4-trans-(4-trans-Propylcyclohexyl)-cyclohexancarbonic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-11-26 to 2002-03-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

07-1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(1r,1's,4r,4'r)-4'-propyl-[1,1'-bi(cyclohexane)]-4-carboxylic acid

EC Number:

613-782-9

Cas Number:

65355-32-0

Molecular formula:

C16 H28 O2

IUPAC Name:

(1r,1's,4r,4'r)-4'-propyl-[1,1'-bi(cyclohexane)]-4-carboxylic acid

Test material form:

solid: particulate/powder

Method

Target gene:

HIS operon (S. thyphimurium)
TRP operon (E. coli)
The strains are constructed to differentiate between base pair (E.coli WP2, TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors

- Source of S9: routinely prepared in the same laboratory following the proposals of Ames et al. (1975)
- Method of preparation of S9 mix: male Wistar HSdCpb:Wu rats from Harlan Winkelmann, Borchen, Germany, aged 6-8 week, were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bw), diluted in Migyol 812 oil (MERCK). Five to seven days after application of Aroclor, the rats were sacrificed and the livers collected in ice--cooled, sterilized beakers containing 0.15 M KCl. The livers were homogenized in a sterile glass potter homogenizer containing 3 mL of 0.15 M KCl per gram of liver wet-weight. The homogenate was spun at 9000 x g for 10 min at about +4°C and the supernatant fluid was decanted and transferred into sterilized and pre-cooled plastic tubes. the S9 was then frozen in liquid nitrogen and stored at -196°C.
- Volume of S9 mix and S9 in the final culture medium: S9 (1st series): 0.1 mL, S9 (1nd series): 0.3 mL, S9 mix: 0.5 mL in both series
- Quality controls of S9 (e.g., enzymatic activity, metabolic capability): Every S9 batch was tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2- aminoanthracene and benzo[a]pyrene is thus determined once for every S9 batch. Clear increases in the number of revertants for TA98, TA100 and TA1537 with all positive controls and for TA 1535 with 2- aminoanthracene are used as an acceptance criterion for each S9-batch. In this study, 2- aminoanthracene and benzo[a]pyrene for TA102, are used as the concurrent positive controls for the different strains in the presence of S9 mix.

Test concentrations with justification for top dose:

1st series: 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate
2nd series: 5, 15.8, 50, 158 and 500 μg/plate

In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively.

Justification for top dose: limit dose in the 1st series, cytotoxic dose in the 2nd series

Vehicle / solvent:

DMSO (test item, benzo[a]pyrene, cumene hydroperoxide, N-ethyl-N-nitro-N-nitrosoguanidine, 2-aminoanthracene), ethanol (9-aminoacridine) and H2O (daunomycin)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

without S9 mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 mix

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

H2O

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: daunomycin

Remarks:

without S9 mix

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
plate incorporation

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 parallel plates were used for each concentrations step of the test material and the positive controls.

TREATMENT:
Bacteria suspension, test item or negative or postive controls, S9 mix or sodium phosphate buffer and top agar solution were mixed and added to each plate. Incubation of plates was performed at +37°C for 2 to 3 days.

1st series: 7 concentrations seperated by half-log intervals and ranging from 5 to 5000 µg/plate both with and without S9 mix.

2nd series: 5 concentrations, including at least 4 non-toxic concentrations


METHODS FOR MEASUREMENTS OF GENOTOXICIY
Revertant colonies were either scored using an Artek MiniCount colony counter or manually. The presence of a background lawn of non-revertant cells was checked for each plate. Tables of individual mean values are generated by use of a validated, automated data processing program (COLONY V2.31, York Electronic Research, York, UK)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
Sign of cytotoxicity to the bacteria are a reduction in the number of spontaneous revertants or a clearing of background lawn of non-revertant bacteria.

Evaluation criteria:

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established (Table 1).

Statistics:

n.a.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occured at concentrations ≥ 158 µg/plate.

Ames test:
- Signs of toxicity: Clear toxicity to the bacteria was not observed.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
See tables in "Attached background material"

Applicant's summary and conclusion

Conclusions:

With and without addition of S9 mix as an external metabolizing system, the test item was not mutagenic under the experimental conditions described.

Executive summary:

The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA98, TA100, TA102, TA1535 and TA1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10% and 30% S9 in the S9 mix were used in the 1st and 2nd series, respectively. The test item was dissolved in DMSO and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occured at concentrations ≥ 158 µg/plate. Clear toxicity to the bacteria was not observed. Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain-specific positive control compounds in the absence of S9 mix. 2-aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mic used. With and without addition of S9 mix as an external metabolizing system, the test item was not mutagenic under the experimental conditions described.