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Diss Factsheets

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:
from 1999-04-23 to 1999-06-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report date:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
429-290-0
EC Name:
-
Cas Number:
3380-30-1
Molecular formula:
C12 H8 Cl2 O2
IUPAC Name:
5-chloro-2-(4-chlorophenoxy)phenol

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL, CH-4414 Füllinsdorf, Switzerland
- Age at study initiation: 8-12 weeks
- Weight at study initiation: males mean value 35.4 g (SD ±3.1 g), females mean value 28.2 g (SD ± 1.7 g)
- Assigned to test groups randomly: yes
- Housing: single, in Makrolon Type I cages with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum (ALTROMIN 1324, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
PEG 400
Details on exposure:
Approximately 18 hours before treatment the animals received no food but water ad libitum. At the beginning ofthe treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Duration of treatment / exposure:
single exposure
Frequency of treatment:
once
Post exposure period:
24 and 48 h
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
24 h preparation interval: 200, 670, and 2000 mg/kg bw
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
48 h preparation interval: 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Ten animals (5 males, 5 females)
Control animals:
yes, concurrent vehicle
Positive control(s):
yes (CPA; Cyclophosphamide)

Examinations

Tissues and cell types examined:
Preparation of the Animals:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Griinwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.
Details of tissue and slide preparation:
Analysis of Cells:
Evaluation ofthe slides was performed using NIKON microscopes with 100x oil immersion objectives. 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 the PCEs. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one ofthe test points. A test item producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system. This can be confirmed by means ofthe nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.
Statistics:
non parametric Mann-Whitney test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
As estimated by a pre-experiment 2000 mg FAT 80'220/A per kg bw (the maximum guideline-recommended dose) was suitable.
The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test item as compared to the mean value of NCEs ofthe vehicle control indicating that the test substance had no cytotoxic properties in the bone marrow. In comparison to the corresponding vehicle controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test subsatnce were below the value of the vehicle control group.
40 mg/kg bw Cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Applicant's summary and conclusion