Cyclohexylbenzene

Endpoint summary

Description of key information

A key study was conducted for combined repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD guideline 422.  The effects related to repeated-dose toxicity to parental animals was demonstrated by clinical, clinical pathological and pathological changes at 1000 mg/kg bw/day. The liver and urinary tract/kidney are considered as the major target organ in both sexes at the highest dose level, related to some clinical (females) and clinical pathology changes (both sexes). The NOAEL for general, systemic toxicity was 200 mg/kg bw/day for the parental rats because of the clinical findings, the clinical pathology and the pathology. Local irritation in fore- and glandular stomach was observed at all dose levels (only in 1-2 females of low and mid dose group).  

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

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Recent and well documented study in according to OECD and GLP guidelines.

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and services, Germany GmbH
- Age at study initiation: 10-11 weeks old
- Housing: Makrolon type M III cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

+ 1% carbocymethylcellulose with about 5mg/100ml Cremophor.EL

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): a least once a week
- Storage temperature of food: room temperature

VEHICLE
- Concentration in vehicle: 1% carboxymethylcellulose + 5mg/100ml cremophor
:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Detail of analytical dose concentration carry out at the analytical chemistry laboratory of experimental toxicology and ecotoxicology of BASF SE.
The purity was performed by capillary GC with area % evaluation corrected with the content of water.
The concentration analyse of all concentration revealed that the value were in the expected range of the target concentration (about 90-110% of nominal concentration)

Duration of treatment / exposure:

for male: treatment started after acclimatisation (6 days) during 2 weeks before the mating, during the mating and 2 weeks after the end of the mating
for female: treatment started after acclimatisation (6 days) during 2 weeks before the mating, during the mating, during the gestation and 4 days after delivery.

Frequency of treatment:

Administered every day at the same time in the morning

Remarks:

Doses / Concentrations:
40 mg/kg bw
Basis:
actual ingested

Remarks:

Doses / Concentrations:
200 mg/kg bw
Basis:
actual ingested

Remarks:

Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10 per dose per sex

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: random

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily on working day dans once daily on saterday, wunday and public hollidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
- Examined parameters: abnormal behaviour during handling, fur, skin, posture, salivation, respiration, activity / arousal level, tremors, convulsions, abnormal movements, impairment of gait, lacrimation, palpebral closure, exophthalmus, feces (appearance / consistency), urine, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once daily

FOOD CONSUMPTION:
- Food consumption of the F0 parents was determined once weekly before and after the mating perio, as well as in dams during gestation (days 0-7, 7-14, 14-20) and lactation (days 1-4)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals.
- Anaesthetic used for blood collection: Yes (identity): isoflurane
- Animals fasted: Yes
- Parameters examined: leukocyte count (WBC), erythrocyte count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet count (PLT), differential blood count, reticulocytes (RET), clotting test: prothrombin time (Hepato Quick’s test, HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the moring
- Animals fasted: Yes
- Parameters examined: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), sodium (NA), potassium (K), chloride (CL), inorganic phosphate (INP), calcium (CA), urea (UREA), creatinine (CREA), glucose (GLUC), total bilirubin (TBIL), total protein (TPROT), albumin (ALB), globulins (GLOB), triglycerides (TRIG), cholesterol (CHOL), bile acids (TBA)

URINALYSIS: Yes
- Time schedule for collection of urine: overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: pH, protein, glucose, ketones, urobilinogen, blood, specific gravity, sediment, color / turbidity, volume

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: towards the end of the administration period
- Dose groups and number of animals that were examined: 5 animals per sex per test group
- Battery of functions tested: home cage observations, open field observations, sensory motor tests / reflexes, motor activity assessment
- Home cage observations (passive observations without disturbing the animals): posture, tremors, convulsions, abnormal movements, impairment of gait, other findings
- Open field observations: behavior when removed from cage, fur, skin, salivation, nose discharge, lacrimation, eyes / pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements / stereotypes, impairment of gait, activity / arousal level, feces excreted within 2 minutes (number, appearance, consistency), urine excreted within 2 minutes (amount / color), rearin within 2 minutes
- Sensory motor tests / reflexes: reaction to an object being moved towards the face (Approach response), touch sensitivity (Touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (Auditory startle response), coordination of movements (Righting response), behaviour during handling, vocalization, pain perception (Tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test
- Motor activity: according to the TSE Labmaster system, supplied by TSE Systems GmbH, Bad Homburg, Germany

