Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Study

Metabolic activation

Dose levels / Concentrations

Results

Guideline

In vitro bacterial gene mutation (Ames test) in S. typhimurium and E.coli

No

10-5000 µg/ plate

Negative

OECD 471, GLP

Yes (S9)

Negative

In vitro mammalian cell gene mutation (mouse lymphoma assay, TK locus)

No

5-25 µg/mL

Positive (evidence of clastogenicity rather than of gene mutation)

OECD 476, GLP

Yes (S9)

1.3-4.4 µg/mL

Positive (evidence of clastogenicity rather than of gene mutation)

In vitro chromosome aberration (CHO cells)

No

10-30 µg/mL

Positive (clastogenicity, polyploidy)

OECD 473, GLP

Yes (S9)

10-25 µg/mL

Positive (clastogenicity, polyploidy, endo-reduplication)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Peer-reviewed assessment report (attached in section 13)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
not applicable
GLP compliance:
yes
Remarks:
self-certified to US EPA regulations [40 CFR Part 160]; and OECD regulations [ENV/MC/CHEM (98) 17]
Type of assay:
bacterial reverse mutation assay
Test material information:
Composition 1
Target gene:
histidine locus (Salmonella strains)
tryptophan locus (Escherichia coli tester strain WP2uvrA)
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 mix that was obtained from Aroclor-induced rat livers.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix that was obtained from Aroclor-induced rat livers.
Test concentrations with justification for top dose:
10.0, 33.3, 100, 333, 1,000, 2,000, and 5,000 µg/plate (±S9, Salmonella strains)
33.3, 100, 333, 1,000, 3,330 and 5,000 µg/plate (±S9, Escherichia coli tester strain WP2uvrA)
Vehicle:
- Vehicle/solvent used.
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
not specified
Details on test system and conditions:
The doses tested in the mutagenicity assay were selected based on the results of a dose range finding study using tester strains TA100 and WP2uvrA and ten doses of test article ranging from 6.67 to 5,000 µg/plate one plate per dose, both in the presence and absence of S9 mix that was obtained from Aroclor-induced rat livers.
The tester strains used in the mutagenicity assay were Salmonella typhimurium test strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA. The assay was conducted in both the presence and absence of S9 mix along with concurrent vehicle and positive controls using three plates per dose. The doses tested in the mutagenicity assay with all Salmonella tester strains in both the presence and absence of S9 mix were 10.0, 33.3, 100, 333, 1,000, 2,000, and 5,000 µg/plate. The doses tested in the mutagenicity assay with tester strain WP2uvrA in both the presence and absence of S9 mix were 33.3, 100, 333, 1,000, 3,330 and 5,000 µg/plate. The results of the initial mutagenicity assay were confirmed in an independent experiment.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
not specified
Vehicle controls valid:
not specified
Positive controls valid:
yes

The test article did not cause an increase in the mean number of revertants per plate with any of the tester strains either in the presence or absence of microsomal enzymes (S9). The positive control substances gave the expected increase in mutant frequencies.

Conclusions:
As information was provided via a peer-reviewed assessment report, it can be considered as sufficiently reliable to assess the potential of ethoxyquin for the induction of gene mutations in bacteria. Under the conditions of this study, ethoxyquin proved negative in a reverse mutation assay (Ames test) in different tester strains of Salmonella typhimurium and Escherichia coli.
Executive summary:

In a reverse gene mutation assay in bacteria (OECD 471), Salmonella typhimuriumtest strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2uvrA  were exposed to ethoxyquin at concentrations of 10.0, 33.3, 100, 333, 1,000, 2,000, and 5,000 µg/plate resp. 33.3, 100, 333, 1,000, 3,330 and 5,000 µg/plate in the presence and absence of mammalian metabolic activation (Aroclor-induced rat liver S9)

