Cyclopentane, methoxy-

Endpoint summary

Description of key information

28 day oral toxicity study by gavage - NOAEL = 150 mg/kg/day

90 day oral toxicity study by gavage - NOAEL = 32 mg/kg/day

90 day inhalation study by whole body exposure - NOAEC = 840 mg/m3

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

02 April 2002 - 10 February 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD, EC, and MHW guidelines, and in compliance with GLP; on this basis, the data is considered reliable without restrictions.

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Ministry of Health and Welfare for Japan - Part II of the Chemical Substance Control Law (1986).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: 99.9%
- Lot/batch No.: 020618

Species:

rat

Strain:

other: Crl:CD (SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Approximately 6 weeks old
- Weight at study initiation: 208.4 - 242.7 g (males); 164.2 - 203.1 g (females)
- Housing: Caged in groups of five. Five animals of a single sex per group.
- Diet (e.g. ad libitum): free access / ad libitum
- Water (e.g. ad libitum):free access / ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23°C
- Humidity (%):Nominally 40 - 70%; actually 31 - 66% (Protocol deviation not considered to have affected the outcome of the study).
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark.

IN-LIFE DATES: From: 18 September (animal arrival) To: 07 November 2002 (last terminal sacrifice date)

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations were freshly prepared weekly and stored at 4°C in the dark.

VEHICLE
- Concentration in vehicle: 3.0, 30, and 140 mg/mL.
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the start of dosing, an analytical method for the determination of NZE in corn oil formulations was developed and validated at 1 mg/mL and 200 mg/mL, and a stability trial conducted, which demonstrated that formulations at 2 mg/ml and 200 mg/mL were stable and homogeneous when re-suspended after 2 days' storage at ambient temperature and up to 8 days' refrigerated storage.

During the course of the study, samples from all formulations prepared at weeks 1 and 3 were analysed and found to be within +10/-15% of the nominal concentration for each dose group, confirming accurate preparation.

Analysis of samples was by GC-FID.

Duration of treatment / exposure:

Test duration: 28 days with the exception of the highest dose group males (700mg/kg/day) which received 15 days of treatment due to poor clinical condition.

Frequency of treatment:

Dosing regime: 7 days/week

Remarks:

Doses / Concentrations:
0 mg/kg/day (Vehicle control)
Basis:
actual ingested

Remarks:

Doses / Concentrations:
15 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
150 mg/kg/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
700 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10 males and 10 females at 0 mg/kg/day and 700 mg/kg/day
5 males and 5 females at 15 mg/kg/day and 150 mg/kg/day

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected on the basis of a prior 7-day preliminary study (Huntingdon Life Sciences, report number ZCE094/020184). The preliminary study concluded that treatment at 1000 mg/kg/day was not well tolerated, but that 700 mg/kg/day was; the 15 mg/kg/day and 150 mg/kg/day were chosen on the basis that they are relevant to EU classification and labelling requirements.
- Rationale for animal assignment (if not random): Random
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: During treatment, animals were observed: early in the working day, immediately prior to dosing, about 1 - 2 hours after dosing, and as late in the working day as possible.Throughout recovery period, all animals were checked early in each working day and again in the late afternoon.
- Animals were checked for mortality and clinical signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes - Neurobehavioural screening
- Time schedule: A detailed physical examination and arena based behavioural observations were carried out on all surviving animals during pre-treatment, weeks 1 - 4 of the dosing period, and during weeks 1 and 2 of the recovery period (where applicable).

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed when assigned to dosing groups, prior to dosing on day 1, and on days 8, 15, 22, and 28 (prior to overnight fasting for clinical pathology). Recovery animals were weighed on days 1, 8, and 14 of the recovery period. All bodyweights were recorded prior to necropsy.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
Daily monitoring by visual appraisal. Water consumption was assessed on Days 23 to 25 (during Week 4 of treatment), due to the apparent
haemoconcentration noted in the blood of the Group 4 males terminated early on Day 16 oftreatment.


OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples taken from the surviving main group 4 (700 mg/kg/day) males at day 16, and from all remaining main group animals on day 29. Blood samples were taken from recovery group males on day 30, and all recovery group females on day 15 of the recovery period.
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Food and water were removed overnight. All animals were then allowed access to water for 1 hour prior to blood sampling.
- How many animals: All (see above)
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: As HAEMATOLOGY, above
- Parameters checked in table [No. 2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: All surviving main group animals on day 29, all recovery group males on day 30, all recovery group females on day 15 of recovery.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked in table [No. 3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Refer to "Detailed Clinical Observations", above

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4)
ORGAN WEIGHT
HISTOPATHOLOGY: Yes (see table 5)

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

Food efficiency:

effects observed, treatment-related

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

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

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:

Clinical observations:
There were six unscheduled deaths among males receiving 700 mg/kg/day, which occurred between Day 12 and Day 15 of treatment and were due to poor clinical condition.

Clinical signs noted among males receiving 700 mg/kg/day consisted of salivation, noted on isolated occasions during the treatment period, and underactive behaviour, piloerection, abnormal gait, body tremors, convulsion, hunched posture, fast respiration and thin appearance noted in the period prior to the animal being killed in extremis.

