reaction mass of [(1R*,5S*)-3,3,5-trimethylcyclohexyl] acetate and [(1S*,5S*)-3,3,5-trimethylcyclohexyl] acetate

Endpoint summary

Description of key information

Oral (OECD 422), rat: NOAEL ≥ 500 mg/kg bw/day in males and 150 mg/kg bw/day in females

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Oct 2015 - 5 Feb 2016

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)

Version / remarks:

adopted 22 Mar 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD), SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 9 weeks (male), 8 weeks (female)
- Weight at study initiation: 312.4 - 349.0 g (males), 189.0 - 226.4 g (females)
- Housing: acclimation period and pre-mating: 1 animal per cage; mating: 1:1; lactation: neonates were kept with the dam; animals were kept in stainless wire mesh cages (260W x 350D x 210H mm) and in polycarbonate cages (260W x 420L x 180H mm)
- Diet: Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C (Harlan Laboratories, Inc., USA), ad libitum
- Water: public tap water filtered and irradiated by ultraviolet light, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 - 23.9
- Humidity (%): 47.0 - 57.5
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was weighed and placed in a container. The test substance was mixed with a small amount of vehicle to dissolve using a magnetic stirrer and then, the vehicle was gradually added to yield the desired concentration. The dosing formulations were stored in a refrigerator (5.7 – 7.6 °C). These dosing formulations were used within 7 days.

VEHICLE
- Justification for use and choice of vehicle: Through the preliminary solubility test to determine the solubility and dispersion characteristics of the test substance, corn oil was selected as the vehicle because the test substance was dissolved in it.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dosing formulations were conducted using gas chromatography and samples were taken three times from the middle of each dosing formulation prior to dosing and analyzed for verification of dose level concentration. The results of dose concentration analyses were determined to be 101.64 - 103.74%. These results were within the acceptable limits (± 15% of nominal values).
As a result of homogeneity and stability analyses conducted the 0.5 and 200 mg/mL dosing solutions were confirmed to be homogenous and stable for 4 h at room temperature and for 7 days under refrigeration.

Duration of treatment / exposure:

Main groups:
males: for 6 weeks, starting 2 weeks before mating, during mating and 2 weeks after mating
females: for 2 weeks prior to mating, throughout gestation and for at least 4 days after delivery up to the day before the scheduled terminal necropsy; females showing no evidence of mating were dosed daily for 26 days after the last day of mating

Recovery groups:
males and females: once daily for 6 weeks; animals were not mated and assigned to 2 weeks of recovery period after the completion of administration

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

12 (main groups)
6 (recovery groups; for control and high dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a previously conducted 2-week repeated oral dose range-finding study, an increase in organ weights of the kidney and liver and bilateral enlargement of the kidney were observed in males at 1000 mg/kg bw/day. Therefore, the high dose level was selected at 500 mg/kg bw/day. Then, the low and mid dose levels were selected at 50 and 150 mg/kg bw/day, respectively.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included: general condition and clinical signs, mortality/viability, abortion and pre-mature birth

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations for signs and symptoms of adverse effects, including central and autonomic nervous system effects, motor activity and behavior, were conducted on all animals once before the test and once a week throughout the dosing and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of males of the main group and animals of each sex of the recovery group were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing and recovery periods, the day before necropsy and on the day of necropsy (fasted body weights). Body weights of females of the main group were recorded just prior to dosing on Day 1, once a week throughout the dosing and recovery periods, on Days 0, 7, 14 and 20 of gestation, on Days 0 and 4 post partum and on the day of necropsy (fasted body weights). Fasted body weights recorded on the day of necropsy were presented, but were not included in statistical analysis.

