2-ethylanthraquinone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three Ames tests are available. 2 -Ethylanthraquinone did not induce gene mutations in bacteria in any of these tests. However, 2-ethylanthraquinone induced chromosomal aberrations in cultured cells in an in vitro chromosomal aberration study. Therefore, an in vivo micronucleus test according to OECD TG 474 was conducted.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is described in the SIDS Initial Assessment Report for SIAM 30 which is peer-review by the Japanese government. The study followed OECD TG 471 and was performed under GLP. The Klimisch rating was taken over from the SIDS dossier. However as the full study report and full individual data were not available for full reliability assessment, a Klimisch 2 reliability for this study is proposed in the dossier.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

-S9 mix; 0, 156, 313, 625, 1250, 2500, 5000 μg/plate(all strains)
+S9 mix; 0, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156 μg/plate(TA100)
+S9 mix; 0, 156, 313, 625, 1250, 2500, 5000 μg/plate(TA1535, TA98, TA1537)
+S9 mix; 0, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/plate(WP2 uvrA)
+S9 mix(confirmative test, 1st); 0, 2.44, 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, 5000 μg/plate(TA100)
+S9 mix(confirmative test, 2nd); 0, 156, 313, 625, 1250, 2500, 5000 μg/plate(TA100)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9 mix: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (TA100, TA98 and WP2 uvrA), Sodium azide (TA1535) and 9-Aminoacridine hydrochloride (TA1537). +S9 mix: 2-Aminoanthracene (all strains)

Remarks:

- Vehicle(s)/solvent(s) used: DMSO

Details on test system and experimental conditions:

METHOD OF APPLICATION
pre-incubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

- plate/dose: 3

Evaluation criteria:

The chemicals were considered to be mutagenic when a dose-related increase in revertant colony count was observed and the number of revertant colonies per plate with the test substance was more than twice that of the negative control and when a reproducibility of test result was observed.

Statistics:

Not conducted

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100. E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

See "attached background material"

Conclusions:

Interpretation of results (migrated information):
negative

2-ethylanthraquinone did not induce gene mutations in bacteria under the conditions of this study.

Executive summary:

Reverse mutation assays using microorganisms (Salmonella typhimurium, Escherichia coli) were conducted to assess the potential of 2-ethylanthraquinone to induce gene mutations. 2-ethylanthraquinone did not induce gene mutations in bacteria under the conditions of this study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is described in the SIDS Initial Assessment Report for SIAM 30 which is peer-review by the Japanese government. The study followed OECD TG 473 and was performed under GLP. However as the full study report and full individual data were not available for full reliability assessment, a Klimisch 2 reliability for this study is proposed in the dossier.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: CHL cells

Metabolic activation:

with and without

Metabolic activation system:

Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

Preliminary Test:
Without S9 mix: 23.8, 39.7, 66.1, 110, 184, 306, 510 µg/mL
With S9 mix: 23.8, 39.7, 66.1, 110, 184, 306, 510, 850, 1417, 2362 µg/mL

Main Test:
Without S9 mix(short-term treatment); 0, 40.0, 80.0, 160, 320, 640 μg/mL
With S9 mix(short-term treatment); 0, 591, 1181, 2362 μg/mL
With S9 mix(short-term treatment, confirmative test); 0, 37.5, 75.0, 150, 300, 600 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Without S9: Mitomycin C. With S9: Cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 6 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid solution
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 plates/dose level

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

NUMBER OF CELLS EVALUATED: 100 per plate (200 per dose level)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

PREPARATION OF THE CULTURES
Two hours before preparation of chromosome specimens, Colcemid solution was added to stop cell division in metaphase. Then all of the culture medium was transferred to a centrifuge, then the cells were detached from the plate using 0.25 % trypsin solution and added to the culture medium in the centrifuge. The cell suspension was centrifuged for 5 minutes at 1000 r/min and the culture medium was removed, then 5 mL of 75-mmol/L potassium chloride aqueous solution which had been warmed to 37ºC was added and hypotonic treatment was conducted at 37ºC for 16 minutes. The hypotonic solution was removed by centrifugation, and the cells were fixed in fixative (3 volumes of methanol:1 volume of acetic acid) which had been
cooled to 4ºC. The fixative was replaced 3 times, an appropriate amount of new fixative added to make the cell suspension, of which 1-2 drops were placed on a degreased glass slide. The slide specimen was fully dried, then stained for 12 minutes with 1.2 % Giemsa stain diluted with 1/100-mol/L sodium phosphate buffer solution. The slide was gently rinsed with water and dried.