Salivation and wet coats were noted frequently among females receiving 700 mg/kg/day during the second half of the treatment period. Salivation was also noted on two isolated occasions among males receiving 150 mg/kg/day during the treatment period. No treatment-related clinical signs were noted among females receiving 150 mg/kg/day or among rats receiving 15 mg/kg/day.

Neurobehavioural investigation carried out in Week 4 revealed higher mean activity among females receiving 700 mg/kg/day. Activity among recovery females previously treated at 700 mg/kg/day was comparable with that of the controls.

Lower than control bodyweight gain was noted for males receiving 700 mg/kg/day during Week 1 and it was severely impaired, with actual bodyweight loss noted, during Week 2.

Bodyweight gain for females receiving 700 mg/kg/day was progressively lower than that of control during Weeks 3 and 4 of treatment. Bodyweight gain for the recovery group male and female rats previously treated at 700 mg/kg/day was comparable with that of the control. Bodyweight gain for both sexes receiving 15 or 150 mg/kg/day was unaffected by treatment.

Food consumption for males receiving 700 mg/kg/day was lower than control during Week 2 of treatment. The food consumption of females receiving 700 mg/kg/day was unaffected by treatment. Food intake for the recovery group male and female rats previously treated at 700 mg/kg/day was comparable with that of controls. Food consumption for both sexes receiving 15 or 150 mg/kg/day was comparable with that of controls.

Food conversion efficiency was lower than control in week 1 and severely impaired during week 2 for males receiving 700 mg/kg/day. Food utilisation was also lower than control for females receiving 700 mg/kg/day during weeks 2 to 4 of treatment, with the degree of impaired increasing as treatment progressed. Food utilisation for the recovery group male and female rats previously treated at 700 mg/kg/day was essentially similar to that of control. Food conversion efficiency was unaffected by treatment for both sexes receiving 15 or 150 mg/kg/day.

Water consumption, measured on Days 23 to 25 (Week 4) of treatment, did not reveal any clear treatment-related changes.

Laboratory findings:
Haematology results obtained from the two males receiving 700 mg/kg/day, killed on Day 16, revealed higher haematocrit, haemoglobin, and red blood cell counts, which resulted in higher MCV and lower MCHC values when compared with the values for the control males terminated on Day 29.

In addition lower lymphocyte and eosinophil countsm which resulted in lower total white blood cell counts, were also noted for the two males receiving 700 mg/kg/day when compared with the Day 29 control males. Moderate hypochromasia was also noted in both of these animals.

Day 29 investigations revealed higher than control group mean haematocrit, haemoglobin, and red blood cell counts; and lower than control lymphocyte, eosinophil and total white blood cell counts for females receiving 700 mg/kg/day.

The assessment of the data for the recovery group male and female rats, previously treated at 700 mg/kg/day, revealed values that were generally comparable to control, indicating recovery from the previous effects of treatment.

Blood chemistry results obtained from the two males receiving 700 mg/kg/day, killed on day 16, revealed higher alkaline phosphate, total cholesterol, sodium, potassium, calcium, phosphorous, albumin, and total protein values, when compared with the values for the control males terminated on day 29. In addition higher alanine aminotransferase and aspartate aminotransferase values were noted in a single animal.

Day 29 investigations revealed higher group mean cholesterol values, total protein and albumin values for females receiving 700 mg/kg/day, when compared with controls.

The assessment of the data for the recovery group male and female rats, previously treated at 700 mg/kg/day, revealed values that were generally comparable with control, indicating recovery from the previous effects of treatment.

Day 29 urinalysis investigations revealed a higher than control group mean urinary protein value and the presence of small amounts of ketones in the urine of females receiving 700 mg/kg/day. In addition lower than control urinary protein and chloride values were noted for males receiving 150 mg/kg/day.

The assessment of the data for the recovery group male and female rats, previously treated at 700 mg/kg/day, revealed values that were generally comparable to control, indicating recovery from the previous effects of treatment.

Effects in organs:
The only organ weight changes of note were higher adrenal weights (relative to bodyweight) and lower spleen weights (both absolute and relative to body weight) noted for the two males receiving 700 mg/kg/day killed on Day 16. The remaining organ weight changes noted among animals receiving 700 mg/kg/day were considered to be due to the lower terminal bodyweights noted for these animals. There were no other organ weight changes attributable to treatment noted among the terminal or recovery animals.

The macroscopic changes were confined to males receiving 700 mg/kg/day killed in extremis or killed on day 16 and consisted of dark adrenals, pale spleens, depressions in the stomach and thin appearance noted in one or more males at this dose level. There were no other macroscopic changes attributable to treatment noted among the terminal or recovery animals.

The microscopic changes were confined to males receiving 700 mg/kg/day killed in extremis or killed on Day 16 and consisted of necrosis of the stomach and lamina propria observed in 4/6 decedent males.