FOOD CONSUMPTION: Yes
- Food consumptions of males of the main group and animals of each sex of the recovery group were recorded just prior to dosing on Day 1, once a week during the dosing and recovery periods and the day before necropsy. Food consumptions of females of the main group were recorded just prior to dosing on Day 0, once a week throughout the dosing and recovery periods, on Days 0, 6, 13 and 19 of gestation, on Days 0 and 3 post partum. Food consumption was not recorded during mating.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 6 males and females were randomly selected from the main study groups in addition to all animals from the recovery groups.
- Parameters examined: erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT), leukocyte count (WBC), neutrophils (NEU), lymphocytes (LYM), monocytes (MONO), eosinophils (EOS), basophils (BASO), reticulocytes (Reti), prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes
- How many animals: 6 males and females were randomly selected from the main study groups in addition to all animals from the recovery groups.
- Parameters examined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine (Crea), total bilirubin (T-Bili), total protein (TP), albumin (Alb), globulin (Glo), A/G ratio, glucose (Glu), total cholesterol (T-Chol), Triglyceride (TG), sodium (Na), potassium (K), chloride (Cl), calcium (Ca)

URINALYSIS: Yes
- Time schedule for collection of urine: 6 males and emales were randomly selected from the main groups in addition to all recovery animals for urinalysis two days before necropsy. Fresh, 3-hour and 24-hour urine samples were collected from the selected animals and analyzed.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Animals were fasted during the fresh urine collection, but were allowed free access to drinking water.
- Parameters examined: in fresh urine samples: pH, protein, glucose, bilirubin, occult blood, color and turbidity, sediment; in 24-hour urine samples: urine volume, specific gravity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Selected animals were examined a few days before necropsy.
- Dose groups that were examined: 6 males and females were randomly selected from the main study groups in addition to all recovery animals
- Battery of functions tested: pinna reflex, auditory (sound) reflex, corneal reflex, pupillary reflex, grip strength test, rotarod test, spontaneous motor activity test

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All males of the main group were sacrificed 2 weeks after mating and females of the main group were sacrificed on Day 6 post-partum. All animals of the recovery group were sacrificed 2 weeks after final dosing. Non-pregnant females were sacrificed on Day 27 after the last day of mating. Complete gross post-mortem examinations were conducted on all animals including the external and internal surfaces. All grossly visible abnormalities were recorded.

ORGAN WEIGHTS: Yes
Paired organs were weighed together. Animals were fasted overnight prior to necropsy and body weights were recorded on the day of necropsy. Organs were weighed and organ-to-body weight ratios were calculated. The testes and epididymides of all adult males were weighed. 6 males and females were randomly selected from the main study animals in addition to all recovery animals for necropsy. Following organs were weighed: brain, heart, liver, thymus, spleen, kidneys, adrenals, ovaries, uterus

HISTOPATHOLOGY: Yes
Tissue preservation and slide preservation
6 males and females were randomly selected from the main groups in addition to all recovery animals for tissue preparation. The testes and epididymides were fixed in Bouin's solution. The eyes with optic nerves were fixed in Davidson’s fixative. All other tissues were preserved in 10% neutral buffered formalin

For the histopathological examination, the preparation of specimens of organs and tissues was carried out and the remaining organs and tissues preserved in 10% neutral buffered formalin: brain, pituitary, thymus, lung with bronchi, trachea, thyroid, esophagus, heart, liver, spleen, kidneys, adrenals, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, testes, epididymides, prostate, ovaries, uterus, submandibular lymph node, mesenteric lymph node, bone marrow (femur and sternum), spinal cord, sciatic nerve, eye and optic nerve, urinary bladder, gross lesions

Besides, from all animals except for six females and males in the main group, the following organs and tissues were harvested and preserved: brain, pituitary, heart, thymus, liver, spleen, kidneys, adrenals, prostate, testes, epididymides, ovaries, uterus

Histopathological examinations were conducted as follows:
- 6 males and females from the control, low, mid and high dose group (especially, focused on spermatogenesis and interstitial testicular cell structure)
- All tissues from animals found dead or killed in a moribund condition
- All gross, macroscopic lesions
- Target organs noted at the high dose were examined for the recovery group

Statistics:

