Reaction mass of (+-)-(1RS,2SR,5SR,7RS,8SR)-5-METHYLTRICYCLO[6.2.1.02,7]UNDECAN-4-ONE and (+-)-(1RS,2SR,5RS,7RS,8SR)-5-METHYLTRICYCLO[6.2.1.02,7]UNDECAN-4-ONE

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to cytotoxic or limit concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E. coli WP2uvrA.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 28 to October 21, 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD test guideline No. 471 without any deviation.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (inspected on December 02, 2002 / Signed on February 13, 2003)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10 % S9: S9-mix from the livers of male Sprague-Dawley rats received three consecutive daily doses of phenobarbitone/β-naphthoflavone (80/100 mg per kg per day).

Test concentrations with justification for top dose:

- Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA100 and WP2uvrA-, with and without S9-mix.
- Mutation test (Experiment-1; Range-finding Test): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains, with and without S9-mix using the direct plate incorporation method.
- Mutation Test (Experiment-2; Main Test): 5, 15, 50, 150, 500 and 1500 µg/plate in all Salmonella strains tested, with and without S9-mix, except TA1537 (with S9-mix) using the direct plate incorporation method; 15, 50, 150, 500, 1500 and 5000 µg/plate in E.coli strain WP2uvrA-, with and without S9-mix and TA1537 with S9-mix using the direct plate incorporation method.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration. Therefore, DMSO was selected as the vehicle of choice.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: Approximately 48 h at 37 °C.

NUMBER OF REPLICATIONS:
- Preliminary toxicity study: One plate/dose
- Mutation study: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn.

Evaluation criteria:

- There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (Kirkland, 1989) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
- A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.

Statistics:

Statistical methods, as recommended by the UKEMS can be used as an aid to evaluation. However, statistical significance will not be the only determining factor for a positive response

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: The test material was immiscible in sterile distilled water at 50 mg/mL.
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: Test material initially exhibited toxicity from 1500 µg/plate to the TA100 and WP2uvrA-strains.

COMPARISON WITH HISTORICAL CONTROL DATA: The comparison was made with the historical control ranges for 2003 and 2004 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the growth of the bacterial background lawn to all of the strains with and without S9-mix initially from 1500 µg/plate.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate and found to be satisfactory.
- Test material formulation, amino acid supplemented top agar and the S9 mix used in the experiments were shown to be sterile.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 7.6.1/2. Test results: Preliminary toxicity test

Metabolic activation	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	82	106	76	62	77	79	85	74	68	0*	0*
+	TA100	93	106	71	87	93	86	86	97	77	33*	0*
-	WP2uvrA-	33	23	21	36	31	30	16	19	24	16*	0*
+	WP2uvrA-	40	37	32	35	32	33	37	31	29	18	0*

Key: *- partial absence of bacterial background lawn

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA- according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP).

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and E.coli strain WP2 uvrA- were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and was ranged between 5 to 5000 µg/plate. The experiment was repeated on a separate day using a modified dose range, 5 to 1500 µg/plate in all Salmonella strains, with and without S9-mix, except TA1537 (with S9-mix) and 15 to 5000 µg/plate in E.coli strain WP2uvrA-, with and without S9-mix and TA1537 with S9-mix.

Negative, vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity tests.

The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test material caused a visible reduction in the growth of the bacterial background lawn to all of the strains both in the presence and absence of S9-mix initially from 1500 µg/plate. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100, and E.coli WP2uvrA- according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Table 7.6/1: Summary of genotoxicity tests

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Safepharm, 2005	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, E. coli WP2	-S9 +S9	Up to cytotoxic or limit concentration	-S9 : non mutagenic +S9 : non mutagenic

Gene mutation Assay (Test n° 1):

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

 

Justification for selection of genetic toxicity endpoint
The key study is GLP-compliant and of high-quality (Klimisch score = 1).

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008 including ATP6.

Self-classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).