Ethoxyquin was tested up to the limit concentration 5000 µg/plate. The positive controls induced the appropriate responses in the corresponding strains.There was no evidence or a concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Peer-reviewed assessment report (attached in section 13)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
not applicable
GLP compliance:
yes
Remarks:
self-certified to US EPA regulations [40 CFR Part 160] and OECD regulations [ENV/MC/CHEM (98) 17]
Type of assay:
other: chromosome aberration test
Test material information:
Composition 1
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
exogenous metabolic activation system
Test concentrations with justification for top dose:
The highest concentration tested in the assay was 1,000 µg/mL, which was above the solubility limit of ethoxyquin after dosing into culture medium.
Concentrations of 1.25, 2.50, 5.00, 10.0; 15.0, 20.0, 30.0, 50.0, 75.0 and 100 µg/mL were tested without metabolic activation; and 5.00, 10.0, 15.0, 20.0, 25.0, 30.0, 40.0 and 50.0 µg/mL were tested with metabolic activation. Cultures treated with concentrations of 10.0, 15.0, 20.0 and 30.0 µg/mL without metabolic activation; and 10.0, 15.0, 10.0 and 25.0 µg/mL with metabolic activation were analysed for chromosomal aberrations.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Negative controls:
yes
Remarks:
McCoy's 5a
Solvent controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3h + S9, 20h -S9
- Fixation time (start of exposure up to fixation or harvest of cells): Cultures were harvested ~20 hours from the initiation of treatment.

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 2x100 per treatment

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Vehicle controls valid:
not specified
Negative controls valid:
not specified
Positive controls valid:
yes
Additional information on results:
Findings:
In the assay without metabolic activation, reductions of 0 %, 0 %, 34 %, 46 %, 54 %, 96 %, 100 % and 100 % were observed in the mitotic indices of the cultures treated with 5.00, 10.0, 15.0, 20.0, 30.0, 50.0, 75.0 and 100 µg/mL, respectively, as compared with the vehicle control cultures. Chromosomal aberrations were analysed from the cultures treated with 10.0, 15.0, 20.0 and 30.0 µg/mL. A significant increase in cells with chromosomal aberrations was observed in the cultures treated with 20.0 and 30.0 µg/mL. A significant increase in polyploidy was observed in the cultures treated with 30.0 µg/mL. No significant increase in endoreduplication was observed in the cultures analysed.
In the assay with metabolic activation, reductions of 15 %, 56 %, 53 %, 65 %, 79 %, 78 % and 74 % were observed in the mitotic indices of the cultures treated with 10.0, 15.0, 20.0, 25.0, 30.0, 40.0 and 50.0 µg/mL, respectively, as compared with the vehicle control cultures. Chromosomal aberrations were analysed from the cultures treated with 10.0, 15.0, 20.0 and 25.0 µg/mL. A significant increase in cells with chromosomal aberrations was observed in the cultures treated with 15.0, 20.0 and 25.0 µg/mL. A significant increase in polyploidy was observed in the cultures treated with 20.0 µg/mL. A significant increase in endoreduplication was observed in the cultures treated with 10.0, 15.0 and 20.0 µg/mL.

Table: Chromosome aberrations in CHO cells, without S9: ~20-hour treatment, ~20-hour harvest

Dose

Group

AB cells scored

MIR %a

PP & ER cells scored

PP cells

ER cells

Judgement (+/-)b

# & % cells showing structural chromosomal aberrations

Judgement (+/-)d

Gaps

Simple breaks

CHTE

CHRE

MAB

Totalsc

g-

g+

Negative Control: McCoy’s 5a

n/a

A

100

 

100

4

0

 

1

 

 

 

 

0

1

 

B

100

 

100

5

0

 

1

 

 

1

 

1

2

 

Total

200

 

200

 

 

 

2

 

 

1

 

1

3

 

Ave

%

--

 

4.5

0.0

 

1.0

 

 

0.5

 

0.5

1.5

 

Vehicle Control: DMSO

10 µl/ml

A

100

 

100

5

0

 

 

 

 

 

 

0

0

 

B

100

 

100

3

0

 

 

 

 

 

 

0

0

 

Total

200

 

200

 

 

 

 

 

 

 

 

0

0

 

Ave

%

0

 

4.0

0.0

 

 

 

 

 

 

0.0

0.0

 

Positive Control: MMC

0.2 µg/ml

A

50

 

100

6

0

 

3

10

19

1

5

29

31

 

B

50

 

100

7

1

 

5

10

14

1

11

30

34

 

Total

100

 

200

 

 

 