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Necropsy: all parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.
- (Organ) weights determined in all animals sacrificed on schedule: anesthetized animals, epididymides, testes
- Organ weights determined in 5 animals / sex / dose (females with litters, same animals as used for the clinical pathology examinations): adrenal glands, brain, heart, kidneys, liver, spleen, thymus
- Organ / tissue fixation for all parental animals in 4% neutral-buffered formaldehyde or in modified Davidson's solution): all gross lesions, adrenal glands, aorta, bone marrow (femur), brain, cecum, cecum, cervix, coagulating glands, colon, duodenum, eyes with optic nerve, esophagus, extraorbital lacrimal glands, epididymides, femur with knee joint, heart, ileum, jejunum (with Peyer’s patches), kidneys, larynx, liver, lungs, lymph nodes (axillary and mesenteric), mammary gland (male and female), nose (nasal cavity), ovaries, oviducts, pancreas, parathyroid glands, pharynx, pituitary gland, prostate gland, rectum, salivary glands (mandibular and sublingual), sciatic nerve, seminal vesicles, skeletal muscle, spinal cord (cervical, thoracic and lumbar cord), spleen, sternum with marrow, stomach (forestomach and glandular stomach), testes, thymus, thyroid glands, trachea, urinary bladder, uterus, vagina

HISTOPATHOLOGY: Yes
- all above described organs were assessed in the control and highest dose group, either on 5 animals or on all animals of that dose group. The forestomach, kidneys and liver were examined additionally for 5 animals of the middle and low dose group.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

treatment-related non-adverse effects

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

not treatment-related

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

rat-specific effects, no human relevance

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

MORTALITY: No animal died prematurely in the present study.

CLINICAL SIGNS:
- Males and females, except gestation and lactation periods: All male animals and nine female animals of test group 3 (1000 mg/kg bw/d), six male animals and eight female animals of test group 2 (200 mg/kg bw/d) showed salivation during the premating period. One male animal of test group 3 showed poor general state on premating day 12. All male animals and nine female animals of test group 3 (1000 mg/kg bw/d), all male animals and eight female animals of test group 2 (200 mg/kg bw/d) showed salivation during the mating period. Nine male animals of test group 3 (1000 mg/kg bw/d), eight male animals and one female animal (No. 124) of test group 2 (200 mg/kg bw/d) showed salivation during the post- mating period. The salivation in different test groups on different time points in both sexes was assessed as substance-related local effect, due to the irritating potential in the stomach of the test substance.
- Females during gestation: Salivation was observed in nine female animals of test group 3 (1000 mg/kg bw/d) and eight female animals of test group 2 (200 mg/kg bw/d) from GD 0 onwards. The salivation in different test groups on different time points in both sexes was assessed as substance-related local effect, due to the irritating potential in the stomach of the test substance. In addition, animal No. 131 of test group 3 (1000 mg/kg bw/d) showed apathy, red encrusted nose, piloerection, poor general state (moderate) on GDs 21 and 22. From GD 23 this animal (No. 131) had slight poor general state and a complete litter loss. Furthermore, animal No. 139 of test group 3 (1000 mg/kg bw/d) showed red encrusted eye and poor general state (moderate) on GD 22, piloerection on GDs 22 and 23, slight poor general state and all pups were stillborn on GD 23. These finding were assessed as being substance related and an adverse effect. One sperm-negative F0 female of test group 2 (No. 124) did not deliver any F1 pups. This finding was assessed as being spontaneous in nature and without biological relevance.
- Females during lactation: Salivation was observed in all female animals of test group 3 (1000 mg/kg bw/d), in eight female animals of test group 2 (200 mg/kg bw/d) and in two female animals of test group 1 (40 mg/kg bw/d) from PND 0 onwards. The salivation in different test groups on different time points in both sexes was assessed as substance-related local effect, due to the irritating potential in the stomach of the test substance. In addition, animal No. 135 of test group 3 (1000 mg/kg bw/d) showed a complete litter loss on PND 2.
Animal No. 131 of test group 3 (1000 mg/kg bw/d) showed poor general state (slight) on PND 0. Furthermore, animal No. 139 of test group 3 (1000 mg/kg bw/d) showed slight poor general state, piloerection and all pups were stillborn on PND 0.
These finding were assessed as being as substance-related and an adverse effect.

