Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

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:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

EC Number:

412-540-8

EC Name:

Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

Cas Number:

22471-55-2

Molecular formula:

C12 H22 O2

IUPAC Name:

ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

Constituent 2

Reference substance name:

Ethyl cis-2,2,6-trimethylcyclohexane-1-carboxylate

Molecular formula:

C12H22O2

IUPAC Name:

Ethyl cis-2,2,6-trimethylcyclohexane-1-carboxylate

impurity 1

Reference substance name:

Unidentified impurities

Molecular formula:

Not specified.

IUPAC Name:

Unidentified impurities

Test material form:

liquid: viscous

Details on test material:

Sample name: ET-344 SP

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals and Animal Husbandry
Sufficient male and female albino CD-1 Strain mice were supplied by Charles River (UK) Limited, Manston, Kent. At the start of the main study the males weighed 23 to 30g and the females 20 to 25g, and were approximately five to seven weeks old. After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by ear punching and a number written on a colour coded cage card.The animals were housed in groups of up to seven in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Rat and Mouse Expanded Diet No. 1, Special Diets Services Limited, Witham, Essex, UK) was allowed throughout the study. The animal room was maintained at a temperature of 19 to 20'C and relative humidity of 44 to 50%. The rate of air exchange was approximately fifteenchanges per hour and the lighting was controlled by a time switch to give twelve hours l ight and twelve hours darkness.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

The vehicle was supplied by Analytical Supplies as follows:
Supplier's identification : Arachis oil
Supplier's lot number : A1581
Safepharm serial number : CO/1029
Date received : 2 February 1996
Description : straw coloured viscous liquid
Storage conditions : room temperature

Details on exposure:

All animals were dosed once only via the intraperitoneal route at the appropriate dose level using a hypodermic needle attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of dosing.
Range-finding Toxicity Study
A range-finding toxicity study was performed to determine a suitable dose level for the micronucleus study. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of cytotoxicity up to a maximum recommended dose of 5000 mg/ kg. Using existing toxicity data the initial maximum dose level used was 2500 mg/ kg.
See table below for the details of the groups. All animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable.

Duration of treatment / exposure:

One day

Frequency of treatment:

Once

Post exposure period:

24 / 48 / 72 hours following treatment

Doses / concentrations

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

See details below.

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control material was supplied by the Sigma Chemical Company, as follows:
Supplier's identification : Cyclophosphamide
Supplier's lot number : 73H0846
Safepharm serial number : CO/911
Date received : 17May1995
Description : white powder
Storage conditions : 4.C
For the purpose of this study the positive control material was freshly prepared as required as a solution at the appropriate concentration in distilled water (Steripak batch no. 501041).The concentration, homogeneity and stability of the positive control material and its preparation were not determined by analysis.

Examinations

Tissues and cell types examined:

Slide EvaluationStained bone marrow smears were coded and examined 'blind' using light microscopy at x1000 magnification. The incidence of micronucleated cel Is per 1 000 polychromatic erythrocytes (PCE-blue stained immature cel Is) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pin k stained mature cel Is) associated with 1 000 polychromatic erythrocytes was counted; these cel Is were also scored for incidence of micronuclei.The ratio of normochromatic to polychromatic erythrocytes was calculated together with appropriate group mean values for males and femalesseparately and combined.

Details of tissue and slide preparation:

Immediately following sacrifice (ie. 24, 48 or 72 hours following dosing), one femur was dissected from each animal, aspirated with foetal calf serumand bone marrow smears prepared following centrifugation and resuspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa, dried and coverslipped using mounting medium.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the three test material groups and the number occurring in the corresponding vehicle control groups.A positive mutagenic response was demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24, 48, or 72-hour kill times when compared to their corresponding control group.If these criteria are not demonstrated, then the test material is considered to be non-genotoxic under the conditions of the test.A positive response for bone marrow toxicity was demonstrated when the dose group mean normochromatic to polychromatic ratio was shown to be statistically significantly higher than the concurrent vehicle control group.

Statistics:

All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a V (X + l) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Range-finding Toxicity Study
Premature deaths were seen in animals dosed with the test material at 2500 mg/ kg (2 deaths / 2 animals) and 1875 mg/kg (4 deaths / 4 animals). Clinical signs were observed in animals dosed with test material at and above 1250 mg/kg, and were as follows: hunched posture, lethargy, ataxia, diuresis and ptosis. With clinical signs occurring at 1250 mg/ kg and premature deaths at and above 1875 mg/ kg, the maximum tolerated dose selected for use in the main study was 1250 mg/kg.Micronucleus Study
Mortality Data and Clinical Observations
In animals dosed with test material there was one premature death in the 72-hour dose group, and clinical signs were observed as follows: hunched posture, ataxia and diuresis. In the animal that died coma, decreased respiratory rate, laboured respiration, ptosis and pal tor of the extremities were also observed at approximately 24 hours.
Evaluation of Bone Marrow Slides
There was a significant increase in the frequency of micronucleated PCEs in the 48-hour test material dose group when compared to the concurrent historical range, therefore it was considered to be spurious and of no toxicologically significance.There was no statistically significant increase in the NCWPCE ratio in any of the test material dose groups when compared to their concurrent vehiclecontrol groups. However the presence of clinical signs and the premature death indicated that systemic absorption had occurred.The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivityof the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Applicant's summary and conclusion

Conclusions:

ET-344-SP was considered to be non-genotoxic under the conditions of the test.

Executive summary:

1. A study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered via the intraperitoneal route to mice. The method followed that described in the OECD Guidelines for Testing of Chemicals No. 474 "Micronucleus Test" and US EPA Test for TSCA of 40 CFR 798 5395.

2. Following a range-finding study to confirm the toxicity of the test material, the micronucleus study was conducted using the test material at the maximum tolerated dose level of 1250 mg/kg.

3. In the micronucleus study, groups of ten mice (five males and five females) were given a single intraperitoneal dose of the test material at 1250 mg/kg. Animals were killed 24, 48 or 72 hours later, the bone marrow extracted and smear preparations made and

stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

4. Further groups of mice were dosed via the intraperitoneal route with arachis oil or orally with cyclophosphamide, to serve as vehicle and positive controls respectively.

5. There was no evidence of a toxicologically significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with test material when compared to the concurrent vehicle control groups. No significant change in the NCE/PCE ratio was observed after dosing with the test material. However, the presence of clinical signs and one premature death would indicate that systemic absorption had occurred.

6. The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes in animals dosed with test material when compared to the concurrent vehicle control groups.

7. The test material, ET-344-SP, was considered to be non-genotoxic under the conditions of the test.