Microscopic examination of females receiving 700 mg/kg/day and of male and female rats receiving 15 or 150 mg/kg/day did not reveal any changes that were considered to be attributable to treatment. In addition, no treatment related findings were observed in the tissues examined from the recovery group animals previously treated a 700 mg/kg/day.

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

gross pathology

Dose descriptor:

NOEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

gross pathology

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Urinalysis: slightly lower than control urinary protein and chloride levels for male rats receiving 150 mglkg/day but no microscopic changes in the kidneys so not considered to be adverse or of biological importance.

Critical effects observed:

not specified

Conclusions:

The No Observable Effect Level (NOEL) following 28-day repeated oral dose administration to rats was 150 mg/kg/day in females and 15 mg/kg/day in males; the effects seen in males rats dosed at 150 mg/kg/day were considered to be minor and not adverse, so 150 mg/kg/day is considered to be a No Observed Adverse Effect Level (NOAEL) in male rats.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2013-07-06 to 2014-08-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study, non GLP, but in compliance with China National Metrology Accreditation, in which the test parameters documented are based on testing guideline.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

no

Remarks:

non GLP, but in compliance with China National Metrology Accreditation

Limit test:

no

Specific details on test material used for the study:

Batch No.: 2820246
Purity: 99.9%

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Zhejiang Center of Laboratory animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 4-5 weeks old
- Weight at study initiation: 92-130 g
- Fasting period before study:
- Housing: Suspended, wire bottom, stainless steel cages, 2 animals per cage by sex
- Diet (e.g. ad libitum): Conventional laboratory diets, ad libitum
- Water (e.g. ad libitum): Tap water by aseptic filtration, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30-70%
- Air changes (per hr): 12 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test substance was emulsified with tween-80 and distilled water by emulsifying machine, and then with distilled water to achieve the required dosages.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 per sex per dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: based on acute oral LD50 values
- Rationale for animal assignment (if not random): randomly
- Rationale for selecting satellite groups: control group and high dose group
- Post-exposure recovery period in satellite groups: 14 days
- Section schedule rationale (if not random):

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded prior to initiation of study, weekly thereafter, and at death or sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / Not specified
- Time schedule for examinations:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at end of study
- Anaesthetic used for blood collection: Yes, 10% chloral hydrate
- Animals fasted: Yes, fasted overnight
- How many animals: all animals
- Parameters checked: white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), hematocrit (HCT), lymphocyte (LYM), granulocyte (GRA), monocyte (MID), red blood cell volume distribution width (RDW), platelet (PLT), prothrombin time (PT), activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at end of study
- Animals fasted: Yes, fasted overnight
- How many animals: all animals
- Parameters checked: total bilitubin (BIL-T), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), globulin (GLB), alkaline phosphatase (ALP), urea nitrogen (BUN), creatinine (CREA), glucose (GLU), potassium (K), natrium (Na), chlorine (Cl)

URINALYSIS: Yes
- Time schedule for collection of urine: at end of study
- Parameters checked: appearance (color and clarity), glucose (uGLU), bilirubin (uBIL), ketone (ket), occult blood (RBC), acidity (PH), protein (PRO), urobilinogen (uBG), nitrite (NIT), and leukocytes (LEU)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals in the study was subjected to a full, detailed gross necropsy which includes careful examination of the external surface of the body, all orifices, the cranial, thoracic and abdominal cavities and their contents. The liver, kidneys, adrenals, testes, epididymis, uterus, ovaries, thymus, spleen, brain and heart of all animals were trimmed of any adherent tissue, and their wet weight taken as soon as possible.

HISTOPATHOLOGY: Yes
Tissues and organs (all gross lesion, brain, spinal cord, stomach, thyroid, thymus, small and large intestines, pancreas, liver, kidneys, adrenals, spleen, heart, trachea and lungs, gonads, uterus, accessory sex organs, prostate, urinary bladder, lymph nodes, peripheral nerve, bone marrow) samples from control and treated rats were cut into thin slices, and then fixed into 4% formaldehyde for more than one week. The tissues and organs were then processed through a graded series of ethanol and xylene, and embedded in paraffin. Organs and tissues sections were stained with hematoxylin and eosin for histopathological examination under a microscope and recorded the abnormal condition.
Full histopathology should be carried out on the preserved organs and tissues of all animals in the control and high dose groups. These examinations should be extended to animals of all other dosage groups, if treatment-related changes are observed in the high dose group.

