Ethyl trans-2,2,6-trimethylcyclohexanecarboxylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a study conducted in accordance with a guideline similar to OECD Guideline 473, ET-344 SP did not demonstrate any increase in the frequency of cells with aberrations in any of the treatment cases used. ET-344 SP was shown to be toxic to CHL cells in vitro in all six treatment cases, with a very steep dose response curve. ET-344 SP was shown to be non-clastogenic to CHL cells in vitro.

In an Ames test conducted in accordance with OECD Guideline 471, ET-344 SP was not mutagenic in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Esherichia coli WP2 uvrA (with and without metabolic activation).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 November 1992 to 12 November 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Preliminary test +/- S9 mix: 5 / 20 / 78 / 313 / 1250 / 5000 ug/ plateMutation test - S9 mix: 5 / 10 / 20 / 39 / 78 / 156 / 313 ug/ plate + S9 mix: 10 / 20 / 39 / 78 / 156 / 313 ug/ plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoanthracene

Details on test system and experimental conditions:

Bacterial Strains :Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Esherichia coli WP2 uvrA were obtained from Institute for Fermentation,JAPAN.Cultures of each strain were prepared by incubating an aliquot of parent stock culture in nutrient broth a t 37°C for 18 hours. Test Procedures: (Pre-incubation Method)After mixing 0.1mL of test material solution, 0.5mL of phosphate buffer or S9 Mix and 0.1 mL of culture of tester strain in a test tube, the mixture was incubated a t 37°C for 20 minutes.Then 2 mL of top agar was added in the test tube, the mixture of each tube was poured on minimal agar plate. The plates were incubated at 37 °C for 2 days and scored for revertants.The test was performed in tripilcate at each concentration.

Statistics:

No

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In case of no growth inhibition, there was no significant difference in the number of the colonies between the test compound dosages and the solvent control with any of the bacteria used here.

Remarks on result:

other: all strains/cell types tested

Conclusions:

The test results indicated that the compound is not mutagenic under the test system used (with and without metabolic activation).

Executive summary:

According to OECD TG 471, the mutagenic activity of Ethyl 2,2,6-trimethylcyclohexanecarboxylate provided by TAKASAGO International Corporation was tested by the in vitro direct and metabolic activation assays using Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Esherichia coli WP2 uvrA.

The Pre-incubation Method was followed.

Vehicule and positive control were included in the study.

The concentrations of test item were as follows:

Preliminary test

+/- S9 mix: 5 / 20 / 78 / 313 / 1250 / 5000 ug/ plate

Mutation test

- S9 mix: 5 / 10 / 20 / 39 / 78 / 156 / 313 ug/ plate

+ S9 mix: 10 / 20 / 39 / 78 / 156 / 313 ug/ plate

The test was performed in tripilcate at each concentration.

In case of no growth inhibition, there was no significant difference in the number of the colonies between the test compound dosages and the solvent control with any of the bacteria used here.

The test results indicated that the compound is not mutagenic under the test system used.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 February 1993 to 22 July 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Safepharm Standard Method Number EEC 21A and was designed to assess the potential chromosomal mutagenicity of a test material, on the metaphase chromosomes of the Chinese hamster lung (CHL) cell line

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: CHINESE HAMSTER LUNG CELLS CHL

Details on mammalian cell type (if applicable):

SUPPLIER: JAPANESE CANCER RESEARCH RESOURCES BANK-CELL BANK

Metabolic activation:

with and without

Metabolic activation system:

from the livers of male Sprague-Dawley rats weighing - 2009. These had received a single i .p. injection of Arocl or 1254 at 500 mg/kg, 5 days before S9 preparation. The S9 was stored at -196°C in a Statebourne liquid nitrogen freezer, model SXR 34

Test concentrations with justification for top dose:

Preliminary Cytotoxicity Test: dose range of 0 to 300 ug/ mL
Chromosome aberration study
a) 24-HOUR HARVEST (6-HOUR TREATMENT, 18-HOUR RECOVERY): 0-40 ug/ mL without S9 and 0-150 with S9
b) 12-HOUR HARVEST ( 4-HOUR TREATMENT, 8-HOUR RECOVERY +S9) 12-HOUR TREATMENT -S9): -40 ug/ mL without S9 and 0-150 ug/ mL with S9
C) 24 AND 48-HOUR HARVESTS (CONTINUOUS TREATMENT): 0-40 ug/ mL

