3a,4,5,6,7,7a-hexahydro-5-methoxy-4,7-methano-1H-indene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the conditions of this study, the test material was not mutagenic to the bacterial strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 in the presence or absence of metabolic activation.

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:

19 October 1985 to 04 November 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

The purpose of this mutagenicity test was to investigate the potential of the test material to induce reverse mutations in histidine dependent strains of Salmonella typhimurium. This was done by incubating the bacteria with the test material on selective media (histidine deficient) which inhibits the growth of histidine dependent bacteria, but permits the growth of histidine independent revertants. Since some chemicals are themselves non­mutagenic but are metabolised to mutagenic metabolites the test was conducted both with and without liver preparation (S-9 mix) which has the function of metabolising the test material. A preliminary toxicity test was required in order to establish dose levels - the test material was tested up to the maximum concentration permitted by toxicity up to a maximum of 5000 µg/plate for non-toxic materials. At least five dose levels were tested together with positive and negative controls.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine dependent strains of Salmonella typhimurium.

Species / strain / cell type:

S. typhimurium, other: TA1537, TA1535, TA100, TA1538 & TA98

Details on mammalian cell type (if applicable):

- The strains carry the uvrB mutation and are therefore repair deficient. This results in an increased sensitivity in detecting mutagens.
- The strains also carry the deep rough mutation (rfa) which causes a partial loss in the polysaccharide coat of the bacteria and therefore allows penetration of large molecules.
- Strains TA98 and TA100 contain the R factor plasmid pKM101 and are more sensitive than the parent strains TA1538 and TA1535 respectively which do not contain the plasmid. The plasmid containing strains are Ampicillin resistant.
- The strains are routinely checked with diagnostic mutagens for strain specificity, for UV sensitivity, for sensitivity to Crystal Violet, and for Ampicillin resistance if appropriate (Ames et al, 1975).

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

- Dose range-finding test: 5000, 500, 50 and 5 μg/plate.
- Main test: 5000, 1500, 500, 150 and 50 μg/plate.
The top dose level was chosen after an evaluation of the results of the preliminary toxicity test. It was either the lowest dose showing toxicity in the preliminary test or the next dose down on a half log10 scale, according to the judgement of the Experimental Supervisor.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

PRELIMINARY TEST
The preliminary toxicity test followed the same test method as the main test (see below), with the exception that four concentrations of test material and duplicate plates were used. The concentrations for the toxicity test were 5000, 500, 50 and 5 μg/plate.

MAIN TEST
- Sub-cultures of bacteria were grown in nutrient broth at 37°C for 16 hrs to provide approximately 2 x 10^9 bacteria per mL. Two mL aliquots of molten soft agar containing 0.5 mM of histidine HCl, and 0.5mM biotin were dispensed into glass tubes and maintained at 45°C. To triplicate plates 0.5 mL of S-9 mix (with metabolism) or 0.5mL of 0.1 M phosphate buffer, pH 7.4 (without metabolism), 0.1 mL of bacterial suspension (2 x 10^9 bacteria/mL) and 0.1 mL of test material in DMSO or other appropriate solvent at each of five concentrations were added.
- The contents of the tubes were thoroughly mixed and poured onto petri dishes containing 30 mL of minimal agar. The plates were incubated at 37°C for 72 hours.
- The number of colonies on each plate was counted and the mean number of revertant colonies per treatment group was calculated.
- The sterility of the S-9 mix and test solutions was confirmed by the incubation of aliquots of the liquids on nutrient agar.
- For the pre-incubation method, 0.5 mL of S-9 mix or 0.5 mL of 0.1 M phosphate buffer, pH 7.4, was incubated for 30 minutes at 37°C with 0.1 mL of bacterial culture and 0.01 mL of test material solution. After incubation, two mL of melted (45°C) soft agar containing 0.5 mM histidine HCl and 0.5 mM biotin was added, mixed, and overlaid onto minimal agar plates. The plates were incubated and colonies counted as above.

Evaluation criteria:

For each strain the mean number of revertants at each treatment group both with and without metabolism was compared with those of the negative control groups. The test material was considered to be a positive mutagen if a dose related increase in revertants, up to at least a doubling of the control values, was obtained in two independent tests.