Statistics:

A parametric or non-parametric test was selected based on the results of normality test and homogeneity of variance test. One-way analysis of variance and Dunnett's t test were used in parameter test. Kruskal-Wallis rank sum test and Wilcoxon-Wilcox rank sum test were used in non-parameteric test. Student's t test was adopted by comparing data between additional high dose and control group, or nonparametric Wilcoxon test. Fisher's exact probability test was used for enumeration data.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

During exposure period and additional 14-day observation, obvious clinical sign of nostril flow fluid was observed in part of high dose group animals (3 females and 1 male) and additional high group (1 female and 1 male), obvious clinical sign of fluffy coat was observed in part of high dose group animals (3 females and 1 male), compared with control or additional control group, there were no statistically significant difference.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For male rats, body weight gain and body weight changes in high dose group were lower than control group, the differences were statistical significance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption per day: for male rats, the average food consumption per day in high dose group were lower than control group.
Relative food consumption: for male and female rats, the average relative food consumption in low, intermediate, high dose groups were closer to control groups.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

For male rats, total food efficiency in high dose groups were lower than control group.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

For female rats, RBC of intermediate and high dose groups were lower than control group, PT and HCT of high dose group were lower than control group, WBC of intermediate and high dose groups were higher than control group, MO and RDW of high dose group were higher than control group, PT of additional dose group were lower than additional control group, RDW of additional high dose group were higher than additional control group.
For male rats, WBC of intermediate dose group was higher than control group, LYM of high dose group was lower than control group, MO and PT of high dose group were higher than control group, PLT of additional high dose group were lower than additional control group.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

For female rats, A/G of intemediate dose group and high dose groups were lower than control group, BIL-T of high dose group were higher than control group, ALP of additional high dose group were higher than additional control group.
For male rats, GLB of additional high dose group were lower than additional control group, A/G of additional high dose group were higher than additional control group.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

For female rats, compared with control group, the coefficients of brain were increased in low dose group but no dose-effect relationship, the coefficients of kidney were increased in intermediate dose group, the coefficients of heart, liver, kidney and ovaries were increased in high dose group. The coefficient of liver and kidney were increased in additional high dose group compared with additional control group.
For male rats, compared with control group, the coefficients of brain, liver and testes were increased in high dose group, the coefficients of adrenals were increased in additional high dose group compared with additional control group.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

haematology

organ weights and organ / body weight ratios

Critical effects observed:

no

Conclusions:

In this repeated dose 90-day oral toxicity study, male and female rats with varying degrees of toxicity effects in intermediate and high dose groups. Obvious toxicity effects were not observed for male and female rats in low dose group. In additional 14 days of observation, no delayed toxicity effects were found in additional high dose group. The NOAEL was estimated to be 32 mg/kg/day in female and male rats in accordance with sample intake of rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

32 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Guideline study, non GLP, but in compliance with China National Metrology Accreditation.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 February 2004 - 14 September 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to OECD test guidelines, and in compliance with GLP, so the data is considered reliable without restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: 99.93%
- Lot/batch No.: 3700546

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Five weeks
- Weight at study initiation:Males: 141 - 174 g; Additional males: 158 - 181 g; females 132 - 152 g
- Housing: Autoclaved polycarbonate cages, replaced once per week after the start of the administration period. Animals were housed in groups of 3 or fewer during the acclimatisation period, and two per cage during the administration period.
- Diet (e.g. ad libitum): Ad libitum, except during the daily dosing period
- Water (e.g. ad libitum): Ad libitum, except during the daily dosing period
- Acclimation period: (Quarantine period) - 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Permissable range: 19.0 - 25.0°C; actual range: 21.6 - 22.7°C
- Humidity (%): Permissable range: 35 - 75%; actual range: 51.3 - 64.4%
- Air changes (per hr): 6 to 20 (10 to 30 per hour during the quarantine period)
- Photoperiod (hrs dark / hrs light): 12 hours light per day

IN-LIFE DATES: From: 02 March 2004 To: 14 July 2004

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Not recorded

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Rectangular parallelpiped chamber, inner volume approximately 90 L.
- Source and rate of air: Compressed air, 20 L/min (c.a. 13 air changes per hour)
- System of generating particulates/aerosols: Test material was passed through nitrogen to generate a gas.
- Air flow rate: 20 L/min
- Air change rate: 13 air changes/hour
- Treatment of exhaust air: Diluted into external air after passing through a charcoal filter.

TEST ATMOSPHERE
- Brief description of analytical method used: Atmosphere samples collected through an impinger (solvent used: methanol). Samples were analysed by GC:FID

VEHICLE (if applicable)
- Justification for use and choice of vehicle: Test material was nebulised using Nitrogen. The test atmosphere was mixed with air prior to exposure to the test animals to achieve the appropriate test material concentration for exposure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Once a week, duplicate atmosphere samples were collected at 0.5, 3.0, and 5.5 hours during the exposure period. Samples were collected using an impinger (solvent = methanol) and analysed by GC-FID.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

Five days per week for 13 consecutive weeks (total of 65 days' exposure).

Dose / conc.:

0 mg/L air

Dose / conc.:

0.2 mg/L air

Dose / conc.:

0.4 mg/L air

Dose / conc.:

0.8 mg/L air

Dose / conc.:

4 mg/L air

No. of animals per sex per dose:

Ten males and ten females per dose level. The control (0 mg/L) and high dose (4.0 mg/L) levels had an additional six males and six females which underwent a recovery period after the end of the dosing phase.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A prior 14-day range-finding study was conducted at targeted concentrations 0.8, 4.0, and 20.0 mg/L. Lethal effects were seen in the 20.0 mg/L level; salivation, body weight change and increased white blood cell count were seen in the 4.0 mg/L level. As toxic effects could be expected at 4.0 mg/L, it was chosen as the high level for the main study; 0.2 and 0.8 mg/L were chosen to give an approximate 5-fold spacing factor between levels. A 0.4 mg/L level was also used.
- Rationale for animal assignment (if not random): Stratified-by-weight randomisation, to obtain approximately mean bodyweights.
- Post-exposure recovery period in satellite groups: 28 Days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice per day on exposure days (before and after exposure), and once per day on non-exposure days.