The statistical analysis of this study was conducted using the SAS program (SAS 9.3). For the data including body weights, food consumption, urine volume and specific gravity, hematology and blood biochemistry parameters, organ weights, mating result, birth and survival rates, sensory reactivity and motor activity, the Bartlett test was conducted to test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test was employed on homogeneity, if significant (significance level: 0.05), followed by Dunnett’s t-test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneity, if significant (significance level: 0.05), followed by Steel’s test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Mating index, fertility index and other data associated with gestation were analyzed utilizing Fisher’s exact test (significance levels: 0.05 and 0.01). For the data of the recovery group, Folded-F test was employed to test homogeneity of variance (significance level: 0.05, two-tailed). Student t-test was employed on homogeneity, if overruled, Aspin-Welch t-test was applied (significance levels: 0.05 and 0.01, two-tailed).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the main groups, soiled perineal region was observed frequently in males and females at 500 mg/kg bw/day from Day 15 to the final dosing day. Also, soiled perineal region was observed once in one male and female at 150 mg/kg bw/day on Day 15. Salivation was observed often in males at 500 mg/kg bw/day from Day 22 and sporadically in females at 500 mg/kg bw/day from Day 6 of gestation. In the recovery groups, soiled perineal region was observed in one male and five females at 500 mg/kg bw/day from Day 21. Salivation was observed in three males and one female at 500 mg/kg bw/day.

No clinical signs were observed in any animal of the control and dosed groups in the detailed examinations once a week.

Mortality:

mortality observed, treatment-related

Description (incidence):

All males of the main group and all animals of the recovery groups survived the duration of the study. Two females of the main group were found dead at 500 mg/kg bw/day on Days 3 and 5 post-partum, respectively. Before females were found dead, clinical signs such as soiled perineal region, staining around mouth and/or hematuria were observed.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No statistically significant differences in body weight changes were noted in males of the main group and in animals of both sexes in the recovery groups, but a statistically significantly high value was noted in females at 500 mg/kg bw/day in the main group on Day 0 post-partum (+9.7% vs. control), which was considered to be a test substance-related effect.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significantly increase in food consumption (+18.5%) was noted in males at 500 mg/kg bw/day in the main group on Day 42. A statistically significant increase in food consumption was noted in females at 150 and 500 mg/kg bw/day in the main group on Day 7 of gestation (+17.8 and +18.2%, respectively) and at 500 mg/kg bw/day on Day 14 of gestation (+14.4%). A statistically significant increase in food consumption was noted in males at 500 mg/kg bw/day in the recovery group on Days 8, 21 and 36 (+9.4, +12.7 and +27.5%, respectively). A statistically significant decrease in food consumption was noted in females in the recovery group on Day 29 (-8.6%).
A statistically significantly high value in food consumption was noted in females at 500 mg/kg bw/day in the main group during the dosing period. It was considered to be a test substance-related effect since it was associated with increase in body weights.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No effects were observed in any animal in the main and recovery groups. Other statistical significances were considered not to be test substance-related changes because of small magnitude and the values were within the range of historical reference data.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No effects were observed in any animal in the main and recovery groups. Other statistical significances were considered not to be test substance-related changes because of small magnitude and the values were within the range of historical reference data.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Proteinuria was observed in two males and two females at 500 mg/kg bw/day in the main groups and in one female at 500 mg/kg bw/day in the recovery group. Occult blood and erythrocyte were observed in one female at 150 mg/kg bw/day in the main group. However, there were no changes in the blood biochemistry and histopathological changes related to proteinuria, occult blood and erythrocytes in urine. Therefore, these changes were considered to be of little toxicological significance since no significant changes were observed in the histopathological examination.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test substance-related effects on auditory reflex, pinna reflex, pupillary reflex and corneal reflex test were observed in animals of both sexes in the main and recovery groups when compared to the control group. In animals of both sexes in the main groups, there were no test substance-related effects in the grip strength test, rotarod test and spontaneous motor activity when compared to the control group. In males of the recovery group, a statistically significant increase in grip strength test was noted at 500 mg/kg bw/day, but it was not considered to be of toxicological significance because of small magnitude.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In absolute and relative organ weights, a statistically significant high value of the thymus was noted in females at 500 mg/kg bw/day in the main group (+120% rel. compared to the control) while no statistically significant differences in the thymus were noted in the other animals (see Table 1). There were no histopathological changes related to increase in thymus organ weights. Therefore, this change was considered to be of little toxicological significance.
Other statistical significances in the absolute and/or relative organ weights were considered not to be test substance-related effects because of small magnitude and/or the values were within the range of historical reference data.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination of the two dead females revealed small thymus (2/2) and spleen (1/2), enlargement of adrenals (1/2) and black focus in the forestomach (1/2). These findings are frequently observed in rats under poor conditions and are effects related to test substance.
No treatment-related changes were observed in surviving animals. Other macroscopic findings observed in this study were considered to be incidental and not test substance-related changes.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Marked thymic lymphoid atrophy (2/2) and mild splenic lymphoid atrophy (2/2) were observed in both dead females at 500 mg/kg bw/day in the main group. These lesions correlated with necropsy findings of small thymus and spleen. In addition, mild adrenal cortical hypertrophy (1/2) and mild erosion/ulceration of stomach (1/2) were observed. These findings are frequently observed in rats under poor conditions and are effects related to test substance.
Histopathological examination of surviving animals revealed hepatocellular hypertrophy in animals of both sexes at 500 mg/kg bw/day in the main groups after six weeks of treatment (see Table 2). Hepatocellular hypertrophy was characterized by increased cytoplasmic volume, which was within the centrilobular zone. At the end of the 2-week recovery period, this finding disappeared in animals of both sexes at 500 mg/kg bw/day. It was considered to have little toxicological significance since hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature in the absence of associated inflammation or necrosis. Furthermore, the effect was completely reversible after the recovery period in this study. No test substance-related histopathological findings were noted in the reproductive organs of either sex.
Minimal and mild to moderate accumulation of hyaline droplets in the cortical tubular epithelium of the kidney was observed in all males of the test substance-dosed groups. Hyaline droplets were not observed in animals after the 2-week recovery period. The increased incidence and severity of this lesion were considered to be induced by the test substance. In addition, the affected tubules were not associated with any other visible evidence of tubular injury such as degeneration, inflammation or necrosis. Hyaline droplets are frequently observed in the cytoplasm of proximal tubules in male rats and characterized by variably sized, refractile, brightly eosinophilic droplets consisted of lysosomal accumulation of alpha-2u-globulin. A large number of agents were reported to result in alpha-2u-globulin nepropathy in males only. Therefore, this male-rat-specific effect was not considered to be of human relevance.
All other microscopic findings in various organs and tissues were considered to be incidental and of no toxicological significance.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse systemic effects up to and including the highest dose

