3,4,5,6,7,8,9,10,11,12,13,14-dodecahydro-2H-cyclododeca[b]pyran

Genetic toxicity: in vitro

Administrative data

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 February 22, 2012 to March 26, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

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

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program, 19-21 July 2011

Type of assay:

bacterial gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bicyclenoxyde

IUPAC Name:

Bicyclenoxyde

Details on test material:

- Name of test material (as cited in study report): Bicyclenoxyde
- Storage condition of test material: approximately room temperature in the dark

Method

Target gene:

Histidine gene for Salmonella and Tryptophan gene 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:

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:

10% rat liver S9 in standard co-factors

Test concentrations with justification for top dose:

0, 50, 150, 500, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) Acetone and DMSO in a confirmatory test
- Justification for choice of solvent/vehicle: the test material was insoluble in sterile distilled water at 50 µg/mL and not fully soluble in DMSO but wassoluble in acetone at the same concentration in a solubility check performed in-house

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ENNG, 9AA and 4NQO

Remarks:

Without S9-mix (see Table 7.6.1/2)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2 AA, BP

Remarks:

With S9-mix (see Table 7.6.1/2)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for experiment 1, pre-incubation for experiment 2

DURATION
- Exposure duration: approximately 48 hours at 37 °C

NUMBER OF REPLICATIONS: triplicate plates per dose level

DETERMINATION OF CYTOTOXICITY
- Method: observation of the growth of non-revertant bacteria (background lawn) and revertant colony numbers

OTHER: ACCEPTANCE CRITERIA: The reverse mutation assay may be considered valid if the following criteria were met:
1. All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (according to historical control).
2. The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
3. All tester strain cultures should be in the approximate range of 0.9 to 9 billion bacteria per mL.
4. Each mean positive control value should be at least two times the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
5. There should be a minimum of four non-toxic test material dose levels.
6. There should be no evidence of excessive contamination.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby (1979)).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response).
A test item 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 making a definite judgement about test item activity. Results of this type will be reported as equivocal.

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 judgment about the test material activity. Results of this type will be reported as equivocal.

Statistics:

As recommended by UKEMS, if needed.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight reduction in TA100 and TA1535 revertant counts at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A precipitate was noted at 5000 µg/plate.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity/range-finding test was carried out to determine the toxicity of the test material and to select the appropriate dose levels for use in the main test. Ten concentrations (0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) and a vehicle control were tested (DMSO). In addition the sterility of the test material was assessed. The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-) (See Table 7.6.1/3). The test material formulation and S9-mix used in this experiment were both shown to be sterile).

COMPARISON WITH HISTORICAL CONTROL DATA: Vehicle and positive control values were within the historical range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a slight reduction in reveratnt colony counts in strains TA100 and TA1535 which was taken as evidence of a weak toxic response at 5000 µg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Preliminary Toxicity Test

With (+) or without (-) S9-mix	Strain	Dose (µg/plate)
		0	0.15	0.5	1.5	5	15	50	150	500	1500	5000
-	TA100	102	68	97	72	95	83	95	86	60	72	60 P
+	TA100	105	100	92	85	110	93	106	106	71	62	61 P
-	WP2uvrA	36	29	24	39	21	24	28	33	38	30	35 P
+	WP2uvrA	42	30	26	28	38	29	22	38	35	28	33 P

P = precipitate

 

Prior to the use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all found to be satisfactory). The amino acid supplemented to agar and the S9-mix used in both experiments was shown to be sterile.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test material is not mutagenic, either with and without metabolic activation in S. typhimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA- according to the criteria of the Annex VI of the of the Regulation (EC) No 1272/2008 (CLP) and of the Directive 67/548/EEC.

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 the test material after dilution in acetone, both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method in Experiment 1 and the pre-incubation method in Experiment 2. The dose range for the first experiment (range-finding test) was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

The vehicle (acetone) 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. In an additional experiment the substance was shown to be non-mutagenic to strain TA98 in the absence of metabolic activation only, when dissolved in DMSO at half the normal concentration and dosed at twice the normal volume.

The test material caused a weak toxic response to the Salmonella strains TA100 and TA1535 at 5000 µg/plate. A precipitate was noted at 5000 µg/plate.

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537 TA98 & TA100, and E.coli WP2 uvrA^-according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP) and of the Directive 67/548/EEC.

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