2-chloroaniline

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:

1988-10-04 to 1988-10-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-chloroaniline

EC Number:

202-426-4

EC Name:

2-chloroaniline

Cas Number:

95-51-2

Molecular formula:

C6H6ClN

IUPAC Name:

2-chloroaniline

Details on test material:

- Name of test material (as cited in study report): o-chloroaniline
- Physical state: yellow to orange liquid
- Analytical purity: 99.6 % (analytical results dated 1988-02-01, see 7.2.3, key, 1, Bomhard, 1988, IUC4 )
- Purity test date: 1988-02-01
- Lot/batch No.: Lagerkessel 15/ 594 488
- Stability under test conditions: stable throughout the test period
- Storage condition of test material: 3-7°C, dark

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen
- Age at study initiation: 8 to 12 weeks
- Weight at study initiation: 29 to 45 g
- Assigned to test groups randomly:yes
- Fasting period before study: no data
- Housing: 3 to 5 mice per macrolon cage I or II, respectively, bedding: wood chips S8/15 (Ssniff)
- Diet: ad libitum, Altromin 1324
- Water: ad libitum, tap water
- Acclimation period:


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22*-2
- Humidity (%): ~50 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: polyethylene glycol (Luterol E400)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

oral gavage

Duration of treatment / exposure:

single

Frequency of treatment:

single

Post exposure period:

48 h (cyclophosphamid 24h)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
500 mg/kg body weight
Basis:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1000 mg/kg body weight
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1500 mg/kg body weight
Basis:
nominal conc.

No. of animals per sex per dose:

5 per sex per dose or control (total 50 mice + 10 exposed mice (1500 mg/kg) in the replacement group)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg body weight (10 mL/kg)

Examinations

Tissues and cell types examined:

bone marrow (femur)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Pilot test for maximal tolerable dose finding with groups of 5 animals, that were orally administered 800 or 1000 mg o-chloroaniline/kg body weight. one animal of the high dose group died. Up to 72 h post-dose apathy, roughend fur, pallor, prone position,
twitching, shivering and difficult breathing were observed

TREATMENT AND SAMPLING TIMES : Animals received vehicle or 500, 1000 or 1500 mg o-chloroaniline/kg body weight single per orale gavage (5 mL/ kg body weight), 10 positive control animals were sacrificed by decaptation after 24 h, the other treated animals were decaptated after 48 h (10 each), 11 of 20 treated animals (high-dose) died during the test

DETAILS OF SLIDE PREPARATION: According to Schmid's method (1975), briefly, femurs were prepared, bone marrow was immersed in calf serum and flushed serveral times from both ends of the femur, the resulting cell suspension was centrifuged 5 min at 1000 rpm, the supernatant was almost totally discarded and the pellet resuspended. one drop was added to a slide, spread, dried over night, stained and covered

METHOD OF ANALYSIS: light microscope (magnification 1000x) The micronuclei apear as stained chromatin in the anucleated erythrocytes. 1000 polychromatic erythrocytes were counted per animal. Ratio of poly- to normochromatic erythrocytes was determined and normochromatic erythrocytes with micronuclei counted as well.

Evaluation criteria:

see above

Statistics:

Wilcoxon's non-parametric rank sum test (p< 0.01)

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

positive

Remarks:

weak significant clastogenic effect after 48 h in high-dose group

Toxicity:

yes

Remarks:

appathy, roughened fur, pallor, prone position, spasm, twitching, shivering, eyelid closure, rapid breathing, ratio between poly- and normochromatic erythrocytes unchanged

Vehicle controls validity:

valid

Negative controls validity:

other: historical

Positive controls validity:

valid

Any other information on results incl. tables

treated animals showed lasting symptoms of toxicity (11/20
animals died at a dose of 1500 mg/kg), the ratio
polychromatic/normochromatic erythrocytes was not altered,
at doses at or above 1000 mg/kg an increased incidence of
micronuclei-bearing polychromatic erythrocytes was observed, thus  indicating a weak clastogenic effect.

