4'-trans-propyl-1,1'-bicyclohexyl-4-trans-carboxaldehyde

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Key, gene mutation, OECD 471, GLP, Salmonella typhimurium TA 98, TA 100, TA 102,  TA 1535 and TA 1537, Escherichia coli WP2  uvrA, +/-S9: negative

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 MAR 2007 - 28 JUN 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD TG 471.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

His operon (Salmonella), Trp operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from Aroclor 1254-pretreated rats Type and composition of metabolic activation system:
- source of S9 : Male Wistar, HSdCpb:Wu rats from Harlan Winkelmann, Borchen, Germany, aged 6-8 weeks, were given a single intraperitoneal injection of Aroclor 1254 (500 mg/kg body weight) dissolved in Miglyol 812 oil (Merck KGaA, Darmstadt, Germany). The animals received drinking water and a standard diet ad libitum. About 16 hours before sacrifice, the rats remained without food. On day 5 to 7, they were sacrificed, the livers were removed and collected in ice-cooled sterilized beakers containing 0.15 M KCl. The livers were homogenized in a sterile glass potter homogenizer with a Teflon pestle containing 3 mL of 0.15 M KCl per gram of liver wet-weight. After homogenization the preparation was transferred to sterilized steel centrifuge tubes and spun at 9000 x g for 10 minutes at about + 4°C and the supernatant fluid was decanted and transferred into sterilized and precooled plastic tubes. The S9 was then frozen and stored in liquid nitrogen at -196°C.
- method of preparation of S9 mix :
The mixture of S9 plus the added cofactors is termed S9 mix:

Quantity per mL S9 mix

Components 1st Series 2nd Series

Liver homogenate (S9) 0.10 mL 0.30 mL
MgCl2/KCI aqueous solution (0.4 M/1.64 M) 0.02 mL 0.02 mL
Glucose-6-phosphate, disodium salt 5 µmol 5 µmol
NADP, disodium salt 4 µmol 4 µmol
Sodium phosphate buffer (0.2 M, pH 7.4) 0.50 mL 0.50 mL
Ultra pure water 0.38 mL 0.18 mL


- concentration or volume of S9 mix and S9 in the final culture medium: 10 % and 30 % S9 in the S9 mix are used in the 1st and 2nd test series, respectively.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Every S9-batch is tested for its metabolic activity by the use of specific substrates, requiring different enzymes of the P450-isoenzyme family. The mutagenicity of 2-aminoanthracene, benzo[a]pyrene, and 3-methylcholanthrene is thus determined once for every S9-batch. Clear increases in the number of revertants for S. typhimurium TA 98, TA 100, and TA 1537 with all positive controls and for TA 1535 with 2-aminoanthracene are used as an acceptance criterion for each S9-batch.

Test concentrations with justification for top dose:

1st series: 8.89, 15.8, 28.1, 50, 88.9, 158, 281 µg/plate
2nd series: 28.1, 50, 88.9, 158, 281 µg/plate (due to limited solubility, the current test material was tested up to only 281 µg/plate)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone, Purity ≥ 99 %

- Justification for choice of solvent/vehicle: Preferentially distilled water or dimethyl sulfoxide (DMSO), alternatively acetone or ethanol, are used as solvents. Analysis of the historical data of the laboratory and experience of other research groups showed that the amounts of the selected solvents used have no influence on the number of spontaneous revertants of any strain. For this reason, only the respective solvent controls are used as the negative control in these studies.

- Justification for percentage of solvent in the final culture medium: Since organic solvents may have diverse effects on e.g. gene regulation usually the maximum amount of solvent is limited to 10 µL per plate for DMSO, ethanol, acetone or other organic solvents. Only the highest test material concentration may be plated with 31.6 µL organic solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

benzo(a)pyrene

cumene hydroperoxide

other: Daunomycine, 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 2 to 3 days

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

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 and mean values are generated by use of a validated, automated data processing program (Perceptive Instruments, Haverhill, Suffolk, UK).

Rationale for test conditions:

Salmonella typhimurium and Escherichia coli strains were tested in accordance with the plate incorporation method described by Ames et al. (1975) and the OECD and EEC guidelines for this test system. The methods were extensively harmonized to follow the requirements of the Labor Ministry of Japan -Notification dated June 13, 1979 and the Notification No. 1-24 of the Pharmaceutical Affairs

Evaluation criteria:

A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if
• a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
• "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

Statistics:

no

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

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity at two highest doses without S9

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

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

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

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

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 at highest concentration of 281 µg/plate from beginning until end of experiment

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The negative control mutant frequencies were all in the regular range. The strain specific positive control test materials, namely daunomycin, sodium azide, 4-nitroquinolin-N-oxide, 9-aminoacridine, and cumene hydroperoxide in the absence of S9 mix, yielded the expected mutant frequencies that were greatly in excess of the negative controls.

Ames test:
- Signs of toxicity : cytotoxicity in strain S. typhimurium TA102 at two highest doses without S9
- Mean number of revertant colonies per plate and standard deviation : please refer to attached file under 'Attached background material'

Conclusions:

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

Executive summary:

Study Design

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102,  TA 1535 and TA 1537,  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% S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively.
The procedures used in this study were in accordance with OECD Guideline 471 (adopted 1997), EEC Annex V Test B 13B 14 (2000), UKEMS Guidelines (1990) and ICH Harmonised Tripartite Guideline (1997). The study was performed in compliance with GLP.

Results

The test material was dissolved in acetone and tested at concentrations ranging from 8.89 to 281 µg/plate. Precipitation of the test material on the agar plates occurred at the highest concentration tested, i.e. 281 µg/plate. Weak toxicity to the bacteria was observed in the concentration range between 158 and 281  µg/plate in the absence of S9 mix..
Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials 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 test materials used 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 mix used. In both series performed, there were no treatment-related increasing in the mutation frequencies observed with and without metabolic activation.

Conclusion

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

in vitro gene mutation in bacteria

Study Design

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102,  TA 1535 and TA 1537, 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% S9 in the S9 mix were used in the 1st and 30 % in the 2nd series, respectively.
The procedures used in this study were in accordance with OECD Guideline 471 (adopted 1997), EEC Annex V Test B 13B 14 (2000), UKEMS Guidelines (1990) and ICH Harmonised Tripartite Guideline (1997). The study was performed in compliance with GLP.

Results

The test material was dissolved in acetone and tested at concentrations ranging from 8.89 to 281 µg/plate. Precipitation of the test material on the agar plates occurred at the highest concentration tested, i.e. 281 µg/plate. Weak toxicity to the bacteria was observed in the concentration range between 158 and 281  µg/plate in the absence of S9 mix..
Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials 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 test materials used 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 mix used. In both series performed, there were no treatment-related increasing in the mutation frequencies observed with and without metabolic activation.

Conclusion

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

Justification for classification or non-classification

Based on the result of the available studies, the registered substance is not subject to classification in accordance with Regulation (EC) No 1272/2008.