8

20

33

2

16

59

65

 

Ave

%

--

 

6.5

0.5

-

8.0

20.0

33.0

2.0

16.0

59.0

65.0

+

Test article: Ethoxyquin

10 µg/ml

A

100

 

100

4

0

 

1

 

 

1

 

1

2

 

B

100

 

100

5

0

 

 

1

 

 

 

1

1

 

Total

200

 

200

 

 

 

1

1

 

1

 

2

3

 

Ave

%

0

 

4.5

0.0

-

0.5

0.5

 

0.5

 

1.0

1.5

-

15 µg/ml

A

100

 

100

4

0

 

1

1

 

 

 

1

2

 

B

100

 

100

4

0

 

2

 

 

 

 

0

2

 

Total

200

 

200

 

 

 

3

1

 

 

 

1

4

 

Ave

%

37

 

4.0

0.0

-

1.5

0.5

 

 

 

0.5

2.0

-

20 µg/ml

A

100

 

100

8

0

 

4

2

 

1

 

3

7

 

B

100

 

100

7

0

 

1

4

2

 

 

5

5

 

Total

200

 

200

 

 

 

5

6

2

1

 

8

12

 

Ave

%

46

 

7.5

0.0

-

2.5

3.0

1.0

0.5

 

4.0

6.0

+

30 µg/ml

A

100

 

100

12

0

 

11

8

3

4

 

13

19

 

B

78

 

100

13

0

 

4

12

3

 

1

13

17

 

Total

178

 

200

 

 

 

15

20

6

4

1

26

36

 

Ave

%

54

 

12.5

0.0

+

78.4

11.2

3.4

2.2

0.5

14.6

20.2

+

Notes:

ab=aberrations;

MIR=mitotic index reduction;

pp=polyploidy;

er=endoreduplication;

chte=chromatid exchange;

chre=chromosome exchange; mab=multiple aberrations, greater than 4 aberrations

a% Mitotic index reduction as compared to the vehicle control.

bSignificantly greater in % polyploidy than the vehicle control, p < 0.01.

cg- = # or % of cells with chromosome aberrations; g+ = # or % of cells with chromosome aberrations + # or % of cells with gaps.

dSignificantly greater in g- than the vehicle control, p < 0.01.

 

Table: Chromosome aberrations in CHO cells, with S9: ~3-hour treatment, ~20-hour harvest

Dose

Group

AB cells scored

MIR %a

PP & ER cells scored

PP cells

ER cells

Judgement (+/-)b,c

# & % cells showing structural chromosomal aberrations

Judgement (+/-)e

Gaps

Simple breaks

CHTE

CHRE

MAB

Totalsc

g-

g+

Negative Control: McCoy’s 5a

n/a

A

100

 

100

6

0

 

 

 

 

1

 

1

1

 

B

100

 

100

7

0

 

1

 

 

 

 

0

1

 

Total

200

 

200

 

 

 

1

 

 

1

 

1

2

 

Ave

%

-

 

6.5

0.0

 

0.5

 

 

0.5

 

0.5

1.0

 

Vehicle Control: DMSO

10 µl/ml

A

100

 

100

2

0

 

 

 

1

1

 

2

2

 

B

100

 

100

5

0

 

1

1

1

 

 

2

3

 

Total

200

 

200

 

 

 

1

1

2

1

 

4

5

 

Ave

%

0

 

3.5

0.0

 

0.5

0.5

1.0

0.5

 

2.0

2.5

 

Positive Control: CP

7.5 µg/ml

A

50

 

100

8

0

 

4

10

14

 

5

25

28

 

B

50

 

100

10

0

 

3

11

21

1

1

29

30

 

Total

100

 

200

 

 

 

7

21

35

1

6

54

58

 

Ave

%

-

 

9.0

0.0

 

7.0

21.0

35.0

1.0

6.0

54.0

58.0

+

Test article: Ethoxyquin

10 µg/ml

A

100

 

100

3

5

 

1

 

 

 

 

0

1

 

B

100

 

100

6

6

 

2

1

1

 

 

2

4

 

Total

200

 

200

 

 

 

3

1

1

 

 

2

5

 

Ave

%

15

 