BODY WEIGHT AND WEIGHT GAIN:
During the lactation period the mean body weight of test group 3 (1000 mg/kg bw/d) was significantly lower on PND 0 as well as on PND 4, with a maximum of -8.2% (compared to the control group) on PND 0. These finding were assessed as being substance-related and an adverse effect.
Body weights and body weight changes were not statistically affected in male animals, but the lower mean body weight of the F0 males during the premating period were likely to be related to the test substance.

FOOD CONSUMPTION: Male and female animals of test group 1000 mg/kg bw/d showed significantly decreased food consumption on premating day 0-7 and significantly decreased food consumption in females on premating day 0-13 compared to the control group..

HAEMATOLOGY: No treatment-related changes among hematological parameters were observed.
In males of test group 3 (1000 mg/kg bw/d) platelet counts were higher compared to controls and in females of the same test group relative reticulocyte counts were increased. However, both parameter were within historical control ranges (platelet 782-946 Giga/L, relative reticulocyte counts 1.2-2.9 %, PART III, Supplement) and therefore, these alterations were regarded as incidental and not treatment-related. In females of test groups 2 and 3 (200 and 1000 mg/kg bw/d) absolute neutrophil counts were higher compared to controls. The means were within (test group 3) or only marginal above (test group 2) the historical control range (absolute neutrophil counts 0.37-0.93 Giga/L, PART III, Supplement) and neither any other differential blood cell count nor total white blood cell counts were changed. Therefore, this alteration was regarded as treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).

CLINICAL CHEMISTRY: In male and females rats of test group 1000 mg/kg bw/d, glucose and chloride levels were decreased. Additionally, in females of the same test group cholesterol, triglyceride and potassium levels were increased. This was also true for potassium levels in females of test group 2 (200 mg/kg bw/d).
In males of test groups 200 and 1000 mg/kg bw/d triglyceride levels were increased (in test group 1000mg/kg/d not statistically significantly). In females of the same test groups total protein and globulin concentrations were higher compared to controls.
In females of test group 3 (1000 mg/kg bw/d) alanine aminotransferase (ALT) activities and calcium levels were increased.
In males of test group 2 (200 mg/kg bw/d) total bile acid levels were lower compared to controls, but the values were not dose-dependently changed.Therefore, all mentioned alterations were regarded as incidental and not treatment-related.

URINALYSIS: In male and female rats of test group 1000 mg/kg bw/d) incidences of higher protein levels in urine were increased.
Additionally in the urine sediment of males of test group 1000 mg/kg bw/d, more squamous and transitional epithelial cells as well as granular and epithelial cell casts were found. These effects were typical for male rats of this age. They were not found in the corresponding females. These findings were often observed in α2u-globulinuria which was confirmed by histopathology investigations and which was regarded as a rat specific finding without human relevance (Hard et al., 1993)
In females of test groups 1, 2 and 3 (40, 200 and 1000 mg/kg bw/d) urine specific gravity was higher compared to controls. This was due to a low specific gravity values in the study controls compared to the historical control range. Therefore, the altered urine specific gravity in urine of all dosed females was regarded as incidental and not treatment-related.

FUNCTIONAL OBSERVATIONAL BATTERY:
No test substance-related effects were observed.

ORGAN WEIGHTS: The increase in absolute and relative kidney weights in males of test group 3 (1000 mg/kg bw/day) and the increase of the relative kidney weight in females of test group 3 (1000 mg/kg bw/day) were thought to be treatment-related. The increase in absolute liver weight in males of test group 3 (1000 mg/kg bw/day), the increase in male relative liver weight in test groups 2 and 3 (200 and 1000 mg/kg bw/day) and the increase of absolute and relative liver weight in females of test groups 2 and 3 (200 and 1000 mg/kg bw/day) were regarded to be treatment-related.
The decrease in the relative heart weight of males of test group 2 (200 mg/kg bw/day) and the increase of the relative weight of testes in males of test group 3 (1000 mg/kg bw/day) were not regarded to be related to treatment as there was no dose-response relationship (heart) or because they were secondary to the terminal body weight reduction (testes) in test group 3 (1000 mg/kg bw/day). There were no histopathologic findings observed that could explain the weight increase in the testes. The reduction in absolute brain weight was also regarded to be secondary to the decrease in terminal body weight in males of test group 3 (1000 mg/kg bw/day).

