Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one

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:

The study was conducted between 12 December 2018 and 24 December 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one

EC Number:

950-718-1

Molecular formula:

C14H22O

IUPAC Name:

Reaction mass of (E)-1-(3,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one and (E)-1-(4,6-dimethylcyclohex-3-en-1-yl)-2-methylpent-1-en-3-one

Test material form:

liquid

Specific details on test material used for the study:

Identification: PG-RAW-90-032
Description: Clear colourless liquid
Storage Conditions: Approximately 4 °C, in the dark

Method

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

10% liver S9 in standard co-factors

Test concentrations with justification for top dose:

Experiment 1 – Plate Incorporation Method
The maximum concentration was 5000 µg/plate (the OECD TG 471 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.

Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 µg/plate.
Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of 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 and acetone at 100 mg/mL in solubility checks performed in house.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

untreated plates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

In the absence of S9-mix

Controls 2

Untreated negative controls:

yes

Remarks:

untreated plates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene (2AA)

Remarks:

In the presence of S9-mix

Details on test system and experimental conditions:

Microsomal Enzyme Fraction
The S9 Microsomal fractions (CD Sprague-Dawley) were pre-prepared using standardized
in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 28 October 2018 was used in this study.

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.

Media
Top agar was prepared using 0.6% Bacto agar (lot number 7193746 04/2022) 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 49292 01/2019 and 49449 01/2019).

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration and acetone at 100 mg/mL in solubility checks performed in house. Dimethyl sulphoxide was selected as the vehicle.
The test item was accurately weighed and, on the day of each experiment, approximate
half-log dilutions prepared in pre-dried dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at 40 °C. No correction for purity was required.
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.

Test for Mutagenicity: Experiment 1 – Plate Incorporation Method
Dose selection
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. 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 ± 3 °C for between 48 and 72 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 – Pre-Incubation Method
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace 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 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 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).

Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Typical ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
These values will also be confirmed against current in-house historical control profiles to further validate acceptability. Although the number of spontaneous revertants can be expected to fall within the ranges, they may occasionally fall outside these. Combined historical negative and solvent control ranges for 2016 and 2017 are presented.
All tester strain cultures should be in the range of 0.9 to 9 x 10^9 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation (S9-mix). The historical ranges of the positive control reference items for 2016 and 2017 are presented.
There should be a minimum of four non-toxic test item dose levels.
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. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

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.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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 presented and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1 (plate incorporation)
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate.
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no 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 (S9-mix).

Experiment 2 (pre-incubation)
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate).
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no 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 (S9-mix).

Any other information on results incl. tables

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
121		10		14		21		5
142	(129)	10	(10)	19	(18)	24	(21)	15	(12)
123		9		20		18		17

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
106		14		26		27		10
101	(99)	10	(11)	34	(26)	31	(26)	7	(9)
90		8		18		19		10

 

 Test Results: Experiment 1 – Without Metabolic Activation(Plate Incorporation)

Test Period		From: 14 December 2018					To: 17 December 2018
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)	106 102 121	(110) 10.0#	12 9 17	(13) 4.0	16 28 24		(23) 6.1	17 18 20	(18) 1.5	16 13 12	(14) 2.1
	1.5 µg	119 121 103	(114) 9.9	17 14 13	(15) 2.1	27 23 23		(24) 2.3	19 12 28	(20) 8.0	10 14 23	(16) 6.7
	5 µg	107 118 111	(112) 5.6	14 15 14	(14) 0.6	24 17 22		(21) 3.6	13 26 26	(22) 7.5	16 15 16	(16) 0.6
	15 µg	98 125 124	(116) 15.3	24 12 12	(16) 6.9	26 23 33		(27) 5.1	24 18 26	(23) 4.2	15 10 9	(11) 3.2
	50 µg	114 121 122	(119) 4.4	17 20 15	(17) 2.5	22 28 30		(27) 4.2	16 17 20	(18) 2.1	5 15 14	(11) 5.5
	150 µg	120 105 111	(112) 7.5	12 11 15	(13) 2.1	14 26 25		(22) 6.7	18 12 21	(17) 4.6	20 11 6	(12) 7.1
	500 µg	120 115 105	(113) 7.6	11 9 11	(10) 1.2	18 25 21		(21) 3.5	21 31 11	(21) 10.0	10 13 19	(14) 4.6
	1500 µg	104 97 109	(103) 6.0	16 14 14	(15) 1.2	21 26 33		(27) 6.0	21 13 21	(18) 4.6	6 13 6	(8) 4.0
	5000 µg	120 107 106	(111) 7.8	9 9 14	(11) 2.9	21 21 23		(22) 1.2	22 17 15	(18) 3.6	6 6 12	(8) 3.5
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
		644 640 675	(653) 19.2	778 859 658	(765) 101.1	792 935 1077		(935) 142.5	188 187 198	(191) 6.1	326 505 481	(437) 97.2