4.5

5.5

+c

1.5

0.5

0.5

 

 

1.0

2.5

-

15 µg/ml

A

50

 

100

11

7

 

4

3

6

 

12

21

24

 

B

50

 

100

6

4

 

4

2

7

2

13

22

23

 

Total

100

 

200

 

 

 

8

5

13

2

25

43

47

 

Ave

%

56

 

8.5

5.5

+c

8.0

5.0

13.0

2.0

25.0

43.0

47.0

+

20 µg/ml

A

50

 

100

12

9

 

5

4

10

 

23

35

37

 

B

50

 

100

10

5

 

2

7

4

 

23

32

33

 

Total

100

 

200

 

 

 

7

11

14

 

46

67

70

 

Ave

%

53

 

11.0

7.0

+b,c

7.0

11.0

14.0

 

46.0

67.0

70.0

+

25 µg/ml

A

50

 

100

5

0

 

1

3

6

1

30

38

38

 

B

50

 

100

5

0

 

10

4

9

 

32

42

47

 

Total

100

 

200

 

 

 

11

7

15

1

62

80

85

 

Ave

%

65

 

5.0

0.0

-

11.0

7.0

15.0

1.0

62.0

80.0

85.0

+

Notes:

ab=aberrations;

MIR=mitotic index reduction;

pp=polyploidy;

er=endoreduplication;

chte=chromatid exchange;

chre=chromosome exchange;

mab=multiple aberrations, > 4 aberrations

a% Mitotic index reduction as compared to the vehicle control.

bSignificantly greater in % polyploidy than the vehicle control, p ≤ 0.01.

cSignificantly greater in % endoreduplication than the vehicle control, p ≤ 0.01.

dg- = # or % of cells with chromosome aberrations; g+ = # or % of cells with chromosome aberrations + # or % of cells with gaps.

eSignificantly greater in g- than the vehicle control, p ≤ 0.01.

Conclusions:
As information was provided via a peer-reviewed assessment report, it can be considered as sufficiently reliable to assess the potential of ethoxyquin for the induction of chromosome aberrations in mammalian cells.
Under nonactivation conditions, the sensitivity of the cell cultures for induction of chromosomal aberrations was shown by the increased frequency of aberrations in the cells exposed to mitomycin C, the positive control agent. The test article, ethoxyquin, has to be considered positive for inducing chromosomal aberrations and polyploidy without metabolic activation.
The successful activation by the metabolic system was illustrated by the increased incidence of cells with chromosomal aberrations in the cultures induced with cyclophosphamide, the positive control agent. The test article, ethoxyquin, induced chromosomal aberrations, polyploidy, and endoreduplication with metabolic activation.
Ethoxyquin was considered to be positive for inducing structural and, as suggested by polyploidy and endoreduplication, possibly also numerical chromosomal aberrations in CHO cells with and without metabolic activation.
Executive summary:

In a mammalian cell cytogenetics assay (Chromosome aberration)(OECD 473), CHO cell cultures were exposed to Ethoxyquin in DMSO at concentrations of 0 - 100 µg/ml with and without metabolic activation.

Ethoxyquin was tested up to cytotoxic concentrations.  Positive controls induced the appropriate response.  There was evidence of Chromosome aberrations induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Peer-reviewed assessment report (attached in section 13)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
EC Directive 2000/32/EC B.17
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPP 84-2
Version / remarks:
EPA Assessment guidelines subdivision F, 84-2
Deviations:
not applicable
Qualifier:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
The study also meets the requirements of Japan.
Deviations:
not applicable
GLP compliance:
yes
Type of assay:
other: in vitro mammalian forward mutation assay (mouse lymphoma assay)
Test material information:
Composition 1
Target gene:
thymidine kinase locus: from tk+tk- to tk-tk-.
Species / strain:
mouse lymphoma L5178Y cells
Details on mammalian cell lines (if applicable):
CELLS USED: mouse lymphoma L5178Y cell line, clone 3.7.2.C
Metabolic activation:
with and without
Metabolic activation system:
post-mitochondrial supernatant fraction obtained from Aroclor 1254-induced livers of adult, male rats and the co-factors required for mixed-function oxidase activity (S9 mix)
Test concentrations with justification for top dose:
5 - 25 µg/mL in the absence of S9 mix
1.3 - 4.4 µg/mL in the presence of S9 mix
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
Solvent controls:
yes
Remarks:
DSMO
Positive controls:
yes
Positive control substance:
not specified
Details on test system and conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity:
yes
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
Preliminary cytotoxicity tests showed that ethoxyquin exhibited a moderate to high level of toxicity. When exposed to the cells for a 4 h period, a concentration of 15 µg/mL reduced the relative suspension growth (RSG, a measure of toxicity) to 49.1 % in the absence of S9 mix. When the exposure period was increased to 24h, ethoxyquin reduced RSG to 49 % at 5 µg/mL. In the presence of S9 mix, ethoxyquin was more toxic, reducing RSG to 55.5 % at 1.5 µg/mL and to 0.5 % at 5 µg/mL. In all instances, higher concentrations than those mentioned were lethal to cells.