GROSS PATHOLOGY: The light brown discoloration in kidneys of all males of test group 3 (1000 mg/kg bw/day) and the enlarged liver (graded mostly slight) of all males and females of test group 3 (1000 mg/kg bw/day) were regarded to be treatment-related.
All other findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

HISTOPATHOLOGY:
Treatment-related findings were observed in kidneys, liver and forestomach of males and liver and forestomach of females.

Kidneys:
Treated males of test groups 2 and 3 (200 and 1000 mg/kg bw/day) revealed a dose- response related increase in eosinophilic droplets. The eosinophilic droplets were proven to be alpha 2u globulin by immunohistochemistry. As secondary effects to the alpha 2u globulin storage in the kidney granular casts and an increase in basophilic tubules were observed in males of test group 3 (1000 mg/kg bw/day). Granular casts were characterized by marked dilation of solitary tubules with lightly staining, granular eosinophilic debris, which is derived from exfoliated cortical cells engorged with protein. These findings were thought to be treatment-related.
Male animals of test groups 1 and 2 (40 and 200 mg/kg bw/day) showed basophilic tubules with grading and incidence comparable to control animals and therefore this was not considered a treatment-related effect.

Liver:
Males and females of test group 3 (1000 mg/kg bw/d) revealed a diffuse liver cell hypertrophy which was centrilobularily accentuated. Males of test groups 1 and 2 (40 and 200 mg/kg bw/day) and females of test group 2 (200 mg/kg bw/day) showed a centrilobular hypertrophy of hepatocytes. These findings were regarded to be treatment-related.

Forestomach:
Males and females of test group 3 (1000 mg/kg bw/day) showed a diffuse hyperplasia of the squamous epithelium of the forestomach which was accompanied by hyperkeratosis. Females revealed in addition a multifocal ballooning degeneration of squamous epithelium cells, especially of the margo plicatus, an edema of the submucosa and inflammatory cell infiltrates in the submucosa and/or squamous epithelium. Multifocal ballooning degeneration and inflammatory cell infiltrates were still observed in one female of test group 1 (40 mg/kg bw/day) and 2 females of test group 2 (200 mg/kg bw/day). These findings were regarded to be treatment-related.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Dose descriptor:

NOAEL

Remarks:

for general toxicity of parents

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Clinical findings, clinical pathology, pathology.

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Local irritation in forestomach (noted only in 1-2 females of 40 and 200 mg/kg dose groups)

Critical effects observed:

not specified

Conclusions:

The NOAEL for general, systemic toxicity was 200 mg/kg bw/day for the parental rats beacause of the clincical findings, the clinical pathology and the pathology. Local irritation in fore- and glandular stomach was observed at all dose levels (only in 1-2 females of low and mid dose group).
The NOAEL for fertility and reproductive performance was 1000 mg/kg bw/day.
The NOAEL for developmental toxicity in the F1 progeny of the test substance-treated groups was found to be 200 mg/kg bw/day.

Executive summary:

Cyclohexylbenzene was administered daily as an aqueous suspension to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 40, 200 and 1000 mg/kg body weight/day. Control animals were dosed daily with the vehicle only (drinking water containing 1% Carboxymethylcellulose with about 5 mg/100 mL Cremophor EL).

The duration of treatment covered a 2 week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

The effects related to repeated-dose toxicity to parental animals was demonstrated by clinical, clinical pathological and pathological changes at 1000 mg/kg bw/day. Regarding clinical examinations, salivation was observed at more dose levels and was considered to be due to local irritation in the stomach, therefore it was not of systemic nature. However signs of general systemic toxicity (poor general state and piloerection) were observed in some female parental animals at 1000 mg/kg bw/day. Most relevant clinical pathological changes were decreased serum glucose and chloride levels in both sexes, increased serum cholesterol, triglyceride and potassium levels in females, and higher incidences of urinary protein in both sexes. Major macroscopic and microscopic pathological changes were seen in the kidneys (increased absolute/relative weight, light brown discoloration and eosinophilic droplets, alpha 2u globulin and intratubular casts in males) and liver (increased absolute/relative weight and enlarged size in both sexes, diffuse hepatocellular hypertrophy with centrilobular accentuation in both sexes). Further findings seen at all dose levels (with increasing incidence) were findings of the forestomach (1 to 2 animals with multifocal ballooning degeneration of squamous epithelial cells and inflammatory cell infiltrates at 40 and 200 mg/kg bw/day; the same findings in most females as well as squamous cell hyperplasia and hyperkeratosis in all animals at 1000 mg/kg bw/day). The latter forestomach findings findings were considered to be local irritation in rat stomach due to gavage, however humans do not have a forestomach. The kidney findings in male rats are also known to be of no human relevance, whereas the urinary protein changes may indicate urinary tract pathology, therefore the liver and urinary tract/kidney are considered as the major target organ in both sexes at the highest dose level, related to some clinical and clnical pathology changes as mentioned above.