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA1538, TA98 & TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- The test material was toxic towards the tester strains.
- The test material did not induce significant number of revertant colonies with any of the five strains either in the presence or absence of S-9 mix.
- It was concluded that no evidence of mutagenic potential of the test material was obtained in the bacterial test system at the concentrations used. However, toxicity was observed at 1500 μg/plate towards all strains without activation. In assays with activation, the toxicity was observed at 1500 µg/plate towards TA 100 and at 5000 µg/plate towards all strains.

Table 1: Summary of First Main Experiment

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		TA100	TA1535	TA1538	TA98	TA1537
-	Solvent 50 150 500 1500 5000	208 205 202 176 - -	47 42 44 44 - -	17 17 16 16 - -	49 50 50 41 - -	17 19 17 17 - -
+	Solvent 50 150 500 1500 5000	154 148 150 134 - -	27 27 24 23 19 -	22 23 23 22 18 -	32 32 29 30 25 -	19 19 18 18 12 -
Positive Controls
-	Name	SA	SA	2NF	2NF	9AA
	Concentration (µg/plate)	5	5	10	10	20
	Mean no. colonies/plate	>1000	>1000	>1000	>1000	123
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2	2	2	2	2
	Mean no. colonies/plate	825	96	390	489	134

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = Sodium azide

2NF = 2-Nitrofluorene

Table 2: Summary of Repeat Main Experiment

± S9 Mix	Concentration (µg/plate)	Mean number of colonies/plate
		TA100	TA1535	TA1538	TA98	TA1537
-	Solvent 50 150 500 1500 5000	177 177 168 156 - -	49 47 49 41 - -	20 18 23 19 - -	56 62 64 60 - -	18 24 20 17 - -
+	Solvent 50 150 500 1500 5000	145 146 147 134 - -	28 27 22 20 14 -	28 25 29 23 19 -	34 32 34 31 23 -	18 16 19 18 13 -
Positive Controls
-	Name	SA	SA	2NF	2NF	9AA
	Concentration (µg/plate)	5	5	10	10	20
	Mean no. colonies/plate	>1000	>1000	>1000	>1000	132
+	Name	2AA	2AA	2AA	2AA	2AA
	Concentration (µg/plate)	2	2	2	2	2
	Mean no. colonies/plate	755	92	430	444	106

9AA = 9-aminoacridine

2AA = 2-aminoanthracene

SA = sodium azide

2NF = 2 -nitrofluorene

Conclusions:

Under the conditions of this study, the test material was not mutagenic to the bacterial strains in the presence or absence of metabolic activation.

Executive summary:

The genetic toxicity of the test material was investigated in a bacterial reverse mutation assay with Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100.

The test material was formulated in DMSO and tested on the bacteria at dose levels of: 5000, 1500, 500, 150 and 50 μg/plate.

The dose levels were selected following a preliminary dose range-finding test. DMSO was used as the solvent control and appropriate positive controls were included to show the suitability of the test system. The test material was examined in the presence and absence of metabolic activation in the form of S-9 mix.

Under the condition of the study the test material was toxic towards all of the tester strains. The test material did not induce significant number of revertant colonies with any of the five strains either in the presence or absence of S-9 mix. It was concluded that no evidence of mutagenic potential of the test material was obtained in the bacterial test system at the concentrations used. However, toxicity was observed at 1500 μg/plate towards all strains without activation. In assays with activation, the toxicity was observed at 1500 µg/plate towards TA 100 and at 5000 µg/plate towards all strains.

The test material was therefore considered not mutagenic to the bacterial strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 in the presence or absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The genetic toxicity of the test material was investigated in a bacterial reverse mutation assay with Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

The test material was formulated in DMSO and tested on the bacteria at dose levels of: 5000, 1500, 500, 150 and 50 μg/plate.

The dose levels were selected following a preliminary dose range-finding test. DMSO was used as the solvent control and appropriate positive controls were included to show the suitability of the test system. The test material was examined in the presence and absence of metabolic activation in the form of S-9 mix.

Under the condition of the study the test material was toxic towards all of the tester strains. The test material did not induce significant number of revertant colonies with any of the five strains either in the presence or absence of S-9 mix. It was concluded that no evidence of mutagenic potential of the test material was obtained in the bacterial test system at the concentrations used. However, toxicity was observed at 1500 μg/plate towards all strains without activation. In assays with activation, the toxicity was observed at 1500 µg/plate towards TA 100 and at 5000 µg/plate towards all strains.

The test material was therefore considered not mutagenic to the bacterial strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 in the presence or absence of metabolic activation.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to genetic toxicity.