1-chloro-2-nitrobenzene

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant guideline study, published in peer-reviewed literature, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of test material (as cited in study report): ortho-chloronitrobenzene, o-CNB
- Analytical purity: > 99%
- Supplier: Wako Pure Chemical Industries, Ltd. (Osaka, Japan)

Test animals

Species:

mouse

Strain:

other: Crj:BDF1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc. (Kanagawa, Japan)
- Age at initiation of treatment: 6 weeks
- Housing: individually in stainless steel wire hanging cages (112 mm [w] x 212 mm [d] x 120 mm [h])
- Diet (ad libitum): γ-irradiation-sterilized powdered diet (CRF-1, Oriental Yeast Co., Tokyo, Japan)
- Water (ad libitum): filtered, UV-irradiation-sterilized water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1
- Humidity (%): 54 ± 5
- Air changes (per hr): 15-17
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): 2 times at an interval of 7 weeks
- Mixing appropriate amounts with (Type of food): a diet containing o-CNB was prepared by blending o-CNB with γ-irradiation-sterilized powdered diet (CRF-1, Oriental Yeast Co., Tokyo, Japan) in a spiral mixer.
- Storage temperature of food: 4 ºC
- Feed in the food hoppers was refreshed once per week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of o-CNB in the powdered diet were determined by gas chromatography, and were found to range from 96.0% to 101% for o-CNB compared to the target concentrations at the time of preparation, and to decrease to 80.4% to 88.9% for o-CNB 8 days after preparation upon storage at room temperature, when the initial observed concentrations were taken as 100%

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily in feed

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
78, 313, 1250, 2500 and 5000 ppm
Basis:
nominal in diet

Doses / concentrations 2

Remarks:

Doses / Concentrations:
10.4, 43.6, 170.4, 345.1 and 684.1 mg/kg bw/day (males); 12.2, 49.5, 196.5, 400.3 and 762.5 mg/kg bw/day (female)
Basis:
actual ingested

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: the dietary concentrations of o-CNB were determined on the basis of a preliminary 2-week oral administration study.
- Rationale for animal assignment (if not random): stratified randomization into 6 body weight-matched groups.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for clinical signs and mortality

BODY WEIGHT: Yes
- Time schedule for examinations: once a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Time schedule for examinations: once a week
Daily intake of o-CNB was calculated from the daily amount of the consumed food, multiplied by the time-averaged, observed concentration of o-CNB in the diet, and divided by the body weight and expressed as mg/kg bw/day.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of 13-wek period
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, overnight
- How many animals: all surviving animals
- Parameters examined: as specified in the OECD test guideline (reported: red blood cell, hemoglobin, hematocrit, MCV)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of 13-wek period
- Animals fasted: Yes, overnight
- How many animals: all surviving animals
- Parameters examined: as specified in the OECD test guideline (reported: total bilirubin, AST, ALT)

Sacrifice and pathology:

ORGAN WEIGHTS: Yes
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes; the tissues specified in the OECD test guideline were examined for histopathology.

Statistics:

Body weights, organ weights and hematological and blood biochemical parameters were analysed by the following algorism. Bartlett's test was used to test whether the variance was homogeneous or not. When variance was homogeneous, one-way ANOVA was performed. When variance was not homogeneous, the Kruskal-Wallis rank sum test was performed by arranging all data of the control and exposed groups in descending order. Statistical differences in the means and the rank means among the group were analyzed by Dunnett's multiple comparison test and the same multiple comparison test by rank, respectively. Histopathological findings were analysed by Fisher's exact test. A two-sided analysis with p-values of 0.05 and 0.01 was performed to determine statistical significance.

Results and discussion

Results of examinations

Details on results:

CLINICAL SIGNS AND MORTALITY
There was no treatment-related mortality (1 female of the 1250 ppm group died intercurrently).
There were no anemic signs of external appearance in any of the mice of either sex fed o-CNB.

HAEMATOLOGY
The most sensitive, significant hematological change was a decrease in MCV for the female mice fed 313 ppm.

CLINICAL CHEMISTRY
Serum total bilirubin was significantly increased in the female mice fed 5000 ppm.
ALT was significantly increased from 2500 ppm in male mice and from 1250 ppm in female mice.

ORGAN WEIGHTS
The relative spleen and liver weights of mice of both sexes were linearly increased with an increase in dietary concentration of o-CNB.

HISTOPATHOLOGY: NON-NEOPLASTIC
Spleen: The incidence of congestion and increased extramedullary hematopoiesis was significantly increased in male and female mice fed 1250 ppm and above. The incidence of hemosiderin deposition was significantly increased from 313 ppm in both sexes.
Liver: The incidence of hemosiderin deposition in Kupffer's cells was significantly increased in the male and female mice fed 1250 ppm and above. A significantly increased incidence of centrilobular hypertrophy of hepatocytes was noted in male mice fed 313 ppm and above and in the female mice fed 1250 ppm and above. The incidence of nuclear enlargement with atypia of centrilobular hepatocytes, which was characterized by cell enlargement, varying nuclear size and shape and coarse chromatin in the nucleus, was significantly increased in the male mice fed 313 ppm and above and in the female mice fed 1250 ppm and above.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 10.4 - <= 12.2 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the increased MCV in female mice, hemosiderin deposit in spleen in both sexes, and centrilobular hypertrophy and nuclear centrilobular enlargement with atypia in the liver in male mice.

Target system / organ toxicity

Critical effects observed:

not specified