The effects related to fertility and developmental toxicity are described under the reproductive toxicity endpoint.

Under the conditions of the present the NOAEL for general systemic toxicity was 200 mg/kg bw/d for the parental rats because of the clinical findings, the clinical pathology and the pathology findings. Local irritation inforestomach and glandular stomach was observed at all dose levels, therefore a LOAEL for local irritation in forestomach of 40 mg/kg bw/day was considered.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Reliable

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

A key study was conducted for combined repeated dose toxicity study with reproduction/developmental toxicity screening test according to OECD guideline 422 (BASF SE, 2013).

Cyclohexylbenzene was administered daily as an aqueous suspension to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 40, 200 and 1000 mg/kg body weight/day. Control animals were dosed daily with the vehicle only (drinking water containing 1% Carboxymethylcellulose with about 5 mg/100 mL Cremophor EL).

The duration of treatment covered a 2 week premating period and mating in both sexes (mating pairs were from the same dose group) as well as entire gestation and 4 days of lactation period in females up to one day prior to the day of schedule sacrifice of the animals.

The effects related to repeated-dose toxicity to parental animals was demonstrated by clinical, clinical pathological and pathological changes at 1000 mg/kg bw/day. Regarding clinical examinations, salivation was observed at more dose levels and was considered to be due to local irritation in the stomach, therefore it was not of systemic nature. However signs of general systemic toxicity (poor general state and piloerection) were observed in female parental animals at 1000 mg/kg bw/day. Most relevant clinical pathological changes were decreased serum glucose and chloride levels in both sexes, increased serum cholesterol, triglyceride and potassium levels in females, and higher incidences of urinary protein in both sexes. Major macroscopic and microscopic pathological changes were seen in the kidneys (increased absolute/relative weight, light brown discoloration and eosinophilic droplets, alpha 2u globulin and intratubular casts in males) and liver (increased absolute/relative weight and enlarged size in both sexes, diffuse hepatocellular hypertrophy with centrilobular accentuation in both sexes). Further findings seen at all dose levels (with increasing incidence) were findings of the forestomach (1 to 2 animals with multifocal ballooning degeneration of squamous epithelial cells and inflammatory cell infiltrates at 40 and 200 mg/kg bw/day; the same findings in most females as well as squamous cell hyperplasia and hyperkeratosis in all animals at 1000 mg/kg bw/day). The latter forestomach findings were considered to be local irritation in rat stomach due to gavage, however humans do not have a forestomach. The kidney findings in male rats are also known to be of no human relevance, whereas the urinary protein changes may indicate urinary tract pathology, therefore the liver and urinary tract/kidney are considered as the major target organs in both sexes at the highest dose level, related to some clinical and clnical pathology changes as mentioned above.

Under the conditions of the present the NOAEL for general systemic toxicity was 200 mg/kg bw/d for the parental rats because of the clinical findings, the clinical pathology and the pathology findings. Local irritation in the forestomach and glandular stomach was observed at all dose levels, therefore a LOAEL for local irritation in forestomach of 40 mg/kg bw/day was considered .

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Key study

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

Systemic effects were observed at the highest dose level (1000 mg/kg bw/d) of the repeated dose toxicity study. It concerns clinical, clinical pathology and pathological findings in the liver and kidneys of male and female animals. The kidneys effects in male animals were known to happen through a mechanism typically for male rats only with no human relevance.

As these systemic effects only occur at a dose level of 1000 mg/kg bw/d, according to the criteria described in section 3.9 of EU Regulation n° 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP), classification for Specific Target Organ Toxicity following Repeated Exposure (STOT-RE) is not required.

According to the criteria described in section 3.2.3 of Annex VI to Council Directive 67/548/EEC on the Classification, Packaging and Labelling of Dangerous Substances (DSD), the results of the repeated dose toxicity tests do not require a classification for danger of serious damage to health by prolonged exposure.