All four mutation experiments gave evidence of mutagenic activity. All experiments contained at least one concentration that tested significant for increase in log mutant fraction at P < 0.05. In addition, all four experiments had a highly significant (P < 0.001) linear trend in mutant fraction with dose level of ethoxyquin. the effective dose levels ranged from 16 to 25 µg/mL in the absence of S9 mix, and from 1.4 to 4.4 µg/mL in the presence of S9 mix. the largest increase obtained in any experiment was 526 mutants per million above the background level in the second experiment in the presence of S9 mix. This occurred at a concentration of 3.9 µg ethoxyquin/mL, which gave a relative total growth (measure of overall toxicity) of 12 % of the control value.

Conclusions:
As information was provided via a peer-reviewed assessment report, it can be considered as sufficiently reliable to assess the potential of ethoxyquin for the induction of gene mutations in mammalian cells. It is concluded that ethoxyquin was mutagenic in mouse lymphoma L5178Y cells, in the absence and presence of S9 mix. Since colony size (small/large) ratios were always > 1 and greater than that of the concurrent vehicle control group value, the activity of ethoxyquin appears more closely associated with large-scale chromosomal damage, than with small scale damage or point mutations.
Executive summary:

In a mammalian cell gene mutation assay (mouse lymphoma assay, thymidine kinase)(OECD 476), mouse lymphoma L5178Y cell line, clone 3.7.2.C,cells cultured in vitro were exposed to ethoxyquin in DMSO at concentrations of 5 and 25 µg/mL in the absence of S9 mix, and between 1.3 and 4.4 µg/mL in the presence of S9 mix (Aroclor 1254-induced rat liver S9).

Ethoxyquin was tested up to cytotoxic concentrations.  The positive controls did induce the appropriate response. There was evidence and a concentration related positive responseof induced mutant colonies over background.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Study

Metabolic activation

Dose levels / Concentrations

Results

Guideline

In vivomouse bone marrow micro-nucleus assay

Yes(in vivotest)

0-1500 mg/kg bw (single dose)

Negative

OECD 474, GLP

UDSex vivo(rat hepatocytes)

Yes(in vivotest)

2x 0-750 mg/kg bw (two doses with 14-hr interval)

Negative

OECD 486, GLP

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Peer-reviewed assessment report (attached in section 13)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
not applicable
GLP compliance:
yes
Remarks:
self-certified to US EPA regulations [40 CFR Part 160]; and OECD regulations [ENV/MC/CHEM (98) 17]
Type of assay:
other: mammalian cell cytogenicity test
Test material information:
Composition 1
Species:
mouse
Strain:
other: Crl:CD-1®(ICR)BR mouse
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: 37.5, 75, or 150 mg/ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article was formulated in corn oil.
Duration of treatment / exposure:
Single gavage, up to 48h post-exposure
Frequency of treatment:
once
Post exposure period:
24h or 48 h
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
375 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 males (all doses, 24h, or 0 and 1500 mg/kg, 48h)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 80 mg(kg
Tissues and cell types examined:
bone marrow, polychromatic erythrocytes (PCEs) / normochromatic erythrocytes (NCEs)
Details of tissue and slide preparation:
Bone marrow was extracted.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls valid:
not specified
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Maximum tolerated dose was estimated to be 1,500 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Ethoxyquin did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (375, 750 and 1,500 mg/kg bw)
- Ratio of PCE/NCE (for Micronucleus assay): Ethoxyquin was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose of the test article.

