Reference type

Author

Year

Title

Bibliographic source

Testing laboratory

Report no.

Owner company

Company study no.

Report date

study report

MHLW (Ministry of Health, Labour and Welfare), Japan

2007

A combined repeated-dose/reproductive-developmental toxicity study of chlorocyclohexane by oral administration in rats.

Japan Existing Chemical Data Base (JECDB)

Mitsubishi Safety Institute Ltd.

B041794

2007-01-23

data published

7.8.1 Toxicity to reproduction: Toxicity to reproduction.001

combined repeated dose and reproduction / developmental screening

no

Qualifier

Guideline

Deviations

according to

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

no

yes

yes

- Name of test material (as cited in study report): Chlorocyclohexane
- CAS No.: 542-18-7
- Molecular weight: 118.60
- Lot No.: 5C1114
- Purity: 99.7%
- Supplier: Junsei Chemical Co., Ltd.
- Vapor pressure: 6.73 mmHg (25°C)
- Vapor density : 4.12 (air = 1)
- Boiling point : 142°C
- Melting point: -44°C
- Flash point: 29°C
- Specific gravity: 1.004 (20/4°C)
- Solubility: Insoluble in water and miscible with organic solvents such as alcohol and ether etc.
- Odor: Peculiar odor
- Physical state: Colorless liquid to slightly pale yellow
- Storage condition of test material: Dark and sealed in refrigerator (2.8 – 8.4°C)

rat

Crj: CD(SD)

male/female

TEST ANIMALS
- Source: Atsugi Breeding Center, Charles River Laboratories Japan, Inc.
- Age at study initiation: 9 weeks of age
- Weight at study initiation: 325-386 g for males and 193-231 g for females
- Housing: bracket-type metallic wire-mesh cages/males and females excluding gestation and lactation periods (W 195 × D 325 × H 180 mm),
and polycarbonate cage during gestation and lactation periods/females (W 265 × D 426 × H 200 mm)
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.4 to 23.7°C
- Humidity (%): 48.8 to 65.0%
- Air changes (per hr): 6 to 20 times per hour
- Photoperiod (hrs dark / hrs light):12-hour lighting per day

oral: gavage

olive oil

PREPARATION OF DOSING SOLUTIONS: Test substance was dissolved in olive oil for injection.
VEHICLE
- Justification for use and choice of vehicle: No data
- Amount of vehicle (if gavage): 5 ml/kg bw
- Lot/batch no. (if required): No data
- Dosing volume: 5 mL/kg
- Stability (test solutions): For 8 days
- Storage condition of test solution: Stored in a dark place at room temperature

yes

Test suspensions at each concentration of initial and final preparations were analyzed by the GC method at Mitsubishi Safety Institute Ltd. Results showed that the concentration of the test article in each suspension was 95.0 to 106.2% of the nominal concentration and both values were within the acceptable range (concentration: percentage of the nominal concentration, 100 ± 10%)

(P) Males: 42 days including 14 days pre-mating and mating periods
(P) Females: Days including 14 days pre-mating, mating and gestation periods and the days until day 4 of lactation

Daily: 7 times / week

0 (vehicle), 10, 60 and 300 mg/kg bw/day

Basis

actual ingested

12 females/dose (0, 10, 60, 300 mg/kg), 7, 12, 12, 7 males of 0, 10, 60, 300 mg/kg, respectively, 5 males and 5 females at 0 and 300 mg/kg bw/day (recovery group)

yes, concurrent vehicle

- Dose selection rationale: A preliminary study was conducted to determine the doses to be employed. Three males and three female SD rats receiving 0, 30, 100, 300, and 1000 mg/kg groups of the substance were administered for 14 days. As a result, death or moribundity was observed in all males and females receiving 1000 mg/kg groups. Salivation and increases in absolute and relative adrenal weights were observed in females receiving 300 mg/kg group. No clear changes related to the test substance were observed in males receiving 300 mg/kg group. Therefore, the high dose was set at 300 mg/kg/day, and the middle and low dose were set at 60 and 10 mg/kg/day using common ratio 5. Vehicle control groups were set using olive oil only.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
Males and females: once before the start of administration, during the administration period, and during the recovery period
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Males: once before the start of administration, once every week until Week 6 during the administration
Females: once before the start of administration, once every week until Week 6 during the administration and once during the lactation period.
No observation performed during the recovery period because no observed clinical changes during the administration period.
BODY WEIGHT: Yes
- Time schedule for examinations:
Males in the main and recovery groups were weighed on Day 1, 8, 15, 22, 29, 36, 42, and 43 of administration, and males of recovery groups were weighed on Day 50 and 56. Female satellite groups were weighted same frequencies to male recovery groups. Females in the main groups were weighed on Day 1, 8 and 15 of administration and copulated females were weighed on Day 0, 7, 14 and 20 of gestation, and days 0 and 4 of lactation.
FOOD CONSUMPTION : Yes
- Food consumption (g/day/rat) for each animal determined from the difference of the of the previous day's feeding amount: Yes
Measurement of food consumption was conducted on all animals at the following frequencies:
Males in the main and recovery groups on Day 1-8, 8-15, 15-22, 22-29, 29-36, 36-38, 43-50, and 50-52. Female satellite groups on Day 1-8, 8-15, 15-22, 22-29, 29-36, 36-42, 43-50, and 50-56. Females in the main group on Day 1-8, 8-15, 22-29, 29-36, 36-38, 43-50, and 50-52.
FOOD INTAKE: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On the day after the final day of administration and on the final day of the recovery period
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters examined red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocyte count, platelet count, white blood cell count, differential white blood cell count, prothrombin time, activated partial thromboplastin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On the day after the final day of administration and on the ending day of the recovery period
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked: ASAT (GOT), ALAT (GPT), γ-GT, ALP, total bilirubin, blood urea nitrogen, creatinine, glucose, total cholesterol, triglyceride, total protein, albumin, A/G ratio, calcium, inorganic phosphorus, sodium, potassium, chloride
URINALYSIS: Yes
- Time schedule for collection of urine: final week of administration (Day 38 of administration) in males
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: 5 animals/males/group
- Parameters checked: pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen
BLOOD HORMONE: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested:
1) Open field observation: Aerial righting reaction, arousal, urination, defecation, posture and body position, breathing, co-ordination movement, gait, tremor, clonic convulsion, tonic convulsion, stereotypy, and bizarre behavior.
2) Manipulative Test: Approach response, touch response, auditory response, tail pinch response, and aerial righting reflex
3) Measurement of Grip Strength: Grip strength of forelimb and hind limb
4) Measurement of Motor Activity.Motor activity: 10-minute intervals from 0 to 60 minutes

