1,3,4,6,7,8a-hexahydro-1,1,5,5-tetramethyl-2H-2,4a-methanonaphthalen-8(5H)-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genotoxic potential of the test substance was assessed according to the OECD Guidelines for Testing of Chemicals No. 471 using the Bacterial Reverse Mutation test with and without metabolic activation. Under the conditions of the test, the test substance was considered to be non-mutagenic.

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:

The study was conducted between 02 April 2014 and 13 May 2014.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimirium strains: Histidine
E. coli strains: tryptophan

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, 500, 1500 and 5000 g/plate

Vehicle / solvent:

The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Untreated

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 metabolic activation

Untreated negative controls:

yes

Remarks:

Untreated

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 metabolic activation

Details on test system and experimental conditions:

Tester strains
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB-bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).

The bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 and from the British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987. All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1369241 07/18) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

Test Item
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.

The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (30.1%) of the test item. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4E-04 microns.

Microsomal Enzyme Fraction
Lot No. PB/betaNF S9 02 March 2014 was used in this study. The S9 Microsomal fraction was prepared in house from male rats induced with Phenobarbitone/ betaNaphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed by centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

This procedure was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives and in accordance with the Harlan Laboratories Ltd, Shardlow, UK policy on animal welfare and the requirements of the United Kingdom’s Animals (Scientific Procedure) Act 1986 Amendment Regulations 2012. The conduct of the procedure may be reviewed, as part of the Harlan Laboratories Ltd, Shardlow, UK Ethical Review Process.

S9-Mix and Agar
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

S9: 5.0 mL
1.65 M KCl/0.4 M MgCl2: 1.0 mL
0.1 M Glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Top agar was prepared using 0.6% Bacto agar (lot number 3218431 04/18) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 36347 04/14 and 36631 05/14).


Test for Mutagenicity (Experiment 1 - Range-Finding Test) – Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.


Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, vehicle or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.


With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.


Incubation and Scoring
All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).


Test for Mutagenicity (Experiment 2 - Main Test) – Pre-Incubation Method
As Experiment 1 (the range-finding test) was deemed negative, Experiment 2 (main test) was performed using the pre-incubation method in the presence and absence of metabolic activation.


Dose selection
The dose range used for Experiment 2 (main test) was determined by the results of Experiment 1 (the range-finding test) and was 5 to 5000 µg/plate.
Seven test item dose levels were selected in Experiment 2 (main test) in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 °C± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.


With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 °C± 3 °C for 20 minutes (with shaking) and addition of molten amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 °C± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Occasional plates were manually counted for accuracy.

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 (Mahonet 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 (Cariello and Piegorsch, 1996)).
 
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 judgment about test item activity. Results of this type will be reported as equivocal.

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:

other: Yes, but not sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mutation Test
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.

Results for the negative controls (spontaneous mutation rates) are considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) although reductions in TA100 and TA1537 revertant colony frequency were noted at the upper dose levels in both the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate in the second mutation test. Experiment 2 (pre-incubation method) results showed that the test item induced a visible reduction in the growth of the bacterial background lawns of TA100 and TA1535 at and above 500 µg/plate in both the absence and presence of S9-mix. A reduction in TA1537 revertant colony frequency was also noted at 5000 µg/plate in the absence of S9-mix. No further toxicity was noted to any of the remaining bacterial tester strains. The sensitivity of the tester strains to the toxicity of the test item varied both between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (globular in appearance) was noted under an inverted microscope at 5000 g/plate, this observation did not prevent the scoring of revertant colonies.

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the second mutation test (pre incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 5 and 50 µg/plate (TA1535) in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.8 times the concurrent vehicle control.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1 (Range-finding Test) 

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
80		16		12		15		7
96	(95)	13	(18)	25	(22)	15	(16)	16	(11)
108		24		28		17		9

 

Experiment 2 (Main Test) 

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
155		23		31		32		16
171	(156)	29	(28)	31	(26)	31	(32)	7	(13)
143		31		17		33		15

 

Test Results: Range-Finding Test – Without Metabolic Activation

Test Period		From: 08 April 2014						To: 11 April 2014
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains								Frameshift strains
		TA100			TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	65 82 82		(76) 9.8#	12 8 8	(9) 2.3	28 23 15		(22) 6.6	13 20 15	(16) 3.6	8 16 13	(12) 4.0
	1.5 µg	64 69 68		(67) 2.6	12 21 13	(15) 4.9	23 21 12		(19) 5.9	13 19 28	(20) 7.5	17 8 7	(11) 5.5
	5 µg	84 86 83		(84) 1.5	15 15 21	* (17) 3.5	24 25 11		(20) 7.8	23 20 16	(20) 3.5	17 8 7	(11) 5.5
	15 µg	71 79 64		(71) 7.5	9 13 17	(13) 4.0	17 17 16		(17) 0.6	21 20 17	(19) 2.1	17 21 8	(15) 6.7
	50 µg	86 91 72		(83) 9.8	19 16 16	* (17) 1.7	13 17 25		(18) 6.1	15 15 17	(16) 1.2	15 17 17	(16) 1.2
	150 µg	64 75 74		(71) 6.1	13 11 15	(13) 2.0	13 12 12		(12) 0.6	17 27 15	(20) 6.4	16 4 12	(11) 6.1
	500 µg	71 76 74		(74) 2.5	12 9 12	(11) 1.7	17 24 15		(19) 4.7	25 21 21	(22) 2.3	15 7 4	(9) 5.7
	1500 µg	82 72 72		(75) 5.8	19 12 8	(13) 5.6	24 17 17		(19) 4.0	17 16 23	(19) 3.8	13 4 3	(7) 5.5
	5000 µg	47 49 55		(50) 4.2	8 11 12	(10) 2.1	19 19 24		(21) 2.9	19 12 13	(15) 3.8	5 3 3	(4) 1.2
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG			ENNG		ENNG			4NQO		9AA
		3 µg			5 µg		2 µg			0.2 µg		80 µg
		520 608 569	(566) 44.1		396 315 380	(364) 42.9	464 571 529		(521) 53.9	195 163 156	(171) 20.8	680 660 691	(677) 15.7