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

mortality

Critical effects observed:

no

Table 1. Absolute and relative thymus weights.

	Dose level (mg/kg bw/day)
	0		50		150		500
	Male	Female	Male	Female	Male	Female	Male	Female
Thymus weight
Absolute (g)	0.35 ± 0.04	0.14 ± 0.09	0.33 ± 0.06	0.19 ± 0.07	0.32 ± 0.02	0.24 ± 0.05	0.35 ± 0.08	0.34 ± 0.07
Relative (%)	0.07 ± 0.01	0.05 ± 0.03	0.07 ± 0.01	0.07 ± 0.02	0.07 ± 0.01	0.08 ± 0.01	0.07 ± 0.01	0.11 ± 0.02

*Significantly different from control group.

 

Table 2. Incidence and severity of treatment-related microscopic findings in the liver

	Dose level (mg/kg bw/day)
	0		50		150		500
	Male	Female	Male	Female	Male	Female	Male	Female
Liver
Hypertrophy, hepatocellular, centrilobular	0/6	0/6	0/6	0/6	0/6	0/6	6/6	4/6

Conclusions:

Based on the results of this study, the NOAEL for systemic toxicity was set at ≥500 mg/kg bw/day in males and at 150 mg/kg bw/day in females due to mortality observed in two females at 500 mg/kg bw/day on Days 3 and 5 post-partum.

Executive summary:

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (2016). Twelve Sprague Dawley rats per sex and dose were treated via gavage with test substance at concentrations of 50, 150 and 500 mg/kg bw/day, respectively. The control group received the vehicle corn oil. Additionally, a recovery group of 6 rats per sex was allocated to the control and high dose group. Males were treated for 6 weeks, starting 2 weeks before the mating period, during mating and 2 weeks after mating. Females were treated for 2 weeks prior to mating, throughout gestation and for at least 4 days after delivery up to the day before the scheduled terminal necropsy. Females showing no evidence of mating were dosed daily for 26 days after the last day of mating. The doses were selected on the basis of a 2-week repeated oral dose range-finding toxicity study in which increased kidney and liver weights and bilateral enlargement of the kidney were observed in males at 1000 mg/kg bw/day.