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cell Line
The Chinese hamster lung (CHL) cell line, isolated by Koyama et al. 1970 and cloned by Ishidate and Sofuni (1985) was used. The cell population doubling time for CHL cells in this laboratory is approximately 11 hours.
Cell Culture
Cells were grown in Eagle's Minimal Essential medium with Earle's Salts (MEM), supplemented with 10% foetal bovine serum and anti-biotics, at 37°C with 5% C02 in air.
Preparation of Test and Control Materials
ET-344 SP was accurately weighed and prepared in DMSO and appropriate dilutions made. The concentration, homogeneity and stability of the test material preparations were not determined by analysis.
Negative and positive controls were used in parallel with the test material. Solvent treatment groups were used as the negative controls and the positive control materials were as follows:
Mitomycin C (MMC) 0.075 ug/ml (Sigma Batch No. 32H0326) for cultures treated for 12, 24 or 48 hours in the absence of metabolising enzymes.
Cyclophosphamide (CP) 10 ug/ml (Sigma Batch No. 70H0948) for cultures treated for 6 (18) hours both with and without S9 mix, and the 4 (8) hour culture with S9 mix.
Preliminary Cytotoxicity Test
A preliminary cytotoxicity test was performed on cell cultures using 12, 24 and 48-hour continuous exposure times without metabolic activation and a 6(18) -hour and 4(8) -hour exposure period with metabolic activation. The 6(18)-hour and 4(8)-hour exposure groups with metabolic activation were exposed for 6 hours and 4 hours followed by an 18-hour and 8-hour culture period respectively, in treatment-free media. Growth inhibition was estimated by counting the number of cells at the end of the culture period on an electronic cell counter (Coulter) and expressing the cell count as a percentage of the concurrent negative control value. Slides were also prepared from the cells in order to check for the presence of cells in metaphase.Culture ConditionsCultures were established approximately 24 hours prior to treatment, 0.55 x 10^6 cells were seeded per flask for the 12-hour treatment cultures, 0.4 x 10^6 cells were selected per flask for the 6 and 24-hour treatment cultures and 0.1 x 10^6 cells were seeded per flask for the 48-hour treatment cultures. The cells were exposed to four doses of the test material, negative and positive controls, both with and without metabolic activation.The dose levels selected for analysis in the main study for the 24-hour treatment without S9 were 5, 10, 20 and 40 ug/ml. All treatments were performed in duplicate (A + B). Cultures were maintained at 37°C in a humidified atmosphere of 5% C02 in air.
The treatment regimens were as follows:
a) Without Metabolic Activation
i) 6 hours exposure to the test material, a phosphate buffered saline wash and then a further 18 hours culture in treatment-free media prior to cell harvest.
ii) 12 hours continuous exposure to the test material prior to cell harvest.
iii) 24 hours continuous exposure to the test material prior to cell harvest.
iv) 48 hours continuous exposure to the test material prior to cell harvest.
b) With Metabolic Activation:
i ) 6 hours exposure to the test material and S9 mix (0.5 ml per 4.5 ml culture medium, of 50% S9 in standard cofactors). A phosphate buffered saline wash and then a further 18 hours in treatment-free media prior to cell harvest.
ii ) 12 hour treatment group has a pulse 4 hours exposure to the test material and S9 mix (0.5 ml per 4.5 ml culture medium, of 50% S9 in standard cofactors). A phosphate saline wash and then a further 8 hours in treatment free media prior to cell harvest.
Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 ug/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were trypsinised to detach them from the tissue culture flask and suspended in 5 ml of culture medium. A sample of the cell suspension from each harvest time was counted to measure growth inhibition at each concentration.The cells were centrifuged (1000 - 1300 rpm for five minutes), the culture medium drawn off and discarded, and the cells resuspended in 4 ml 0.075M KCl. After thirteen minutes (including five minutes centrifugation) , all but approximately 0.5 ml of hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the KCl suspension into 3 ml fresh methanol/glacial acetic acid (3:1 v / v ) . The fixative was changed at least twice and the cells stored at 4°C for at least four hours to ensure complete fixation.Preparation of Metaphase SpreadsThe cells were resuspended in fresh fixative prior to slide preparation. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.
Staining
When the slides were dry they were stained in 2% Gurrs Giemsa R66 for 5 minutes, rinsed, dried and mounted in Depex mounting medium.CodingAfter checking that the slide preparations were of good quality, the slides were coded using a computerised random number generator.

Evaluation criteria:

Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 23 to 27 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the UKEMS guidelines for mutagenicity testing.Aberrations recorded by the slide scorer are checked by a senior cytogeneticist. Cells with 27 - 31 chromosomes are scored aneuploid cells. Cells with greater than 31 chromosomes are classified as polyploid cells and the incidence of polyploid cells % reported. The percentage of cells showing structural chromosome aberrations (gaps, breaks and exchanges) are calcul ated and reported as both including and excluding those with gaps.Mitotic IndexA total of 1000 cells were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Key result

Species / strain:

mammalian cell line, other: Chinese hamster lung cell line

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Detailed results are presented in the attached pdf document

Conclusions:

ET-344 SP did not induce any dose-related increases in the frequency of cells with chromosome aberrations either in the presence or absence of a liver enzyme metabolising system or after various exposure times.
ET-344 SP is therefore considered to be non-clastogenic to CHL cells in vitro.

Executive summary:

Chinese hamster lung (CHL) cells were treated with ET-344 SP at four dose levels in each treatment case, in duplicate, together with negative and positive controls. Six treatment regimens were used: 6 hours exposure both with and without the addition of an induced rat liver homogenate metabolising system at 50% in standard co-factors (followed by 18 hours incubation); 4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 50% in standard co-factors (followed by 8 hours incubation); 12 hours continuous exposure, 24 hours continuous exposure and 48 hours continuous exposure. Three of the four dose levels were scored for chromosome aberrations.

The dose range for the chromosome aberration test was selected on the basis of the results of a preliminary toxicity test and was 25 to 100 ug/ ml for the 6(18)-hour and 4(8)-hour treatments with S9, 5 to 20 ug/ ml for the 12-hour treatment without S9 and the 24-hour continuous treatment and 10 to 40 ug/ ml for the 6(18)-hour treatment without S9 and the 48-hour treatment.

The negative (solvent) controls gave frequencies of aberrations within the range expected for the CHL cell line.

All the positive control treatments except cyclophosphamide without S9 gave highly significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

ET-344 SP did not demonstrate any increase in the frequency of cells with aberrations in any of the treatment cases used. ET-344 SP was shown to be toxic to CHL cells in vitro in all six treatment cases, with a very steep dose response curve. ET-344 SP was shown to be non-clastogenic to CHL cells in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In an in vivo micronucleus study conducted in accordance with OECD Guideline 474, ET-344-SP, was considered to be non-genotoxic under the conditions of the test.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

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

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.

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

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.

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The substance is not classified for genetic toxicity in accordance with the CLP Regulation.