2-ethylanthraquinone

Genetic toxicity: in vivo

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 guideline

Test guideline 1

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.

Test guideline 2

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.

Test guideline 3

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

Test material information

Constituent 1

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 / concentrations

Doses / concentrations 1

Dose / conc.:

400 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Doses / concentrations 3

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 Test–Summary 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.