All males of the main group and all animals of the recovery groups survived until scheduled study termination. Two females of the main group were found dead at 500 mg/kg bw/day on Days 3 and 5 post-partum, respectively. Before females were found dead, clinical signs such as soiled perineal region, staining around mouth and/or hematuria were observed. In the main groups, soiled perineal region was observed frequently in males and females at 500 mg/kg bw/day from Day 15 until study termination. Also, soiled perineal region was observed once in one male and female at 150 mg/kg bw/day on Day 15. Salivation was observed often in males at 500 mg/kg bw/day from Day 22 and sporadically in females at 500 mg/kg bw/day from Day 6 of gestation. In the recovery groups, soiled perineal region was observed in one male and five females at 500 mg/kg bw/day from Day 21. Salivation was observed in three males and one female at 500 mg/kg bw/day. No statistically significant differences in body weight changes were noted in males of the main group and in animals of both sexes in the recovery groups, but a statistically significantly high value was noted in females at 500 mg/kg bw/day in the main group on Day 0 post-partum (+9.7%), which was considered to be a test substance-related. A statistically significantly high value in food consumption was noted in females at 500 mg/kg bw/day in the main group during the dosing period. It was considered to be test substance-related since it was associated with increase in body weights. No test substance-related adverse effects were noted in the results of detailed clinical signs, sensory function, motor activity, urinalysis, hematology and blood biochemistry in animals of both sexes in the test substance-dosed groups. Significant increased thymus weights were noted in females at 500 mg/kg bw/day in the main group (+120% rel. compared to the control) while no statistically significant differences in the thymus were noted in the other animals. However, no histopathological changes related to increase in thymus organ weights were observed. Therefore, this change was considered to be of little toxicological significance. Histopathology of the two dead females at 500 mg/kg bw/day revealed marked thymic lymphoid atrophy (2/2) and mild splenic lymphoid atrophy (2/2). These lesions correlated with necropsy findings of small thymus and spleen. In addition, mild adrenal cortical hypertrophy (1/2) and mild erosion/ulceration of stomach (1/2) were observed. These findings are frequently observed in rats under poor conditions and are effects related to test substance. Histopathological examination of surviving animals revealed hepatocellular hypertrophy in animals of both sexes at 500 mg/kg bw/day in the main groups after six weeks of treatment. Hepatocellular hypertrophy was characterized by increased cytoplasmic volume, which was within the centrilobular zone. At the end of the recovery period, this finding disappeared in animals of both sexes at 500 mg/kg bw/day. Therefore, this finding was considered to have little toxicological significance since hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature in the absence of associated inflammation or necrosis. Furthermore, the effect was completely reversible after the recovery period in this study. Minimal and mild to moderate accumulation of hyaline droplets in the cortical tubular epithelium of the kidney was observed in all males treated with test substance. Hyaline droplets were not observed in animals after the recovery period. The increased incidence and severity of this lesion were considered to be induced by the test substance. In addition, the affected tubules were not associated with any other visible evidence of tubular injury such as degeneration, inflammation or necrosis. Hyaline droplets are frequently observed in the cytoplasm of proximal tubules in male rats and characterized by variably sized, refractile, brightly eosinophilic droplets consisted of lysosomal accumulation of alpha-2u-globulin. Therefore, this rat-specific effect was not considered to be of human relevance. No test substance-related findings were observed on reproductive organs in males and females at any dose group.

Based on the results of this study, the NOAEL for systemic toxicity was set at 500 mg/kg bw/day in males and 150 mg/kg bw/day in females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No 1907/2006.

