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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Based on the available data, test article was found negative inducing gene mutagenicity under the experimental conditions.
Link to relevant study records
Reference
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
Qualifier:
equivalent or similar to
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
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals 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.
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
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.)
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).
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.
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.

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

Additional information

Additional information from genetic toxicity in vivo:

In vivo

There is one in vivo study performed to investigate the mutagenicity by using the micronucleus test. In this study, 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 were prepared at 24, 48 and 72 hours after treatment. Each group comprised 5 males and 5 females, as well as control groups. 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. Based on the available data, test article cannot cause the chromosome damage in the vivo micronucleus test in mice.

In vitro

The conclusion made from the in vivo test was supported by four in vitro studies (Study No. 31717 MMO, Project No.: AM15388N, Study Number: TA544. 337006, Study Number: 1485900), all conducted in the presence and absence of metabolic activation. The first two of the four studies are Ames tests that studied with five mutant strains of Salmonella typhimurium (TA98, TA100, TA102, TA 1535 and TA 1537 in the key study and TA1535, TA1537, TA1538, TA98, and TA100 in the supportive study) with and without liver homogenate (S9).

Test article was tested in concentrations of up to 5000 µg/plate in the key study and at 0.1 to 75 µl per plate in the presence of S9-mix, and of 3 to 75 µl per plate in the absence of S 9-mix in the supporting study respectively. In the key study strong emulsion was observed in the Petri plates when scoring the revertants mainly at dose-levels ≥ 1250 µg/plate. In the supporting study test article was not bacteriotoxic at the concentrations tested in the absence of S9-mix.In the presence of S9-mix a bacteriotoxic effect to strain TA1537 was observed at 3 µl per plate, and to TA1535, TA1538 and TA100 at 10 µl per plate. Precipitation of the test compound on the plates was observed with all concentrations.

The test compound did not increase the spontaneous mutation frequency in any of the five tester strains in the key and in the supporting study and at any dose tested. Similarly, the estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level, revealed no significant difference at any one of the test points.

In conclusion, these results indicated that test article was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98, TA100 and TA102 in the absence and presence of a metabolic system.

The third in vitro study was performed to evaluate the clastogenic potential of a test article based upon its ability to induce chromosome aberrations in Chinese hamster ovary (CHO) cells according to OECD guideline 473.The test article was applied up to 5000 µg/ml. In the initial and main assay, the percentage of cells with structural aberrations in the test article-treated groups was not significantly increased above that of the solvent control. Thus the test item did not cause clastogenic effects in CHO cells.

The fourth in vitro study was performed to investigate the potential of test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD guideline 476.The test article was applied up to 2600 µg/ml in the pre-test and up to 320 µg/ml in the two main tests. No substantial and reproducible dose dependent increase of the mutation frequency was observed in either of the main experiments. Thus, the test item did not induce gene mutation effects at the HPRT locus in V79 cells.


Justification for selection of genetic toxicity endpoint
The results in in vivo test should be given more weight than in vitro test.

Justification for classification or non-classification

2-ethylhexyl salicylate does not have to be classified for mutagenicity according to the criteria laid down in the EU Dangerous Substances Directive (67/548/EEC) and in the EU Classification Labelling and Packaging Regulation (1272/2008/EC) because 2-ethylhexyl salicylate did not reveal any mutagenic effect in the bacterial reverse mutation assay in the presence or absence of metabolic activation, in the mammalian cell gene (HPRT) mutation test in V79 cells, in an in vitro chromosomal aberration test in CHO cells and in an in vivo micronucleus test in mice.