the results of the 90 d repeated dose toxicity study and the combined repeated dose toxicity and reproduction-development screening study in rats, it is proposed to classify the test substance as Rep. Cat. 2 (H361f: Suspected of damaging fertility) according to EU CLP (1272/2008/EC) criteria. There were no additional adverse effects that would trigger classification for repeated dose toxicity according to CLP criteria.