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

Additional information

The test substance was tested in a combined repeated dose oral toxicity study with the reproduction/developmental toxicity screening study according to OECD Guideline 422 and in compliance with GLP (2016). Twelve Sprague Dawley rats per sex and dose were treated via gavage with test substance at concentrations of 50, 150 and 500 mg/kg bw/day, respectively. The control group received the vehicle corn oil. Additionally, a recovery group of 6 rats per sex was allocated to the control and high dose group. Males were treated for 6 weeks, starting 2 weeks before the mating period, during mating and 2 weeks after mating. Females were treated for 2 weeks prior to mating, throughout gestation and for at least 4 days after delivery up to the day before the scheduled terminal necropsy. Females showing no evidence of mating were dosed daily for 26 days after the last day of mating. The doses were selected on the basis of a 2-week repeated oral dose range-finding toxicity study in which increased liver weights in females and increased kidney weights and bilateral enlargement of the kidney in males were observed at 1000 mg/kg bw/day.

All males of the main group and all animals of the recovery groups survived until scheduled study termination. Two females of the main group were found dead at 500 mg/kg bw/day on Days 3 and 5 post-partum, respectively. Before females were found dead, clinical signs such as soiled perineal region, staining around mouth and/or hematuria were observed. In the main groups, soiled perineal region was observed frequently in males and females at 500 mg/kg bw/day from Day 15 until study termination. Also, soiled perineal region was observed once in one male and female at 150 mg/kg bw/day on Day 15. Salivation was observed often in males at 500 mg/kg bw/day from Day 22 and sporadically in females at 500 mg/kg bw/day from Day 6 of gestation. In the recovery groups, soiled perineal region was observed in one male and five females at 500 mg/kg bw/day from Day 21. Salivation was observed in three males and one female at 500 mg/kg bw/day. No statistically significant differences in body weight changes were noted in males of the main group and in animals of both sexes in the recovery groups, but a statistically significantly high value was noted in females at 500 mg/kg bw/day in the main group on Day 0 post-partum (+9.7%), which was considered to be a test substance-related. A statistically significantly high value in food consumption was noted in females at 500 mg/kg bw/day in the main group during the dosing period. It was considered to be test substance-related since it was associated with increase in body weights. No test substance-related adverse effects were noted in the results of detailed clinical signs, sensory function, motor activity, urinalysis, hematology and blood biochemistry in animals of both sexes in the test substance-dosed groups. Significant increased thymus weights were noted in females at 500 mg/kg bw/day in the main group (+120% rel. compared to the control) while no statistically significant differences in the thymus were noted in the other animals. However, no histopathological changes related to increase in thymus organ weights were observed. Therefore, this change was considered to be of little toxicological significance. Histopathology of the two dead females at 500 mg/kg bw/day revealed marked thymic lymphoid atrophy (2/2) and mild splenic lymphoid atrophy (2/2). These lesions correlated with necropsy findings of small thymus and spleen. In addition, mild adrenal cortical hypertrophy (1/2) and mild erosion/ulceration of stomach (1/2) were observed. These findings are frequently observed in rats under poor conditions and are effects related to test substance. Histopathological examination of surviving animals revealed hepatocellular hypertrophy in animals of both sexes at 500 mg/kg bw/day in the main groups after six weeks of treatment. Hepatocellular hypertrophy was characterized by increased cytoplasmic volume, which was within the centrilobular zone. At the end of the recovery period, this finding disappeared in animals of both sexes at 500 mg/kg bw/day. Therefore, this finding was considered to have little toxicological significance since hepatocellular hypertrophy in the centrilobular zone is generally considered to be an adaptive response in nature in the absence of associated inflammation or necrosis. Furthermore, the effect was completely reversible after the recovery period in this study. Minimal and mild to moderate accumulation of hyaline droplets in the cortical tubular epithelium of the kidney was observed in all males treated with test substance. Hyaline droplets were not observed in animals after the recovery period. The increased incidence and severity of this lesion were considered to be induced by the test substance. In addition, the affected tubules were not associated with any other visible evidence of tubular injury such as degeneration, inflammation or necrosis. Hyaline droplets are frequently observed in the cytoplasm of proximal tubules in male rats and characterized by variably sized, refractile, brightly eosinophilic droplets consisted of lysosomal accumulation of alpha-2u-globulin. Therefore, this rat-specific effect was not considered to be of human relevance. No test substance-related findings were observed on reproductive organs in males and females at any dose group.

Based on the results of this study, the NOAEL for systemic toxicity was set at 500 mg/kg bw/day in males and 150 mg/kg bw/day in females.

Justification for classification or non-classification

The available data on repeated oral dose toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.