Findings:

The test article, ethoxyquin, induced mortality in three 1,500 mg/kg bw animals and signs of clinical toxicity in the treated animals at 750 and 1,500 mg/kg bw. The clinical signs of toxicity included hypoactivity, squinted eyes, irregular respiration, ataxia, and/or a temporary trace (standing on hind limbs with head in upright position and little movement). Ethoxyquin did not induce statistically significant increases in micronucleated PCEs at any test article dose examined (375, 750 and 1,500 mg/kg bw). Additionally, ethoxyquin was not cytotoxic to the bone marrow (i.e., no statistically significant decreases in the PCE:NCE ratios) at any dose of the test article.

The positive control substance, cyclophosphamide, gave the expected increase in the percentage of micronucleated polychromatic erythrocytes.

 

Table Summary of results in the micronucleus assay

Treatment

Dose

Harvest time

% Micronucleated PCEs

Mean of 2,000 per animal (males)

Ratio PCE:NCE Mean (males)

Vehicle control

Corn oil 10 mL/kg

24 hr

0.04 ± 0.01

0.69 ± 0.02

48 hr

0.02 ± 0.01

0.69 ± 0.04

Positive control

Cyclophosphamide 80 mg/kg bw

24 hr

1.60 ± 0.08*

0.60 ± 0.04

Ethoxyquin

375 mg/kg bw

24 hr

0.05 ± 0.02

0.72 ± 0.10

750 mg/kg bw

24 hr

0.03 ± 0.02

0.58 ± 0.07

1,500 mg/kg bw

24 hr

0.03 ± 0.02

0.56 ± 0.05

48 hr

0.02 ± 0.01

0.54 ± 0.08

Notes: * Significant greater than the corresponding vehicle control, p < 0.01

Conclusions:
As information was provided via a peer-reviewed assessment report, it can be considered as sufficiently reliable to assess the potential of ethoxyquin for the induction of micronuclei in mammalian cells, i.e. in the bone marrow in mice in vivo.
Ethoxyquin proved negative in the mouse bone marrow micronucleus assay under the conditions of this study up to the highest dose level of 1500 mg/kg bw that was already toxic to the animals. So it can be concluded that the positive result of the available chromosome aberration test in vitro has no relevance in the living organism, and can hence be neglected.
Executive summary:

In the Crl:CD-1®(ICR)BR mouse bone marrow micronucleus assay (OECD 474), six males per dose were treated orally by gavage with ethoxyquin at doses of 0, 375, 750 or 1500 mg/kg bw.  Bone marrow cells were harvested at 24h or 48h post-treatment. The vehicle was corn oil.

There were signs of toxicity (hypoactivity, squinted eyes, irregular respiration, ataxia, and/or a temporary trace (standing on hind limbs with head in upright position and little movement)) during the study.  Ethoxyquin was tested at an adequate dose based on toxicity. The positive control induced the appropriate response.  There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable.  This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Peer-reviewed assessment report (attached in section 13)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: EPA OPPTS Guideline 870.5550
Deviations:
not applicable
GLP compliance:
yes
Remarks:
self-certified by the performing laboratory to US EPA regulations [40 CFR Part 160]; and OECD regulations [ENV/MC/CHEM (98) 17] but no formal certificate, QAU statement included
Type of assay:
unscheduled DNA synthesis
Test material information:
Composition 1
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
Duration of treatment / exposure:
Administration of ethoxyquin was done on two separate occasions. The second dose was given 14 hours after the first dose and 2 hours before sacrifice and liver perfusion.
Frequency of treatment:
Administration of ethoxyquin was done on two separate occasions. The second dose was given 14 hours after the first dose and 2 hours before sacrifice and liver perfusion.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
vehicle control, corn oil
Dose / conc.:
225 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
No. of animals per sex per dose:
The ethoxyquin-treated and vehicle control groups comprised five animals each and three rats received the positive control substance. However, only four animals from the treatment and vehicle control groups and two animals from the positive control group were assessed.
Control animals:
yes, concurrent vehicle
Positive control(s):
2-acetylaminofluorene (2-AAF)
- Route of administration: oral
- Doses / concentrations: 75 mg/kg bw on one occasion, 16 hours before perfusion
Tissues and cell types examined:
Hepatocytes, isolated by enzymatic dissociation
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A preliminary toxicity test had previously confirmed that a two subsequent doses of 750 mg/kg bw each with a gap of 14 hours between were sufficiently tolerated by male rats although clear signs of toxicity occurred.

