Aniline

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2001- 5 June 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Reference 2

Reference Type:

publication

Title:

High-dose clastogenic activity of aniline in the rat bone marrow and its relationship to the carcinogenicity in the spleen of rats

Author:

Bomhard EM

Year:

2003

Bibliographic source:

Arch. Toxicol. 77, 291-297

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Anilinium chloride

EC Number:

205-519-8

EC Name:

Anilinium chloride

Cas Number:

142-04-1

IUPAC Name:

anilinium chloride

Constituent 2

Reference substance name:

aniline, hydrochloride

IUPAC Name:

aniline, hydrochloride

Details on test material:

- Name of test material (as cited in study report): Aniline hydrochloride
- Substance type: white crystalline
- Physical state: crystalline solid
- Analytical purity: 100% w/w
- Purity test date: 10 September 1999 and 10 April 2002
- Lot/batch No.: 99-048
- Storage condition of test material: Ambient temperature (under N2)

Test animals

Species:

rat

Strain:

other: PVG

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (Bicester, UK)
- Age at study initiation: 9-11 weeks
- Housing: 3-4 per cage
- Diet (e.g. ad libitum): Rat and Mouse No. 1 Maintenance Diet; Special Diets Services, Witham, UK
- Water (e.g. ad libitum): tap water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- Humidity (%): 30-70%
- Air changes (per hr): 15 times/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: no data

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Application volume in each case was 10 ml/kg body weight; no further specification

Duration of treatment / exposure:

single dose

Frequency of treatment:

single dose

Post exposure period:

Up to 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
300, 400 and 500 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

7

Control animals:

other: vehicle and positive control

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): positive in micronucleus test
- Route of administration: oral
- Doses / concentrations: 3 animals received a single oral administration of 7.5 mg cyclophosphamide/kg body weight; these animals were killed and the bone marrow sampled 24 h later.

Examinations

Tissues and cell types examined:

polychromatic and normochromatic erythrocytes of the bone marrow

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
sampling after 24 and 48 h in test groups, 24 hr in positive control group. For each animal both femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paintbrush was rinsed in saline, wiped to remove the excess and wetted with a solution of albumin (6% w/v in physiological saline)

DETAILS OF SLIDE PREPARATION:
The iliac end of the femur was then dipped into the marrow canal and four smears were painted on a slide. Four slides were prepared for each animal. Initially, one slide from each animal was dipped in phosphate buffer then stained with a solution of acridine orange for 1 min, placed in phosphate buffer for 10 min and then in a fresh batch of buffer for a further 15 min. After staining, the des were air-dried and wet-mounted in buffer prior to analysis. Additional slides were stained for animals for which extended analysis was require

METHOD OF ANALYSIS:
The incidence of micronucleated immature erythrocytes per 1000 immature erythrocytes and the percentage of immature erythrocytes in the erythrocyte sample were evaluated by analysis of variance, separately at 24 and 48 h.
Slides were coded and scored blind. Initially for each animal, 2000 (2 x 1000) immature erythrocytes were examined for the presence of micronuclei. The slides were also examined for evidence of cytotoxicity, which may be manifest by alterations in the ratio of different cell types in the bone marrow. This was assessed by determining the ratio of immature to mature erythrocytes in a sample of 1000 erythrocytes. To investigate increases in the incidence of micronucleated immature erythrocytes at the 24-h-sampling time, a further 2000 (2 x 1000) immature erythrocytes were examined on slides from animals treated with the vehicle control and aniline hydrochloride at 500 mg/kg at the 24-h sampling time.

Evaluation criteria:

Each treatment group mean was compared with the control group mean at the corresponding sampling time using a one-sided Student's t-test, based on the error mean square in the analysis. The criteria for identification of cell types and micronuclei are based on those of Tinwell and Ashby (1989).

Statistics:

ANOVA; the data for the incidence of micronucleated immature erythrocytes were transformed using a square-root transformation, prior to analysis. The data for the percentages of immature erythrocytes were transformed using the double-arcsine transformation of Freeman and Tukey prior to analysis. Analyses were carried out separately for the aniline hydrochloride-treated groups and positive control group using the MIXED procedure in SAS software. Each treatment group mean was compared with the control group mean at the corresponding sampling time using a one-sided Student's t-test, based on the error mean square in the analysis.

Results and discussion

Test results

Sex:

male

Genotoxicity:

positive

Remarks:

at 400 and 500 mg/kg bw, sampling time 24 h

Toxicity:

yes

Remarks:

cyanosis at all dose levels

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Any other information on results incl. tables

Toxicity:

The clinical signs of animals dosed with 300 mg/kg and above included cyanosis, coloured urine (light brown) and being cold to touch. Examinations of the internal organs showed no abnormalities. None of the animals died during the observation period. The clinical signs indicate that the dose levels chosen can be considered to be in the range of the maximum tolerated dose of aniline hydrochloride for the route of administration used.

Genetic toxicity:

The group means incidences of micronucleated immature erythrocytes together with the percentages of immature erythrocytes are shown in Table 1. A small but statistically significant and dose-related increase in the incidence of micronucleated immature erythrocytes over the vehicle control values was observed at the 24-h sampling time. No such increase was observed at the 48-h sampling time. No statistically significant differences in the percentage of immature erythrocytes between the vehicle control and aniline hydrochloride-treated animals were observed at either sampling time. The test system positive control, cyclophosphamide, induced statistically significant increases in the frequency of micronucleated immature erythrocytes at the 24-h sampling time. One of the three animals of this group was a non-responder for unknown reasons.

Table 1a: Mean incidence of micronucleated immature erythrocytes

Treatment	Dose (1)	Mean icidence of MPE/1000 IE ± SD
		24 h	48 h
Aniline hydrochloride	500 mg/kg	4.9 ± 2.2	2.1 ± 0.9
	400 mg/kg	3.0 ± 2.2	2.0 ± 0.8
	300 mg/kg	2.7 ± 1.6	2.5 ± 0.7

Increase was statistically significant

Table 1b: Mean percentage of immature erythrocytes

Treatment	Dose (1)	% Immature erythrocytes ± SD
		24 h	48 h
Aniline hydrochloride	500 mg/kg	50.3±1.0	38.7±12.1
	400 mg/kg	37.8±12.3	44.9±5.9
	300 mg/kg	48.2±18.4	52.6±7.1

(1) dose expressed in terms of aniline base

IE = immature erythrocytes, MPE = micronucleated immature erythrocytes, SD = Standard deviation