Bicyclo[2.2.1]heptane, 2-ethyl-5-methoxy-

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:

Between 5 May 1988 and 26 May 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

403-610-9

EC Name:

-

Cas Number:

122795-41-9

IUPAC Name:

1-ethyl-3-methoxytricyclo[2.2.1.0²,⁶]heptane; 2-ethyl-5-methoxybicyclo[2.2.1]heptane; 2-ethyl-6-methoxybicyclo[2.2.1]heptane

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

other: S. typhimurium TA1535, TA1537, TA1538, TA98, TA100 and E.coli WP2 uvrA, trp

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose range finding test: 5000, 500, 50 and 5 µg/plate
Mutation tests:
Test 1: 5000, 2500, 1250, 625, 312.5 µg/plate
Test 2: 5000, 2500, 1250, 1000, 625, 500, 312.5, 250, 125, 62.5 µg/ml
Test 3: 1000, 500, 250, 125, 62.5, 31.25 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulphoxide

Controls

Controls 1

Positive controls:

yes

Remarks:

0.5µg/plate with TA 1538, 0.5 µg/plate with TA 98, 1 µg/plate with TA100, 2 µg/plate with TA1535, 2 µg/plate with TA 1537 and 20 µg/plate with WP2 uvrA

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With S9 mix

Controls 2

Positive controls:

yes

Remarks:

80 µg/plate with TA1537

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix

Controls 3

Positive controls:

yes

Remarks:

3 µg/plate with TA100, 2µg/plate with WP2 uvrA, 5µg/plate with TA1535

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9 mix

Controls 4

Positive controls:

yes

Remarks:

1 µg/plate with TA98, 2µg/plate with TA1538

Positive control substance:

2-nitrofluorene

Remarks:

Without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

PRELIMINARY TOXICITY TEST:
The following procedure is carried out on each bacterial strain:
Four concentrations of test substance are assessed for toxicity using the six tester strains. The highest concentration is usually 0.05 g of test substance dissolved in 1 ml of solvent. Three 10-fold serial dilutions of the top concentration are also tested. The chosen solvent is used as the negative control.

0.1 ml of a bacterial culture containing approximately 2 x 10^9 cells/ml, and 0.5 ml S-9 mix or 0.5 ml 0.1 M sodium orthophosphate buffer (pH 7.4) are placed in glass bijou bottles. 0.1 ml of the test solution is added followed by 2 ml histidine or tryptophan deficient agar. The mixture is thoroughly shaken and overlaid onto previously prepared plates containing 25 ml minimal agar. Single petri dishes are used for each dose level. They are incubated at 37°C for 72 hours. After this period the plates are examined for the appearance of a complete bacterial lawn. Revertant colonies are counted using a Biotran Automatic Colony Counter. Any toxic effects of the test substance are detected by a substantial reduction in revertant colony counts or by the absence of a complete background bacterial lawn.

MAIN TEST:
WITHOUT METABOLIC ACTIVATION
The following procedure is carried out on each tester strain.

0.1 ml aliquots of bacterial suspension and 0. 5 ml of sterile 0.1 M sodium orthophosphate buffer (pH 7. 4) are added to each of one set of sterile hijou bottles.
0.1 ml of the test compound is added to cultures at five concentrations separated by a two-fold interval. A top dose level of 5000 µg/plate will be used; however if toxicity ls observed at this concentration, 2000 µg/plate will be chosen as the top dose level. The negative control is the chosen solvent. The appropriate positive control is also included. Three bottles are used at each dose level.

2. 0 ml of histidine or tryptophan deficient agar is added to each of the bottles, thoroughly mixed and then overlaid onto previously prepared plates containing 25 ml of minimal agar. Plates are incubated for 72 hours at 37°C.

Colonies are counted using a Biotran Automatic Colony ·Counter, and the mean number of revertant colonies per treatment group assessed

WITH METABOLIC ACTIVATION
Methodology is as described in above except that 0.5 ml of liver homogenate 5-9 mix (see Section 3) is added to bijou bottles in place of sterile buffer. Plates will be prepared without the adition of bacteria in order to assess the sterility of the test compound and the S-9 mix.

ASSESSMENT OF RESULTS:
The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group.

A compound is deemed to provide evidence of mutagenic potential if a statistically siqnificant dose-related increase in the number of revertant colonies is obtained in two separate experiments.

Evaluation criteria:

The mean number of revertant colonies for all treatment groups is compared with those obtained for negative and positive control groups. The effect of metabolic activation is assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group.

A compound is deemed to provide evidence of mutagenic potential if a statistically siqnificant dose-related increase in the number of revertant colonies is obtained in two separate experiments.

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:

not examined

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit 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:

cytotoxicity

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:

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit 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:

The revertant colony counts for ethyl methoxy norbornane obtained in the dose range finding test are shown in Table 1. Ethyl methoxy norbornane was toxic towards the tester strains at the highest dose level. However 5000 µg/plate was chosen as the top dose level in the mutation tests.

The mean number of revertant colonies, obtained in the first mutation test with tester strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 and WP2 uvrA are shown in Table 2.

The mean number of revertant colonies, together with the individual plate counts for ethyl methoxy norbornane obtained in the first mutation test with all six tester strains are shown in Table 3.

Positive control mutability checks are shown in Table 4.

In the first mutation test ethyl methoxy norbornane was toxic towards tester strains TA 100, TA 1535 and TA 98, in the presence and absence of metabolic activation, and TA 1537, in the presence of metabolic activation only. The toxicity was observed at most of the dose levels tested, therefore a lower dose range was used for these tester strains in the second mutation test.

The mean number of revertant colonies, obtained in the second mutation test with tester strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and WP2 uvrA are shown in Table 5.

The mean number of revertant colonies, together with the individual plate counts for ethyl methoxy norbornane obtained in the second mutation test with all six tester strains are shown in Table 6. Positive control mutability checks are shown in Table 7.

Due to the toxicity observed in the first mutation test, a third test was performed using tester strains TA 100, TA 1535, TA 98 and TA 1537. The mean number of revertant colonies obtained in this test are shown in Table 8. The mean and individual colony counts obtained with ethyl methoxy norbornanp. with the four tester strains are shown in Table 9. Positive control mutability checks are shown in Table 10.

No substantial increases in revertant colony numbers of any of the six tester strains were observed following treatment with ethyl methoxy norbornane at any dose level, either in the presence or absence of metabolic activation (S-9 mix).

Any other information on results incl. tables

Please refer to attached background material for tables of results.

Applicant's summary and conclusion

Conclusions:

It is concluded that no evidence of mutagenic potential of Neoproxen was obtained in this bacterial test system at the dose levels used.

Executive summary:

Salmonella typhimuriums trains TA1535, TA1537, TA1538, TA98, TA100 and Escherichia coli strain WP2uvrA^-were tested according to OECD TG 471. The strains were treated with the test material (Neoproxen) using the plate incorporation method both with and without the addition of a rat liver homogenate metabolizing system (S9). Neoproxen was toxic towards tester strains TA l00, TA 1535 and TA 98, in the presence and absence of metabolic activation, and TA 1537, in the presence of metabolic activation only. Therefore the test concentrations in three independent tests ranged from 31.25 to 5000 µg/plate. No substantial increases in revertant colony numbers of any of the six tester strains were observed following treatment with Neoproxen at any dose level, either in the presence or absence of metabolic activation (S-9 mix). The test material was considered to be non-mutagenic under the conditions of this test.