DETAILS OF SLIDE PREPARATION: The isolated hepatocytes were allowed to attach to glass coverslips and were cultured in vitro with (methyl-3H) thymidine at 10 iCi/mL for four hours to 'radiolabel' DNA undergoing repair replication. The hepatocytes were 'chased' for approximately 20 hours with unlabelled thymidine.

METHOD OF ANALYSIS: They were fixed on slides and processed for autoradiography. Two cultures per animal were assessed. 50 morphologically intact cells that were not in the S-phase per slide were analysed. Thus, grain count enumeration was based on 100 cells per animal.

OTHER: DNA repair was evaluated by comparing the grain count of hepatocyte nuclei with the accompanying cytoplasmic grain count. The gross nuclear grain count and the net nuclear grain count from treated cultures (ethoxyquin and 2-AAF) were compared by means of Student's t test with the vehicle control values.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls valid:
not specified
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
A preliminary toxicity test had previously confirmed that a two subsequent doses of 750 mg/kg bw each with a gap of 14 hours between were sufficiently tolerated by male rats although clear signs of toxicity occurred. Accordingly, this dose level was therefore selected as the maximum dose in this test system. Thus, the single high dose was about 43 % of the LD50 while the total amount applied was approaching this level of about 1726 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
At both dose levels, signs of toxicity such as reduced activity, irregular respiration, unsteady gait or reduced body tone were observed but all animals survived until scheduled termination at 2 hours after the second dose.
Ethoxyquin did not cause any statistically significant increases in the net nuclear grain count at any dose level compared to vehicle control values.
At both dose levels, however, the test compound did cause a significant increase in the gross nuclear grain count when compared to vehicle control values. As an accompanying increase (although not statistically significant) in the cytoplasmic grain count was also observed, there was no resulting increase in the net nuclear grain count and, therefore, no indication of unscheduled DNA repair was obtained.
The positive control substance gave the expected increase in the mean net nuclear grain count.

Table Results of an ex vivo unscheduled DNA synthesis (UDS) test using rat hepatocytes

Group

Dose level (mg/kg/bw)

Animal number

Mean (gross) nuclear grain count

Mean cytoplasmic grain count

Mean net nuclear grain count

% cells in repair

Mean

SD

Mean

SD

Mean

SD

Vehicle control

-

201

8.13

4.26

12.87

5.08

-4.74

4.01

2

202

6.60

3.21

10.35

4.09

-3.75

4.01

3

204

6.96

2.96

10.97

4.33

-4.01

4.07

0

205

7.81

4.56

15.26

6.11

-7.45

5.47

0

Ethoxyquin

225

211

10.67**

5.40

15.66

6.52

-4.99

5.01

2

212

11.50**

6.86

17.96

8.34

-6.46

6.75

3

213

10.10**

4.12

16.90

7.46

-6.80

6.92

1

215

10.15**

4.73

17.87

8.15

-7.72

7.03

2

Ethoxyquin

750

221

11.92*

7.76

20.31

10.30

-8.39

7.97

1

222

12.19*

5.74

19.47

8.64

-7.28

6.82

2

223

8.88*

5.12

15.89

7.46

-7.01

6.47

0

225

10.88*

5.17

18.97

8.85

-8.09

6.98

2

2-Acetyl-aminoflu-rene (2-AAF)

