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Toxicological information

Genetic toxicity: in vivo

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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

Constituent 1
Chemical structure
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 / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg body weight
Basis:

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