GROSS PATHOLOGY AND ORGAN WEIGHTS：Brain, heart, liver, kidneys, adrenals, thymus, spleen, testes, and epididymis.
HISTOPATHOLOGY: Cerebrum, pituitary, thymus, lymph nodes (including mesenteric and mandibular lymph nodes), trachea, lung, stomach, intestinal tract (duodenum, jejunum, ileum, cecum, colon, rectum), thyroids, parathyroid, heart, liver, spleen, kidneys, adrenals, urinary bladder, testes, epididymis, seminal vesicles (including the coagulating gland), prostate (ventral lobe), ovaries, uterus, vagina, bone marrow (one side femur), Sciatic nerve (one side femur), spinal cord, and gross abnormal sites.

The data were analyzed for homogeneity of variance by the Bartlett test. If variances were homogeneous, data was analyzed by one-way analysis of variance, whereas heterogenous data was analyzed by Kruskal-Wallis ranking test. When a significant difference was observed, Dunnett’s multiple comparison test was conducted between control and treated groups. If not homogenous, analysis was performed using the Kruskal-Wallis ranking test. Qualitative value as the pathological findings was analyzed by Wilcoxon test and Fisher’s exact test. Urinalyses data were analyzed by Kruskal-Wallis and Dunnet’s type mean rank test. Statistical significance was set at < 5% by two-sided.

Endpoint

Effect level

Based on

Sex

Basis for effect level / Remarks

NOAEL

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

male

Effects of lesions in the hyperplasia of mucosal epithelium of urinary bladder in males receiving 60 mg/kg .

NOAEL

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

female

Effects of lesions in the hyperplasia of mucosal epithelium of urinary bladder in females receiving 300 mg/kg.

yes

yes

no effects

not examined

no effects (Significant increase in reticulocyte counts was observed in males receiving 300 mg/kg bw/day, but it was not considered to be toxicological effects.)

yes (Significant decrease in inorganic phosphorus concentration was observed in females receiving 300 mg/kg bw/day, but it was not considered to be toxicological effects.)

no effects

no effects

yes

yes

yes

no data

CLINICAL SIGNS AND MORTALITY: No animal died in any group. Salivation was observed in males and females receiving 300 mg/kg from Day 9 to the end of dosing. No abnormal changes were observed in both sexes with recovery animals during the recovery period.
DETAILED CLINICAL OBSERVATIONS, MANIPULATIVE TEST, GRIP STRENGTH TEST AND LOCOMOTOR ACTIVITY MEASUREMENT: There were no changes related to the test substance in any group during the dosing.
BODY WEIGHT: Depression of body weight gains was observed in males and females receiving 300 mg/kg from Day 8 to 42.
ORGAN WEIGHTS: A significant increase in relative kidney weight was observed in males receiving 300 mg/kg bw/day at the end of both administration and recovery periods. HISTOPATHOLOGY: Hyaline droplet of proximal tubular epithelium in kidneys was observed in all males receiving 300 mg/kg. Minimal hyperplasia of mucosal epithelium in urinary bladder was observed in males receiving 60 mg/kg and males and females receiving 300 mg/kg at the end of the administration. This pathological change was found in females receiving 300 mg/kg after the recovery period. Minimal cell infiltration was present in one male and one female receiving 300 mg/kg at the end of administration and recovery periods.

Based on the effects of chlorocyclohexane on the urinary bladder, the no observed adverse effect level (NOAEL) for repeated oral dosing was determined to be 10 mg/kg bw/day in male rats and 60 mg/kg bw/day in female rats.

UUID

IUC5-6d4f5ddf-6d29-41e4-b901-cef79aa25950

Dossier UUID

0

Author

dra / National Institute of Health Sciences / Tokyo / Japan

Date

2017-01-04 16:42:56 JST

Remarks

Purpose flag

key study

Study result type

experimental result

Reliability

1 (reliable without restriction)

Rationale for reliability incl. deficiencies

OECD Test Guideline study under GLP condition