Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 7 to June 27, 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: guideline test with GLP, but only 1000 instead of 2000 erythrocytes were evaluated

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
GLP compliance:
yes
Remarks:
Quality Assurance certificate as pre-GLP study
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-ethylhexyl salicylate
EC Number:
204-263-4
EC Name:
2-ethylhexyl salicylate
Cas Number:
118-60-5
Molecular formula:
C15H22O3
IUPAC Name:
2-ethylhexyl 2-hydroxybenzoate
Test material form:
other: liquid
Details on test material:
chemical name: Benzoic acid, 2-hydroxy-, 2-ethylhexyl ester

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals and Animal Management
Adult male and female NMRI mice, 25-30 g in weight and 6-10 weeks old were obtained from SAVO GmbH, D-7964 Kisslegg F.R.G.
The animals were acclimatized for at least 5 days before use in the test. They were examined for signs of disease. Animals suspected of being diseased were culled from the study and by others. The animals were separated according to sex, marked for identification, and allocated by randomization to cages and groups.
The animals were group housed up to five per cage in MAKROLON cages of size II with ALTROMIN woodshaving. The air-conditioned surrounding had a temperature of 22 ± 2 °C and a relative humidity of ca. 55 %.
Artificial light was provided in a 12/12 h light/dark cycle. The animals were given standard laboratory diet and tap-water ad libitum. The cages and beddings were changed with clean ones twice a week.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
Three groups, each comprising 5 males and 5 females, were treated with the test compound dissolved in arachis oil (5 animals per sex and term). A dose volume of 10 ml/kg body weight was given to the mice by oral intubation using a stainless steel ravage tube.
Duration of treatment / exposure:
72 hours
Frequency of treatment:
once
Post exposure period:
24, 48, 72 hours after dosing
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
50 mg/kg body weight 9,10-Dimethyl-X,2-benzanthracene (lot 116C-0271, Sigma chemical company, St. Louis, U.S.A., dissolved in olive oil.)

Examinations

Tissues and cell types examined:
The animals were dosed once at 0 h. Sampling was performed at 24 h, 48 h, and 72 h. The vehicle control group comprised 5 males and 5 females and was treated with 10 ml of arachis oil per kg body weight. Preparations were made at 24 hours. One positive control group was treated with 9,10-dimethyl-l1,2-benzanthracene (DMBA) per os. DMBA was dissolved in olive oil, A dose volume of 10 ml/kg was used in this group. Preparations from the DMBA group were made at 48 h.
Details of tissue and slide preparation:
Preparation of Bone Marrow Smears
The animals were killed by cervical dislocation. Bone marrow was removed from both femora by rinsing: with fetal calf serum. Bone marrow cells were centrifuged at 150 s for 10 min. and the supernatant was discarded. From the pellet, smears were made on slides and air dried, according to hematological routine. Two slides were made per animal.
The preparations were stained by the May-Gruenwald-Giemsa method according to Schmid (1973):
- Stain for 3 minutes in undiluted May-Gruenwald solution.
- Stain for 2 minutes in May-Gruenwaldt diluted with distilled water 1:1
- Rinse briefly in distilled water.
- Stain for 10 minutes in Giemsa, diluted with distilled water 1:6 -Rinse thoroughly in distilled water.
- Dry in air, clean the back side of the slides with methanol
-Clear in Xylene for 5 minutes, and mount in Eukitt, Analysis
The slides were coded and observed blindly under a microscope with a lOOx oil immersion objective lens at 1250 fold magnification. At least 1000 polychromatic erythrocytes per animal were scored for the incidence of micronuclei. The number of micronucleated normo-chromatic erythrocytes was also recorded. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.
Evaluation criteria:
no data
Statistics:
Statistical significance was determined according to the methods of Kasienbaum and Bowman (1970).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
After dosing, the animals of all groups treated with the test substance showed reduced mobility. In the animals of all groups the ratio of polychromatic to normochromatic erythrocytes was normal,
In none of the different treatment times studied did HR 89/131494 produce any increase in the frequency of micronucleated polychromatic erythrocytes. Both negative and positive control frequencies of micronucleated polychromatic erythrocytes agreed with those previously- established in our laboratory and with previous reports. The incidence of micronucleated normochromatic erythrocytes over all treatment was within the expected level.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It is concluded that test substance did not induce chromosomal damage or damage to the mitotic apparatus in bone marrow cells of mice.
Executive summary:

The study was performed to investigate the mutagenic effect of the compound by means of the micronucleus test in the bone marrow cells of NMRI mice.

Male and female mice were treated with one oral administration of the test substance in arachis oil at a dose level of 2000 mg/ kg body. Bone marrow mears were prepared at 24, 48, and 72 hours after treatment. Each group comprised 5 males and 5 females, as well as control groups. At least 1000 polychromatic erythrocytes per animal were scored for the incidence of micronuclei. The number of micronucleated normochromatic erythrocytes was also recorded. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.Under the test conditions described, the test compound failed to induce an increase in the incidence of micronucleated polychromatic erythrocytes compared with the negative controls at any time tested. The positive control substance, 9,10-dimethyl-1,2-benzanthracene, however, produced a huge increase in the frequency of micronucleated polychromatic erythrocytes.

Based on the available data, test article cannot cause chromosome damage in the vivo micronucleus test in mice.