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed before exposure on days 1, 8, 15, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 91, 92, 99, 106, 113, 119, and 120.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes (Mean food consumption was measured on a weekly basis).

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Performed on the day of animal allocation to groups, and in the final week of the administration period.
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 91 for main group (non-recovery) animals, day 119 for recovery group animals.
- Anaesthetic used for blood collection: Yes (Thiopental sodium)
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: As above (see Haematology)
- Animals fasted: Yes
- How many animals: All
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes (see table 4)

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No rats died during the exposure or recovery period.
Salivation and nasal discharge were seen in the group exposed to 4.0 mg/L after the daily exposure period; these signs were not seen on the non-dosing days.

BODY WEIGHT AND WEIGHT GAIN
During the administration period, the THE SUBSTANCE 4.0 mg/L group had statistically significant lower body weights compared to the control group from Day 22 in males and from Day 36 in females. Males of the THE SUBSTANCE 4.0 mg/L group also indicated statistically significant lower body weight gains compared to the control group. During the recovery period, there were statistically significant lower body weights in males and females of the THE SUBSTANCE 4.0 mg/L group. However, males of the THE SUBSTANCE 4.0 mg/L group had higher body weight gains compared to the control group, but they did not achieve statistical significance.
Females of the THE SUBSTANCE 4.0 mg/L group had a statistically significant higher body weight gain as compared to the control group on Day 113. Additionally, there were statistically significant higher body weights and body weight gains in males of the THE SUBSTANCE 0.2 mg/L group as compared to the control group. However, since there were no similar differences in the other groups exposed to higher concentrations, this difference was considered to be incidental.

FOOD CONSUMPTION
During the administration period, there were statistically significant lower food consumptions in females of the THE SUBSTANCE 4.0 mg/L group on Days 43, 57, and 64 as compared to the control group. There were no statistically significant differences between the control group and the test substance exposed groups during the recovery period.
Males of the THE SUBSTANCE 0.2 mg/L group sporadically indicated statistically significant higher food consumptions as compared to the control group during the administration period; however, since there were no differences in the groups exposed to higher concentrations, the differences were considered not attributed to the test substance administration. Moreover, statistically significant lower food consumptions compared to the control group were sporadically observed in females of the THE SUBSTANCE 0.2, 0.4, and 0.8mg/L groups; however, since these differences did not show continuity and did not coincide with body weight change, the differences were considered not attributed to the test substance administration.

OPHTHALMOSCOPIC EXAMINATION
In the examination before the start of administration or on the final week of the administration period, there were corneal opacity in males of the control group and were particulate opacity in lens, focal opacity in lens, and persistent hyaloid artery in males and females of each group including the control group. These findings were considered to be spontaneous.

HAEMATOLOGY
Males of the THE SUBSTANCE 0.4 mg/L group indicated statistically higher values of reticulocyte ratio and white blood cell count as compared to the control group, and males of THE SUBSTANCE 0.2 mg/L group also indicated higher white blood cell count at the end of the administration period. However, since there were no dose-related pattern in the incidence or severity, these differences were not considered attributed to the test substance administration.
At the end of the recovery period, males of the THE SUBSTANCE 4.0 mg/L group indicated statistically significant higher eosinophil ratio as compared to the control group. However, since this difference did not occur at the end of the administration period and the difference was slight without variations in white blood cell count, the difference was not considered attributed to the test substance administration.

CLINICAL CHEMISTRY
At the end of the administration period, males of the THE SUBSTANCE 4.0 mg/L group indicated statistically higher values of ALAT (GPT) and potassium as compared to the control group. There were no differences in these parameters at the end of the recovery period. Males of the THE SUBSTANCE 0.2 mg/L group revealed statistically higher potassium as compared to the control group at the end of the administration period; however, since there were no differences in this parameter in the THE SUBSTANCE 0.4 or 0.8 mg/L group, the difference was not considered attributed to the test substance administration. Moreover, males of the THE SUBSTANCE 4.0 mg/L group had statistically significant higher values of ALP and chlorine as well as lower triglyceride at the end of the recovery period; however, since these differences did not occur at the end of the administration period, they were not considered attributed to the test substance administration.

