1-Propanol, 2-methyl-3-[(1,7,7-trimethylbicyclo[2.2.1]hept-2-yl)oxy]-

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:

The experimental phase of the study was conducted between 19 March to 2 April 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Meets the requirements of the Japanese Regulatory Authorities including METI, MHLW and MAFF, OECD Guidelines for Testing of Chemicals No. 471 "and the USA, EPA (TSCA) OPPTS harmonised guidelines.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

416-210-4

EC Name:

-

Cas Number:

128119-70-0

Molecular formula:

C14H26O2

IUPAC Name:

2-methyl-3-({1,7,7-trimethylbicyclo[2.2.1]heptan-2-yl}oxy)propan-1-ol

Test material form:

liquid

Method

Target gene:

Histidine for S. typhimurium
Tryptophan for E. coli

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Not applicable.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver, S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 5, 50, 500, 5000 µg/plate.
Experiment 1: 0, 39.036, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate.
Experiment 2: 0, 19.53125, 39.036, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Sterile distilled water.
- Justification for choice of solvent/vehicle: The test material was fully soluble in sterile distilled water at 50 mg/ml in solubility checks performed in-house

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 1 µg/plate

Remarks:

With S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537 and TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 2 µg/plate

Remarks:

With S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98 and TA1538

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene 0.5µg/plate

Remarks:

With S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene: 10 µg/plate

Remarks:

With S9 mix

Controls 6

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-Ethyl-N -nitro-N-nitrosoguanidine, 3µg/plate

Remarks:

without S9 mix

Controls 7

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-Ethyl-N -nitro-N-nitrosoguanidine, 5µg/plate

Remarks:

Without S9 mix

Controls 8

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Migrated to IUCLID6: 9-Aminoacridine: 80 µg/plate

Controls 9

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of TA98 and TA1538

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Nitrofluorene, 2µg/plate for TA1538 and 1µg/plate for TA98

Remarks:

without S9 mix

Controls 10

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates of WP2uvrA

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-Ethyl-N -nitro-N-nitrosoguanidine, 2µg/plate

Remarks:

Without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) Experiments 1 and 2

DURATION
- Preincubation period for bacterial strains: 10h
- Exposure duration: 48 - 72 hrs

NUMBER OF REPLICATIONS: Triplicate plating.

DETERMINATION OF CYTOTOXICITY
- Method: plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn.

BACTERIAL STRAINS
Batches of the strains were stored at -80°C. Each batch of frozen strain was tested for cell membrane permeability and, where applicable, for the pKM101 plasmid which confers resistance to ampicillin. The response of the strains to a series of diagnostic mutagens was also assessed. For use in tests an aliquot of frozen culture was added to 25 ml of nutrient broth (DAB 7, Merck) and incubated, with shaking, at 37°C for 10 hours. This culture provided approximately 2 x 109viable cells per ml which was checked photometrically.

PREPARATION OF S9 FRACTION
- Species: Rat
- Sex: Male
- Strain: Sprague-Dawley derived
- Source: Harlan Olac Ltd.
- Age range: 7-8 weeks
- Weight range: <300 g
- Diet: Biosure Rodent Diet LAD 1
Mixed function oxidase systems in the livers of a group of rats were stimulated by Aroclor 1254, administered as a single intra-peritoneal injection in Arachis oil at a dosage of 500 mg/kg bodyweight. On the fifth day after injection, following an overnight starvation, the rats were killed, and their livers aseptically removed.
The following steps were carried out at 0-4°C under aseptic conditions. The livers were placed in 0.15 M KCI (3 ml KCI:1g liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation, the homogenates were centrifuged at 9000 g for 10 minutes. The supernatant fraction (S-9 fraction) was dispensed into aliquots and stored at -80°C until required. The efficacy of each batch of S-9 fraction was tested with the carcinogens 7,12-dimethylbenzanthracene and 2-aminoanthracene before use.

PREPARATION OF S9-MIX
S-9 mix contained: S-9 fraction (10% v/v), MgCl2(8 mM), KCI (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM), NADH (4 mM). All the cofactors were filter-sterilised before use.