75

231

22.37**

11.92

14.25

6.56

8.12**

9.15

62

233

28.43**

14.00

21.48

10.56

6.95**

10.13

61

Statistical significance: ** p < 0.001* P < 0.01

Conclusions:
As information was provided via a peer-reviewed assessment report, it can be considered as sufficiently reliable to assess the potential of ethoxyquin for the induction unscheduled DNA synthesis (UDS) in hepatocytes of treated rats.
It is concluded that ethoxyquin did not show convincing evidence of unscheduled DNA synthesis (UDS) in hepatocytes of male Sprague-Dawley (CD) rats following twofold oral administration of up to 750 mg/kg bw in this ex vivo test system.
As unscheduled DNA synthesis is indicative for DNA damage and repair, and mutations are the results of failed repair after a genotoxic insult, it can be concluded that the positive result of the available forward mutation test in mammalian cells in vitro has no relevance in the living organism, and can hence be neglected.
Executive summary:

In an unscheduled DNA synthesis assay (OECD 486), male Sprague-Dawley rats (5 / dose) were exposed to 0, 225, or 750 mg/kg ethoxyquin in corn oil. The second dose was given 14 hours after the first dose and 2 hours before sacrifice and liver perfusion to obtain primary rat hepatocytes, which were then cultured in vitro with (methyl-3H) thymidine at 10 µCi/mL for four hours to 'radiolabel' DNA undergoing repair replication. The hepatocytes were 'chased' for approximately 20 hours with unlabelled thymidine. 50 morphologically intact cells that were not in the S-phase per slide were analysed. Thus, grain count enumeration was based on 100 cells per animal.

Ethoxyquin was tested up to toxic concentrations. At both dose levels, signs of toxicity such as reduced activity, irregular respiration, unsteady gait or reduced body tone were observed but all animals survived until scheduled termination at 2 hours after the second dose.

The positive controls induced the appropriate response.  There was no evidence that unscheduled DNA synthesis, as determined by radioactive tracer procedures (nuclear silver grain counts) was induced.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 486 for other genotoxic mutagenicity data.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Ethoxyquin was tested for mutagenicity in different systems in vitro as well as in vivo. The in vitro studies revealed contradictory results since the compound proved negative in the Ames test for point mutations by base substitutions or frameshifts in the various Salmonella typhimurium strains and Escherichia coli but induced structural chromosomal aberrations in CHO cells with and without metabolic activation. In addition, polyploidy and endoreduplication were observed in CHO cell cultures. Furthermore, at doses that inhibit cell growth, ethoxyquin induced forward mutations in the mouse lymphoma cell mutation assay in the absence and presence of S9 mix, confirming a genotoxic potential in vitro. By assessing the mutant colonies, this finding may be, again, rather attributed to chromosomal damage than to point mutations.

In spite of the apparent clastogenicity and the increase in numerical aberrations in vitro, ethoxyquin proved clearly negative in a micronucleus assay in mouse bone marrow up to the limit dose of 1500 mg/kg bw that was already toxic to the animals. According to that in vivo test, ethoxyquin did not produce micronuclei in the immature erythrocytes. However, bone marrow cytotoxicity was not seen up to the highest dose and since the bone marrow had not been examined for the presence of ethoxyquin or its metabolites in the toxicokinetic studies, it may be doubted whether that tissue was actually reached by the test substance. However, since, in a similar situation (i.e.,positive in vitro findings in a test for clastogenicity but negative outcome of the in vivo test), the metabolite methylethoxyquin induced bone marrow toxicity at the same dose as evidenced by significantly decreased PCE:NCE ratios, one may assume also the parent compound to have reached the bone marrow. Thus, the negative micronucleus test with ethoxyquin is considered sufficient to outweigh the positive findings in vitro. In addition, the test compound proved negative in an in vivo DNA repair (UDS) assay following twofold oral administration to rats. Even though the latter method is not suitable to evaluate clastogenicity, it gives a clear indiction whether or not direct interaction of a substance with cellular DNA may be expected.

Based on these two negative in vivo assays and taking into consideration that there was no evidence of cancerogenicity in long-term studies, ethoxyquin is considered non-mutagenic in vivo.

There is no indication given that these results are not relevant for humans, so there is no reason to believe that the negative outcome of the in vivo studies cannot be transferred to humans, as well as there was no trigger for additional studies identified.

Additional information

Justification for classification or non-classification

Based on these two negative in vivo assays and taking into consideration that there was no evidence of cancerogenicity in long-term studies, ethoxyquin is considered non-mutagenicin vivo.