ORGAN WEIGHTS
At the end of the administration period, there were the following statistically significant differences as compared to the control group. In the liver, there were higher absolute and relative weight in males of the THE SUBSTANCE 0.2 and 4.0 mg/L groups and higher relative weight in females of the THE SUBSTANCE 4.0 mg/L group and in males of the THE SUBSTANCE 0.8 mg/L group. In the kidneys, there were higher absolute weight in males of the THE SUBSTANCE 0.2 and 4.0 mg/L groups and higher relative weight in males and females of the THE SUBSTANCE 4.0 mg/L group. Males and females of the THE SUBSTANCE 4.0 mg/L group had a lower absolute brain weight. In the salivary gland, there were lower absolute weight in males of the THE SUBSTANCE 0.4, 0.8, and 4.0 mg/L groups and lower relative weight in males of the THE SUBSTANCE 0.2, 0.4, 0.8, and 4.0 mg/L groups. Females of the THE SUBSTANCE 4.0 mg/L group had higher relative heart weight. At the end of recovery period, there were statistically significant higher relative weight in the heart, liver, and kidney in females of the THE SUBSTANCE 4.0 mg/L group among the above differences. Additionally, females of the THE SUBSTANCE 0.8 mg/L group had statistically significant lower absolute and relative ovary weight at the end of the administration period.
Regarding the higher absolute and body weight-relative liver weight and higher absolute kidney weight in males of the THE SUBSTANCE 0.2 mg/L, since there were no similar differences in the THE SUBSTANCE 4.0 mg/L group or dose-related patterns, the differences were not considered to be toxicologically significant. Moreover, since there were no dose-related patterns in higher absolute and body weight-relative ovary weight in females of the THE SUBSTANCE 0.8 mg/L group, the differences were not considered to be toxicologically significant. In the following differences at the end of the recovery period, the lower absolute adrenal weight in males of the THE SUBSTANCE 4.0 mg/L group as well as the higher absolute and body weight-relative thyroid weight and higher body weight relative values of the salivary glands, spleen, and adrenals in females of the THE SUBSTANCE 4.0 mg/L group, there were no differences in these parameters at the end of the administration period. Therefore, these differences were considered to be changes not related to the test substance administration.

GROSS PATHOLOGY
At the end of the administration period, there was crust of the skin observed in clinical observation in males and distention of the uterus in females. Distention of the uterus was observed in one rat each of the control, THE SUBSTANCE 0.2, 0.4, and 0.8 mg/L groups respectively. Since these findings were spontaneously changes in the rats, it was considered not attributed to the test substance administration.
At the end of the recovery period, there was loss of fur observed in clinical observation in males and females. Moreover, scratch wounds were observed in males. Additionally, there were the following findings: small thymus in one female and partial lobe torsion of the liver in one female of the control group, white patch in the lungs in one female of the THE SUBSTANCE 4.0 mg/L group. Since these findings were spontaneous changes, it was considered not attributed to the test substance administration.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the animals subjected to the necropsy at the end of the administration period, a hyaline droplet in proximal tubular epithelium in the kidneys was observed in three males of the THE SUBSTANCE 4.0 mg/L group. Moreover, simple hyperplasia of mucosal epithelium in the urinary bladder was observed in three males and two females of the THE SUBSTANCE 4.0 mg/L group. These findings were not observed in males or females of the THE SUBSTANCE 0.2, 0.4, and 0.8 mg/L groups, as well as the control group. Some histopathological findings were observed in males and females of each group including the control group. However, since these findings were spontaneous findings with no dose-related pattern in their incidence, it was considered not related to the test substance administration.
In the animals subjected to the necropsy at the end of the recovery period, there were no findings in the kidneys and urinary bladder observed in the THE SUBSTANCE 4.0 mg/L group at the end of the administration period.
Accumulation of foam cells was observed in the lungs that showed white patch in the necropsy. However, since this finding was spontaneous finding and was not observed in the animals subjected to the necropsy at the end of the administration period, it was considered to be an incidental change. Moreover, focal necrosis of the liver was observed at the area showing the partial lobe torsion. There was dilatation of hair follicle on the skin that showed loss of fur and there were no histological findings in the small thymus.

Dose descriptor:

NOEL

Effect level:

208 ppm (analytical)

Based on:

test mat.

Remarks:

Value quoted at 1 atmosphere and 20°C; converted from actual value of 0.87mg/L.

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOEL

Effect level:

201 ppm (analytical)

Based on:

test mat.

Remarks:

Value quoted at 1 atmosphere and 20°C; converted from actual value of 0.84mg/L.

Sex:

female

Basis for effect level:

other: Salivation and nasal discharge, lower body weight and bodyweight gain, higher liver and kidney weight, simple hyperplasia in mucosal epithelium of the urinary bladder in female rats treated with 4.67 mg/L

Critical effects observed:

not specified

Conclusions:

Repeated exposure of the substance vapour for 13 consecutive weeks by whole-body inhalation exposure demonstrated toxicological effects in the targeted concentration of 4.0 mg/L. Consequently, the no observed effect level of the substance was concluded to be 208 ppm (20°C, 1 atm, converted from actual value of 0.87 mg/L) for males and 201 ppm (20°C, 1 atm, converted from actual value of 0.84 mg/L) for females under the condition of this study. There was reversibility in all of the adverse effects during the 28 days of the recovery period.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

840 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Guideline study, GLP.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

28 -day repeated oral study:

A subacute oral toxicity study was conducted (Huntingdon Life Sciences, 2003, Study ZCE095) to assess the systemic toxicity of the substance in the rat, over 28 days of treatment by repeated once daily administration. A subsequent 14-day recovery period was allowed for selected animals to assess the potential for reversibility of any treatment-related changes. The study was conducted in accordance with OECD and EC test guidelines, and in compliance with GLP.