negative control 48 h

sex	polychromatic erythrocytes	no. normochromatic erythrocytes per 1000 polychromatic erythrocytes	micronucleated cells per 1000 normochromatic erythrocytes	micronucleated cells per 1000 polychromatic erythrocytes
m	1000	751	1.3	3
m	1000	584	0	2
m	1000	889	1.1	0
m	1000	689	0	4
m	1000	1362	1.5	1
f	1000	991	3.0	2
f	1000	801	2.5	1
f	1000	717	0	3
f	1000	1291	0.8	1
f	1000	995	0	0
mean/SD	1000	907/257	1.0/1.1	1.7/1.3

positive control 20 mg/kg cyclophosphamide per oral 24h

sex	polychromatic erythrocytes	no. normochromatic erythrocytes per 1000 polychromatic erythrocytes	micronucleated cells per 1000 normochromatic erythrocytes	micronucleated cells per 1000 polychromatic erythrocytes
m	1000	1552	1.3	23
m	1000	1298	0.8	13
m	1000	1241	0	23
m	1000	994	1.1	24
m	1000	822	1.2	18
f	1000	827	0	6
f	1000	953	1.0	9
f	1000	753	0	9
f	1000	465	0	9
f	1000	750	0	9
mean/SD	1000	961/318	0.5/0.6	14.3/7.

test substance o-chloroaniline 500 mg/kg 48 h

sex	polychromaticerythrocytes	no. normochromatic erythrocytes per 1000 polychromatic erythrocytes	micronucleated cells per 1000 normochromatic erythrocytes	micronucleated cells per 1000 polychromatic erythrocytes
m	1000	892	1.1	2
m	1000	1373	2.9	0
m	1000	861	2.3	2
m	1000	1262	0.8	1
m	1000	826	1.2	0
f	1000	590	1.7	3
f	1000	565	0	0
f	1000	656	3.0	0
f	1000	451	0	4
f	1000	735	0	3
mean/SD	1000	821/297	1.3/ 1.2	1.5/ 1.5

test substance o-chloroaniline 1000 mg/kg 48 h

sex	polychromatic erythrocytes	no. normochromatic erythrocytes per 1000 polychromatic erythrocytes	micronucleated cells per 1000 normochromatic erythrocytes	micronucleated cells per 1000 polychromatic erythrocytes
m	1000	981	1.0	3
m	1000	1169	0.9	2
m	1000	1130	2.7	5
m	1000	900	1.1	3
m	1000	863	0	2
f	1000	703	0	3
f	1000	1112	1.8	3
f	1000	1119	0	5
f	1000	688	0	9
f	1000	1494	1.3	6
mean/SD	1000	1016/242	0.9/ 0.9	4.1/ 2.2

test substance o-chloroaniline 1500 mg/kg 48 h

sex	polychromaticerythrocytes	no. normochromatic erythrocytes per 1000 polychromatic erythrocytes	micronucleated cells per 1000 normochromatic erythrocytes	micronucleated cells per 1000 polychromatic erythrocytes
m	1000	1875	1.6	2
m	1000	892	0	2
m	1000	1017	1.0	7
m	1000	1116	0	8
m		male died without replacement
f	1000	2265	0.9	6
f	1000	491	0	2
f	1000	1280	0	4
f	1000	1120	0	8
f	1000	578	0	7
mean/SD	1000	1182/573	0.4/ 0.6	5.1*/ 2.6

* p< 0.01 in non-prametric Wilcoxon ranking test

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): positive weakly clastogenic
There were biologically important and statistically significant variations in regard to incidence of micronucleated polychromatic erythrocytes between the negative control and the group orally treated with 1500 mg/kg body weight.

Executive summary:

Herbold, BA (1989)

o-chloroaniline was orally administed (gavage) to NMRI mice at a concentration of 500, 1000 or 1500 mg/kg body weight in polyethylene glycol and the clastogenic effect on polychromatic erythrocytes of the bone marrow (femur) was estimated after 48 h according to EU method B.12 utilizing the method introduced by Schmid (1975). Cyclophosphamide (20 mg/kg per os) served as positive control. A significant increase in micronuclei formation, 5.1/1000 (+/- 2.6), was observed 48 h post-application in bone marrow of mice treated with o-chloroaniline (1500 mg/kg). The positive and vehicle controls were valid, 14.3/1000 (+/- 7.0) and 1.7/1000 (+/- 1.3). Therefore indications of a weak clastogenic effect of o-chloroaniline at high doses were found.