Evaluation criteria:

Final evaluation was conducted excluding cells having only gaps.
And incidence of abnormal cells of less than 5 % was judged to be negative, 5 % or more to less than 10 % was regarded as equivocal, and 10 % or more with reproducibility or dependence on the dose level of the test substance was judged to be positive.

Statistics:

The D20 value is the concentration (mg/mL) of the test substance required to induce some kind of abnormality in 20% of metaphases, which was calculated by the least squares method.

Species / strain:

other: CHL

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9: 47.4 % survival cells at highest concentration. With S9: tested up to limit concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation such as white membrane was observed at the end of exposure to the test substance at all dose levels. Because treatment in the higher dose groups was with suspensions of the test substance, there was residue of white powder of the test substance.

RANGE-FINDING/SCREENING STUDIES: The concentration of 50% cell growth inhibition was calculated as 161 μg/mL for short-term treatment without S9. In treatment with S9, although a weak inhibition of cell growth was observed, inhibition of 50 % or more was not seen even at the highest dose of 2362 μg/mL (equivalent to 10 mM).
At the end of exposure to the test substance, white membrane was observed at 23.8 μg/mL and more without S9 and at 66.1 μg/mL and more with S9, white powder at 306 μg/mL and more without S9 and at 184 μg/mL and more with S9, and precipitation of white clumps at 306 μg/mL and more with S9. Because treatment in the higher dose groups was with suspensions of the test substance, there was residue of the test substance in the culture medium at 510 μg/mL and more by both treatment methods.

COMPARISON WITH HISTORICAL CONTROL DATA: The incidences of chromosome aberrations in the positive control and negative control were both within the range of this center’s historical data, indicating that this study is valid.

Short-term Treatment without S9

The incidence of structural chromosome aberrations in the test substance treatment groups was comparable to the negative control. The incidence of polyploid cells was 1.5% at 40.0 μg/mL, 8.0% (±) at 80.0 μg/mL and 10.5% (+) at 160 μg/mL. Dose-dependent inhibition of cell growth was also observed, and the survival rate decreased to 10.1% at 320 μg/mL, and only 171 cells could be observed for chromosome aberrations. The survival rate at the highest dose of 640 μg/mL increased because some of the precipitate floated. Precipitation such as white membrane was observed at the end of exposure to the test substance at all dose levels. Because treatment in the higher dose groups was with suspensions of the test substance, there was residue of white powder of the test substance.

Short-term Treatment with S9

The incidence of structural chromosome aberrations in the test substance treatment groups was 6.5% (±) at 591 μg/mL, 4.5% at 1181 μg/mL and 5.0% (±) at 2362 μg/mL. The incidence of polyploid cells was comparable to the control at all dose levels. Strong inhibition of cell growth due to treatment with the test substance was not observed.

Precipitation such as white membrane was observed at the end of exposure to the test substance at all dose levels. Because treatment in the higher dose groups was with suspensions of the test substance, there was residue of white powder of the test substance.

Confirmatory test

The incidence of structural chromosome aberrations in the test substance treatment groups was 1.0% at 37.5 μg/mL, 3.0% at 75.0 μg/mL, 7.0% (±) at 150 μg/mL, 7.5% (±) at 300 μg/mL and 6.5% (±) at 600 μg/mL. The incidence of polyploid cells was comparable to the control at all dose levels. Strong inhibition of cell growth due to treatment with the test substance was not observed. Although the positive criterion of exceeding 10% was not met, reproducibility of the induction of structural chromosome aberrations was observed.

Precipitation such as white membrane was observed at the end of exposure to the test substance at 75.0 μg/mL or more. Because treatment in the higher dose groups was with suspensions of the test substance, there was residue of white powder of the test substance.

Conclusions:

Interpretation of results (migrated information):
positive

Slight increase in chromosomal aberrations was observed in the test with the short-term treatment (-S9 and +S9).

