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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 02 March 2016 to 25 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Some of the animals in the main study had body weights that marginally exceeded the upper range quoted in the General Study Plan (200g). However, the animals were within the age range quoted and individual dose group weight variation did not exceed 20% of the mean weight. The animals were therefore considered acceptable for use in the study This minor deviation to the GSP was considered not to have affected the purpose or integrity
of the Study.
Deviations:
yes
Remarks:
This minor deviation to the GSP was considered not to have affected the purpose or integrity of the Study.
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Some of the animals in the main study had body weights that marginally exceeded the upper range quoted in the General Study Plan (200g). However, the animals were within the age range quoted and individual dose group weight variation did not exceed 20% of the mean weight. The animals were therefore considered acceptable for use in the study.
Deviations:
yes
Remarks:
This minor deviation to the GSP was considered not to have affected the purpose or integrity of the Study.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
Some of the animals in the main study had body weights that marginally exceeded the upper range quoted in the General Study Plan (200g). However, the animals were within the age range quoted and individual dose group weight variation did not exceed 20% of the mean weight. The animals were therefore considered acceptable for use in the study.
Deviations:
yes
Remarks:
This minor deviation to the GSP was considered not to have affected the purpose or integrity of the Study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
endogenous gene animal assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylanthraquinone
EC Number:
201-535-4
EC Name:
2-ethylanthraquinone
Cas Number:
84-51-5
Molecular formula:
C16H12O2
IUPAC Name:
2-ethyl-9,10-anthraquinone
Details on test material:
- Name of test material (as cited in study report): 2-ETHYLANTHRAQUINONE
- Physical state: yellow solid flakes
- Analytical purity: 98.83%
- Impurities (identity and concentrations): not reported
- Purity test date: not reported
- Lot/batch No.: 110929
- Expiration date of the lot/batch: 31 December 2012
- Stability under test conditions: not reported
- Storage condition of test material: room temperature in the dark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 7 to 12 weeks old
- Weight at study initiation: 182.6 to 214.4 g
- Assigned to test groups randomly: yes
- Housing: solid-floor polypropylene cages with wood-flake bedding
- Diet (e.g. ad libitum): Free access to mains food was allowed throughout the study
- Water (e.g. ad libitum): Free access to mains drinking water was allowed throughout the study
- Acclimation period: minimum acclimatisation period of five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25ºC
- Humidity (%): 30 to 70%
- Air changes (per hr): fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours light and twelve hours darkness

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Groups, each of five rats, were dosed twice at time 0 and 24 hours via the oral route with the test item at 400, 200 or 100 mg/kg.
Two further groups of rats were included in the study; one group (five rats) was dosed via the oral route with the vehicle alone (arachis oil) and a second group (five rats) was dosed orally with cyclophosphamide.
Duration of treatment / exposure:
24 hours
Frequency of treatment:
0 and 24 hours
Post exposure period:
48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
400 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
cyclophosphamide

Examinations

Tissues and cell types examined:
Bone Marrow
Details of tissue and slide preparation:
Immediately following termination, both femurs were dissected from each animal and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained, allowed to air-dry and a cover slip applied using mounting medium.
Evaluation criteria:
Comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.
A positive mutagenic response is demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared to the vehicle control group.
If these criteria were not fulfilled, then the test item was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.
Statistics:
The data was analysed following a transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
See Table 1.

Any other information on results incl. tables

Table 1            Micronucleus TestSummary of Group Mean Data

Treatment Group Number of PCE with Micronuclei per 4000 PCE PCE/NCE Ratio
Group Mean SD Group Mean         SD
Vehicle Control
10 ml/kg
48-hour Sampling Time
5.0 3.2 2.31 0.81
Positive Control
25 mg/kg
24-hour Sampling Time
61.0*** 16.9 1.78 1.27
2-Ethylanthraquinone (CAS No. 84-51-5)
400 mg/kg
48-hour Sampling Time
2.4 2.5 1.25* 0.23
2-Ethylanthraquinone (CAS No. 84-51-5)
200 mg/kg
48-hour Sampling Time
4.8 1.6 1.02** 0.20
2-Ethylanthraquinone (CAS No. 84-51-5)
100 mg/kg
48-hour Sampling Time
4.8 1.3 1.53 0.45

 

PCE

 

=

 

Polychromaticerythrocytes

NCE

=

Normochromaticerythrocytes

SD

=

Standard deviation

*

=

P<0.05

**

=

P<0.01

***

=

P<0.001

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:
The test item was considered to be non-genotoxic under the conditions of the test.
Executive summary:

The ability of the test item to produce damage to chromosomes or aneuploidy when administered to rats was performed according to OECD TG 474.

The micronucleus test was performed with only male rats using the oral route. Groups of five rats were treated at the maximum tolerated dose of 400 mg/kg, with 200 and 100 mg/kg as the two lower dose levels and using arachis oil as the vehicle. Animals were dosed twice at time 0 and 24 hours. Bone marrow was extracted and smear preparations were made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei and PCE/NCE ratio was calculated as an indicator for toxicity.

There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group and the test item was considered to be non-genotoxic under the conditions of the test.