ENNG4NQO9AA*#

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

*: p ≤ 0.05

#: Standard deviation

Test Results: Range-Finding Test – With Metabolic Activation

Test Period		From: 08 April 2014						To: 11 April 2014
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains								Frameshift strains
		TA100			TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	79 83 75		(79) 4.0#	13 11 12	(12) 1.0	23 28 33		(28) 5.0	17 12 16	(15) 2.6	8 13 9	(10) 2.6
	1.5 µg	76 82 75		(78) 3.8	9 16 11	(12) 3.6	29 28 25		(27) 2.1	23 13 16	(17) 5.1	4 11 13	(9) 4.7
	5 µg	79 96 79		(85) 9.8	12 9 8	(10) 2.1	36 27 16		(26) 10.0	24 20 21	(22) 2.1	20 13 11	(15) 4.7
	15 µg	88 79 100		(89) 10.5	8 8 9	(8) 0.6	28 27 21		(25) 3.8	13 23 8	(15) 7.6	5 5 9	(6) 2.3
	50 µg	75 80 65		(73) 7.6	15 11 8	(11) 3.5	27 27 29		(28) 1.2	17 13 13	(14) 2.3	17 23 16	(19) 3.8
	150 µg	84 98 78		(87) 10.3	19 8 12	(13) 5.6	15 19 27		(20) 6.1	16 15 13	(15) 1.5	7 9 5	(7) 2.0
	500 µg	67 67 63		(66) 2.3	8 9 9	(9) 0.6	16 16 23		(18) 4.0	8 13 13	(11) 2.9	9 11 21	(14) 6.4
	1500 µg	57 61 63		(60) 3.1	9 9 9	(9) 0.0	11 16 17		(15) 3.2	15 13 12	(13) 1.5	11 5 9	(8) 3.1
	5000 µg	41 40 23		(35) 10.1	8 8 7	(8) 0.6	29 21 13		(21) 8.0	12 12 12	(12) 0.0	4 4 7	(5) 1.7
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA			2AA		2AA			BP		2AA
		1 µg			2 µg		10 µg			5 µg		2 µg
		1419 711 1116	(1082) 355.2		152 104 131	(129) 24.1	143 175 183		(167) 21.2	131 175 144	(150) 22.6	381 490 505	(459) 67.7

BP2AA#

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

# : Standard deviation

Conclusions:

Interpretation of results (migrated information):
negative

Isolongifolanone was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

Methods…….

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere treated with the test item using both the Ames plate incorporation and pre‑incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for the range-finding test (Experiment 1) was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of the range-finding test and was 5 to 5000 µg/plate.

 

Seven test item dose levels were selected in Experiment 2 (main test) in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

 

Results…….

The vehicle (dimethyl sulphoxide) 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 maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) although reductions in TA100 and TA1537 revertant colony frequency were noted at the upper dose levels in both the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate in the second mutation test. Experiment 2 (pre-incubation method) results showed that the test item induced a visible reduction in the growth of the bacterial background lawns of TA100 and TA1535 at and above 500 µg/plate in both the absence and presence of S9-mix. A reduction in TA1537 revertant colony frequency was also noted at 5000 µg/plate in the absence of S9-mix. No further toxicity was noted to any of the remaining bacterial tester strains. The sensitivity of the tester strains to the toxicity of the test item varied both between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (globular in appearance) was noted under an inverted microscope at 5000 mg/plate, this observation did not prevent the scoring of revertant colonies.

 

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the second mutation test (pre‑incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 5 and 50 µg/plate (TA1535) in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.8 times the concurrent vehicle control.

 

Conclusion

Isolongifolanonewas considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) although reductions in TA100 and TA1537 revertant colony frequency were noted at the upper dose levels in both the absence and presence of S9-mix. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 µg/plate in the second mutation test. Experiment 2 (pre-incubation method) results showed that the test item induced a visible reduction in the growth of the bacterial background lawns of TA100 and TA1535 at and above 500 µg/plate in both the absence and presence of S9-mix. A reduction in TA1537 revertant colony frequency was also noted at 5000 µg/plate in the absence of S9-mix. No further toxicity was noted to any of the remaining bacterial tester strains. The sensitivity of the tester strains to the toxicity of the test item varied both between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (globular in appearance) was noted under an inverted microscope at 5000 g/plate, this observation did not prevent the scoring of revertant colonies.

 

Results for the negative controls (spontaneous mutation rates) are considered to be acceptable. 

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

 

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the first mutation test (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the second mutation test (pre incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 5 and 50 µg/plate (TA1535) in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.8 times the concurrent vehicle control.

The test substance was therefore considered to be non-mutagenic under the conditions of this test.

Justification for selection of genetic toxicity endpoint
The study was conducted on the target substance according to internationally recognised guidelines.

Justification for classification or non-classification

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

To arrive at a classification, test results are considered from experiments determining mutagenic and genotoxic effects in germ and/or somatic cells of exposed animals and in in vitro tests.

The system is hazard based, classifying substances on the basis of their intrinsic ability to induce mutations in germs cells, and does not give a quantitative assessment of the risk.

To this end, the test substance has been assessed according to internationally recognized guidelines in an in vitro gene mutation study in bacteria (Ames test). 

 

In the Ames test, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested with any dose of the test item, either with or without metabolic activation.

 

Based on the negative results in vitro, the test item is considered non-mutagenic.