Ethoxyquin

Genetic toxicity: in vivo

Administrative data

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)

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

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

Test material information

Constituent 1

Reference substance name:

Ethoxyquin

EC Number:

202-075-7

EC Name:

Ethoxyquin

Cas Number:

91-53-2

Molecular formula:

C14H19NO

IUPAC Name:

ethoxyquin

Details on test material:

Chemical name
IUPAC: 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline
CAS: 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline

Test animals

Species:

mouse

Strain:

other: Crl:CD-1®(ICR)BR mouse

Sex:

male/female

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

375 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

750 mg/kg bw/day (nominal)

Doses / concentrations 4

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

Examinations

Tissues and cell types examined:

bone marrow, polychromatic erythrocytes (PCEs) / normochromatic erythrocytes (NCEs)

Details of tissue and slide preparation:

Bone marrow was extracted.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not specified

Negative controls validity:

not applicable

Positive controls validity:

valid

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.