Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
21 April 1989 - 3 July 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
A category approach is proposed for hexyl salicylate using data from other salicylate substances. The members of this category are demonstrated to be metabolised rapidly by esterases to salicylic acid; systemic exposure will therefore be to the metabolites to a much greater extent. The systemic toxicity of the salicylates is comparable and is attributable to the salicylic acid generated by metabolism. The other product of metabolism is the alcohol liberated from the sidechain following esterase metabolism. In the case of hexyl salicylate, this alcohol will be 1-hexanol which is of low toxicity and is rapidly metabolised and excreted and/or incorporated into normal metabolism. Hexanol is of lower toxicity than other alcohols generated from the metabolism of other category members (e.g. methanol); the source data therefore represents a worst case approach.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1898

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): HR 89/131494
- Molecular formula (if other than submission substance): C15H22O3
- Analytical purity: 99.9%
- Lot/batch No.: 37798

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: SAVO GmbH, D-7964 Kisslegg, F.R.G.
- Age at study initiation: 6 - 10 weeks old
- Weight at study initiation: 25 - 30g
- Assigned to test groups randomly: The animals were separated according to sex, marked for identification and allocated by randomization to cages and groups.
- Fasting period before study: No information provided
- Housing: The animals were group housed, up to 5 per cage in Makrolon cages of size II with Altromin woodshavings. The cages and bedding were changed with clean ones twice a week.
- Diet (e.g. ad libitum): Standard laboratory diet (Altromin 1324) ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): Approximately 55%
- Air changes (per hr): Not documented
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark cycle.

IN-LIFE DATES: From: 21 April 1989 To: 3 July 1989

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The compound was dissolved in arachis oil and the solution was freshly prepared just prior to use.

Duration of treatment / exposure:
Single exposure
Frequency of treatment:
The animals were dosed once
Post exposure period:
72 hours
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle control group
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
Treated group
No. of animals per sex per dose:
5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
9,10-dimethyl-1,2-benzanthracene dissolved in olive oil.
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow polychromatic erythrocytes (PCEs)
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES
The test animals were killed by cervical dislocation and bone marrow removed from both femora by rinsing with fetal calf serum. Bone marrow cells were centrifuged at 150g for 10 minutes and the supernatant discarded. From the pellet, smears were made on slides and air dired, according to haematological routine.

DETAILS OF SLIDE PREPARATION:
2 slides were made per animal. The preparations were stained by the May-Gruenwald-Giemsa method according to Schmid (1973). The slides were stained for 3 minutes in undiluted May-Gruenwald solution. They were then stained for 2 minutes in May-Gruenwald, diluted with distilled in a 1:1 ratio followed by brief rinsing in distilled water. The slides were stained for 10 minutes in Giemsa, diluted with distilled in a ratio of 1:6 and rinsed thoroughly in distilled water. They were dried in air and the back of the slides was dried with methanol. They were cleared in Xylene for 5 minutes and mounted in Eukitt.

METHOD OF ANALYSIS:
The slides were coded and observed blindly under a microscope with a 100x 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 normochromatic erythrocytes was also recorded. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.
Statistical significance was determined according to the methods of Kastenbaum and Bowman (1970).

Evaluation criteria:
No information provided
Statistics:
Statistical significance was determined according to the methods of Kastenbaum and Bowman (1970).

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: According to an initial toxicity test, the maximum tolerated dose was fixed to 2000 mg/kg body weight.

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): No increase in the frequency of micronucleated polychromatic erythrocytes in any treatment group at any treatment time. The incidence of micronucleated normochromatic erythrocytes in all treatment groups was within expected levels. The ratio of polychromatic erythrocytes to normochromatic erythrocytes was considered to be normal.

Afte dosing, the animals of all treated groups showed reduced motility.

Any other information on results incl. tables

No additional information

Applicant's summary and conclusion

Conclusions:
No evidence of micronucleus formation was seen under the conditions of this assay.
Executive summary:

The genetic toxicity of 2 -ethylhexyl salicylate was investigated in a mouse bone marrow micronucleus assay. Groups of five male and five female mice were treated by single oral administration of 2 -ethylhexyl salicylate (in arachis oil) at the limit dose level of 2000 mg/kg bw. The animals were sacrificed and bone marrow smears were prepared at 24, 48 and 72 hours after treatment. A positive (DMBA) and negative (vehicle) control group were also tested. At least 1000 polychromatic erythrocytes (PCEs) from each animal were scored for the incidence of micronuclei. The number of micronucleated normochromatic erythrocytes (NCEs) was also recorded, and the PCE:NCE ratio determined for each animal by counting a total of 1000 erythrocytes. No evidence of micronucleus formation was seen under the conditions of this assay.