The substance was administered by oral gavage in corn oil, once daily, to three groups of five male and five female rats for up to twenty-eight consecutive days at dosage levels of 0, 15, 150 or 700 mg/kg/day. . In addition, a further two groups of five male and five female rats (Recovery groups) rec Neived the test substance at dosage levels of 0 (Control) or 700 mg/kg/day for up to 28 days and surviving animals were then maintained for a further 14 days untreated.

Treatment of males receiving 700 mg/kg/day was stopped on Day 16, due to the deteriorating clinical condition noted among these animals from Day 11. There were 6 unscheduled deaths among males receiving 700 mg/kg/day, which occurred between Days 12 and 15 of treatment and were due to poor clinical condition. The two surviving Main group males were killed and examined on Day 16, and the two surviving Recovery group males were maintained for a further 14 days untreated before undergoing terminal examination.

The main evidence of toxicity seen in males receiving 700 mg/kg/day was noted from Day 11, and consisted of poor clinical condition characterised by under active behaviour, piloerection, abnormal gait, body tremors, convulsions, hunched posture, fast respiration, and thin appearance. These signs were generally noted over a 24 to 48 hour period prior to the animal being killed in extremis. In addition all males receiving 700 mg/kg/day showed low bodyweight gain/actual bodyweight loss in Week 2, and lower relative spleen weights and higher relative adrenal weights were noted for the two males killed on Day 16. Macroscopic examination revealed a higher incidence of pale spleens and dark adrenals among these animals. Microscopic examination revealed a lower level of extramedullary haemopoesis in the spleen and a higher incidence of cortical medullary congestion in the adrenals. However, there were no other abnormal microscopic findings noted in the spleen, and congestion in the adrenals is a common finding observed among decedent animals. Thus, it is unclear whether the changes seen in the spleen and adrenals of males receiving 700 mg/kg/day were a direct effect of the test substance or secondary to other toxic changes. Assessment of the haematological and blood chemistry data obtained from the two males receiving 700 mglkg/day, killed on Day 16, revealed concentration of the blood constituents. This may have been a result of dehydration caused by the animals not drinking due to their poor clinical condition. In addition lower Iymphocyte and eosinophil counts were noted for these males, which was considered likely to be a secondary effect of treatment caused by their poor clinical condition.

Among females receiving 700 mg/kg/day, progressively lower bodyweight gain was noted during Weeks 3 and 4, which was associated with progressively impaired food conversion efficiency noted during Weeks 2 to 4 of treatment. This may indicate that dose administration over a longer period of time with the substance would not be tolerated and might result in deaths at this dosage level in females. In addition treatment-related changes similar to the blood concentration effects noted in the two males killed early on Day 16 were noted among females receiving 700 mg/kg/day. However, the water consumption and urinary volume for these animals were comparable to control. A higher level of motor activity was also observed among females receiving 700 mg/kg/day in Week 4, although the aetiology of this change is unclear, as no other signs of behavioural disturbance were noted in these animals. Assessment of the data obtained from the recovery animals previously treated at 700 mglkg/day showed that once treatment was stopped the animals recovered from the changes caused by treatment.

The only changes associated with treatment observed among rats receiving 150 mg/kg/day were lower than control urinary protein and chloride levels for males. However, these changes were slight, no similar lowering of these urinary parameters were noted among the treated female groups and no microscopic changes were observed in the kidneys of treated animals. Thus, these changes at 150 mg/kg/day were not considered to be adverse in nature and not to be of toxicological importance.

It is concluded that a dosage level of 15 mg/kg/day in males and 150 mg/kg/day in females represents the No Observable Effect Level (NOEL) for the substance, as no treatment related findings were observed at these dose levels when the substance was administered to rats for 28 consecutive days. The changes noted among males receiving 150 mg/kg/day were considered to be minor and not to be adverse in nature. Thus, a dosage level of 150 mg/kg/day in males only is considered to represents the No Observable Adverse Effect Level (NOAEL) on this study.

90 -day repeated oral study:

The evaluation of subchronic oral toxicities in male and female SD rats was conducted on this substance (Zhejiang Academy of Medical Sciences, 2014). Sixty rats per sex were randomly assigned to six groups: one control group, one low dose group, one intermediate dose group, one high dose group, two additional satellite groups in control and high dose, each group was 20 animals with 10 per sex. The substance was used for intragastric administration in the repeated dose 90-day oral toxicity study, the concentrations of the sample in the study were 0, 32, 125, 500, 0 and 500 mg/kg. All test animals were exposed and observed for 90 days, two additional satellite groups were observed for at least 14 days post treatment for observation of reversibility. Clinical signs, mortality, food consumption and body weight change were observed. When the study was terminated, urinalyses, gross necropsy, main organs weight, hematology, clinical biochemistry and histopathology were carried out.