Executive summary:

In order to investigate the mutagenicity of 2-ethylanthraquinone, specifically the potential to induce chromosome aberrations, an in vitro chromosome aberration test was conducted using Chinese hamster lung fibroblast (CHL) cells. The method used was equivalent to the one described in OECD TG 473.

Based on the results of the cell growth inhibition test, doses that inhibit cell growth in short-term treatment without S9 were examined. For treatment with S9, no dose inhibiting cell growth could be determined and doses up to the limit concentration of 2362 μg/mL, (equivalent to 10 mM) were examined. The results showed induction of polyploid cells in treatment with 2 -ethylanthraquinone without S9, and it was judged to be positive since dose-dependence was also observed. For treatment with S9, very slight induction of structural chromosome aberrations was judged to be equivocal and a confirmatory test was conducted. In the confirmatory test, although the positive criterion of exceeding 10% was not met, reproducibility of the induction of structural chromosome aberrations was observed. The incidences of chromosome aberrations in the positive control and negative control were both within the range of this center’s historical data, indicating that this study is valid.

From these results, 2 -ethylanthraquinone was judged to be positive for induction of chromosome aberrations (numerical aberrations) in cultured mammalian cells under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The ability of the test item to produce damage to chromosomes or aneuploidy when administered to rats was determined in an in vivo mouse micronucleus test.

Under the conditions of the test performed 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.

Link to relevant study records

Reference

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

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

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

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

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

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.

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.

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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Three Ames tests and one in vitro chromosomal aberration test using Chinese hamster cells (CHL/IU) are available to assess the genotoxic potential of 2-ethylanthraquinone.

In vitro bacterial tests

In the OECD SIDS report, in vitro reverse mutation assays using tester strains TA 1535, TA 1537, TA 98 and TA 100 and WP2 uvr A are described that were performed in accordance with OECD TG 471 and under GLP. The study is rated as Klimisch 2 in the present dossier due to the fact that the full report is not available to the registrant. However, the Japanese Ministry for Foreign Affairs evaluated the corresponding study as reliable without restrictions. The in vitro studies with Salmonella typhimurium and Escherichia coli showed that the substance did not induce gene mutations in bacteria under the conditions of the studies.

Scheres (1991) tested the substance in the Salmonella/microsome plate test up to 3330 µg/plate in the absence and presence of S9-mix. Only 4 strains were tested instead of 5 as requested by OECD TG 471, adopted on July 21, 1997 (TA1535, TA1537, TA98 and TA100). The test substance did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains. These results were confirmed in a repeated, independent experiment. The test substance can be considered as not mutagenic in this test system in the absence and presence of a metabolic activation system.

Cascieri (1978) performed a Salmonella plate incorporation mutagenesis assay using five tester strains - TA98, TA100, TA1535, TA1537, and TA1538 - with and without metabolic activation by rat liver microsomes. The experimental protocol followed by Cascieri (1978) is a modification of the techniques described by Ames (1975). The test substance in this in vitro assay did not cause a significant increase in the reversion index of any of the tester strains with or without metabolic activation.

In vitro mammalian cell test

In the OECD SIDS report, an in vitro chromosomal aberration test using cultured Chinese hamster cells (CHL/IU) is described. The study is rated as Klimisch 2 in the present dossier due to the fact that the full study report was not available to the registrant. However, the Japanese Ministry of Foreign affairs evaluated the study as reliable without restriction. The chromosomal aberration test followed OECD TG 473 and was performed under GLP. 2-ethylanthraquinone was tested in the absence and presence of the metabolic activation S9-mix and induced chromosomal aberrations in cultured cells under the conditions of this study.

In vivo micronucleus test

An in vivo micronucleus assay was performed according to OECD TG 474. The study is rated Klimisch 1. In this study, carried out on Wistar rats, 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.

Justification for selection of genetic toxicity endpoint
Slight increase in chromosomal aberrations were observed in the selected in vitro study. All other in vitro studies on genotoxicity gave negative results.

Justification for classification or non-classification

The test item did not induce gene mutations in bacteria in any of three Ames tests. Although chromosomal aberrations in cultured cells were seen in an in vitro chromosomal aberration study, the test item gives a negative repsonse in an in vivo cytogenicity study. According to table R.7.7 -1.10 of ECHA Endpoint Specific Guidance Chapter 7., the negative result in an in vivo cytogenicity study indicates that the test item is not genotoxic and no further testing is required.