PRELIMINARY TOXICITY TEST
Four concentrations of test substance were assessed for toxicity using the six tester strains. The highest concentration was 50 mg/ml of test substance in the chosen solvent, which provided a final concentration of 5000 µg/plate. Three 10-fold serial dilutions of the highest concentration were also tested. The chosen solvent, dimethyl sulphoxide, was used as the negative control. An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S-9 mix or 0.5 ml 0.1M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 ml of the test solution was added, followed immediately by 2 ml of histidine/tryptophan deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. A single petri dish was used for each dose level. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period the appearance of the background bacterial lawn was examined. Revertant colonies were counted using a Seescan Automatic Colony Counter. Any toxic effects of the test substance can be detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn. In the absence of any toxic effects the top concentration used in the main tests is the same as that used in the preliminary toxicity test. If toxic effects are observed a lower concentration may be chosen for the main assays. Ideally the concentrations chosen for the mutation tests should include a minimum of four non-toxic concentrations.

MUTATION TEST PROCEDURE
The test substance was added to cultures of the six tester strains at eight concentrations separated by 2-fold dilutions. The highest concentration of Bornafix used was 5000 µg/plate. The negative control was the chosen solvent, dimethyl sulphoxide. The positive control compounds were also included. An aliquot of 0.1 ml of a 10 hour bacterial culture and 0.5 ml S-9 mix or 0.5 ml 0.1 M phosphate buffer (pH 7.4) were placed in glass bottles. An aliquot of 0.1 ml of the test solution was added, followed immediately by 2 ml of histidine/tryptophan deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. Three petri dishes were used for each dose level. A set of plates were also prepared containing only bacterial culture and S-9 mix or phosphate buffer (0 µg/plate). Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S-9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period revertant colonies were counted using a Seescan Automatic Colony Counter. At a later date the main test was repeated using the procedures described above but using a total of nine concentrations of test substance.

Evaluation criteria:

Acceptance Criteria:
The mean number of revertant colonies for all treatment groups is compared with those obtained for solvent control groups. The mutagenic activity of a test substance is assessed by applying the following criteria:
(a) If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(b) If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
(c) If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response given in paragraphs (a) and (b), the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.

(i) Repeat tests may be performed using modifications of the experimental method. These modifications include (but are not restricted to), the use of a narrower dose range than that already tested; the use of different levels of liver homogenate S-9 fraction in the S-9 mix. Should an increase in revertant colony numbers be observed which satisfies paragraph (a) the substance is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
(ii) If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers. The statistical procedures used will be those described by Mahon et al (1989).

Statistics:

Standard deviation

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

In the preliminary toxicity test Bornafix was toxic towards all the tester strains at 5000 µg/plate. 5000 µg/plate was chosen as the top dose level in the first mutation test but a total of eight dose levels were included to ensure that sufficient non-toxic concentrations were tested.

In the first mutation test variable toxicity was shown towards the different tester strains. The lowest dose level at which signs of toxicity were observed was 312.5 µg/plate with strain TA 1538 in the presence of S9 mix. In attempt to obtain at least four non-toxic dose levels for each strain in the second mutation test a lower dose level of 19.53 125 µg/plate was included.

No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with Bornafix at any dose level, either in the presence or absence of S9 mix.

The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

Any other information on results incl. tables

 

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471.

Executive summary:

In this in vitro assessment of the mutagenic potential of Bornafix, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of Escherichia coli (WP2uvrA) were exposed to the test material, diluted in dimethyl sulphoxide which was also used as a negative control. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats. In the preliminary dose range finding study with dose levels of up to 5000 µg/plate toxicity was observed towards all the tester strains at the top dose level. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. To ensure that sufficient non-toxic dose levels were tested other dose levels used in the first mutation assay were: 2500, 1250, 625, 312.5, 156.25, 78.125 and 39.0625 µg/plate. In the first mutation assay some toxicity was observed down to 312.5 µg/plate so an additional lower dose level of 19.53125 µg/plate was included in the second mutation assay. No evidence of mutagenic activity was seen at any dose level of Bornafix in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that, when tested in dimethyl sulphoxide, Bornafix was not mutagenic in this bacterial system.