During exposure and additional 14-day observation period, no detectable clinic signs and death were observed in each female and male rat. No abnormal gross anatomy were observed among the rats. Histopathological changes were no differences between high dose group with the control group or additional control group.

In this repeated dose 90-day oral toxicity study, male and female rats with varying degrees of toxicity effects in intermediate and high dose groups. Obvious toxicity effects were not observed for male and female rats in low dose group. In additional 14 days of observation, no delayed toxicity effects were found in additional high dose group. The NOAEL was estimated to be 32 mg/kg/day in female and male rats in accordance with sample intake of rats.

90 -day repeated inhalation study:

A subchronic inhalation toxicity study was conducted (Mitsubishi Chemical Safety Institute Ltd., 2004, Study B031366) to assess

the systemic toxicity of the substance in the rat, over 90 days of treatment by repeated administration.

The substance was administered by whole-body inhalation exposure, 6 hr/day and 5days/week, to five groups of 10 male and 10 female rats for up to thirteen weeks at concentrations of 0 (Control) , 0.2, 0.4, 0.8 and 4.0 mg/L. In addition, a further two groups of 6 male and 6 female rats received the test substance at concentration of 0 or 4.0 mg/L for up to 90 days and were subjected to a further 28 days untreated as a recovery period.

Salivation and nasal discharge were observed in males and females of the the substance 4.0 mg/L group on clinical observation. These findings were temporary changes after the exposure, as they were not observed on non-exposure days or were not observed in the recovery period.

Males and females of the substance 4.0 mg/L group showed lower body weight which was observed from Day 22 in males and from Day 36 in females and throughout the recovery period. This was associated with lower food consumption in the females. However, since body weight gain of this group recovered during the recovery period, the reversibility from the effect on body weight was concluded.

In the blood chemistry, there was a higher value of ALAT (GPT) in males of the substance 4.0 mg/L group as compared to those of the control group. Males and females of the same group indicated higher liver weight; however, there were no histological findings in the liver. Moreover, there was a higher value of potassium in males of the substance 4.0 mg/L as compared to those of the control group. These differences were not observed at the end of the recovery period.

In the pathological examination, there were higher absolute and body weight-relative kidney weight in males of the substance 4.0 mg/L group. Males of this group showed hyaline droplets in proximal tubular epithelium of the kidneys; this was considered related to the weight difference.This finding is observed specifically in male rats and is due to the administration of various chemicals which accelerate alpha 2u globulin and hyaline droplet formation in proximal tubular epithelium. Consequently, this finding has no toxicological significance in man. Simple hyperplasia in mucosal epithelium of the urinary bladder was also observed in males and females of the substance 4.0 mg/L group. However both findings were reversible as they were not observed in animals subjected to the necropsy at the end of the recovery period.

Besides the above findings, there were higher absolute and body weight-relative liver weight in males and females of the substance 4.0 mg/L group. These differences suggested acceleration of the metabolic function in the liver. However, since there were no histopathological findings in the liver, the higher liver weight was not considered to be toxicologically significant.

The variation in absolute and body weight-relative brain, salivary glands, heart and kidneys weight in the substance 4.0 mg/L group during the treatment or at the end of the administration period was not followed by histopathological findings and was therefore not considered to be toxicologically significant.

From these above results, repeated exposure of the substance gas for 13 consecutive weeks by whole-body inhalation exposure demonstrated toxicological effects in the targeted concentration of 4.0 mg/L. Consequently, the no observed effect level of the substance was concluded to be 208 ppm (20°C, 1 atm, converted from actual value of 0.87 mg/L) for males and 201 ppm (20°C, 1 atm, converted from actual value of 0.84 mg/L) for females under the condition of this study. There was reversibility in all of the adverse effects during the 28 days of the recovery period.

No repeated dose dermal toxicity study was conducted, as reliable results already exist for repeated dose toxicity following administration by oral and inhalation routes.

Justification for classification or non-classification

According to CLP Regulation (Commission Regulation 1272/2008), table 3.9.1 to 3.9.3, Substances should be classified for target organ toxicity (repeat exposure) on the basis of observations from appropriate studies in experimental animals in which significant/severe toxic effects, of relevance to human health, were produced at generally low or moderate exposure concentrations (<100 mg/kg/day by oral, <1 mg/L/day by inhalation).

For this substance, there is no significant toxic effects up to 500 mg/kg/day in 90 -day repeated oral study.

In 90 -day repeated inhalation study, hyaline droplet in proximal tubular epithelium in the kidneys, simple hyperplasia of mucosal epithelium in the urinary bladder was observed in high dose group 4.0 mg/L. Accumulation of foam cells was observed in the lungs that showed white patch in the necropsy. Moreover, focal necrosis of the liver was observed at the area showing the partial lobe torsion. There was dilatation of hair follicle on the skin that showed loss of fur and there were no histological findings in the small thymus. There was reversibility in all of the adverse effects during the 28 days of the recovery period.

On this basis, the substance is not classified for STOT-RE endpoint.