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

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

#               Standard deviation

Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 14 December 2018					To: 17 December 2018
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)	123 136 117	(125) 9.7#	8 14 14	(12) 3.5	33 35 29		(32) 3.1	20 23 20	(21) 1.7	17 20 17	(18) 1.7
	1.5 µg	116 113 121	(117) 4.0	13 13 17	(14) 2.3	34 44 37		(38) 5.1	18 32 24	(25) 7.0	25 8 11	(15) 9.1
	5 µg	136 116 118	(123) 11.0	7 9 11	(9) 2.0	37 26 27		(30) 6.1	25 35 18	(26) 8.5	20 13 14	(16) 3.8
	15 µg	118 129 132	(126) 7.4	14 12 12	(13) 1.2	33 33 26		(31) 4.0	26 29 22	(26) 3.5	26 14 19	(20) 6.0
	50 µg	127 101 126	(118) 14.7	12 12 12	(12) 0.0	28 30 29		(29) 1.0	23 18 30	(24) 6.0	14 16 10	(13) 3.1
	150 µg	96 122 109	(109) 13.0	10 10 10	(10) 0.0	33 29 27		(30) 3.1	31 24 15	(23) 8.0	18 13 7	(13) 5.5
	500 µg	112 119 113	(115) 3.8	12 8 8	(9) 2.3	20 31 35		(29) 7.8	17 25 27	(23) 5.3	16 12 12	(13) 2.3
	1500 µg	106 132 118	(119) 13.0	11 10 13	(11) 1.5	22 27 32		(27) 5.0	25 21 16	(21) 4.5	16 8 6	(10) 5.3
	5000 µg	83 102 79	(88) 12.3	12 8 14	(11) 3.1	31 25 26		(27) 3.2	19 27 29	(25) 5.3	13 12 12	(12) 0.6
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
		1878 2013 2031	(1974) 83.6	149 93 100	(114) 30.5	138 176 125		(146) 26.5	116 146 172	(145) 28.0	224 232 266	(241) 22.3

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

#            Standard deviation

Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 21 December 2018					To: 24 December 2018
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)	110 109 105	(108) 2.6#	21 26 11	(19) 7.6	17 21 20		(19) 2.1	20 8 15	(14) 6.0	11 18 15	(15) 3.5
	15 µg	89 84 86	(86) 2.5	14 7 15	(12) 4.4	16 16 19		(17) 1.7	23 17 9	(16) 7.0	8 11 11	(10) 1.7
	50 µg	87 78 88	(84) 5.5	19 16 11	(15) 4.0	13 14 17		(15) 2.1	8 13 22	(14) 7.1	6 16 11	(11) 5.0
	150 µg	78 66 102	(82) 18.3	17 18 14	(16) 2.1	11 18 12		(14) 3.8	20 12 9	(14) 5.7	12 14 10	(12) 2.0
	500 µg	63 60 87	(70) 14.8	17 11 11	(13) 3.5	15 12 17		(15) 2.5	9 12 19	(13) 5.1	9 11 10	(10) 1.0
	1500 µg	65 65 59	(63) 3.5	19 22 26	(22) 3.5	14 11 8		(11) 3.0	13 15 19	(16) 3.1	9 6 8	(8) 1.5
	5000 µg	62 67 58	(62) 4.5	12 14 13	(13) 1.0	15 7 15		(12) 4.6	14 16 7	(12) 4.7	6 6 4	(5) 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
		1081 1091 944	(1039) 82.1	1864 1171 1735	(1590) 368.6	1164 960 1114		(1079) 106.3	244 261 238	(248) 11.9	497 280 263	(347) 130.5

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

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

#               Standard deviation

Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 21 December 2018					To: 24 December 2018
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)	100 89 93	(94) 5.6#	12 8 8	(9) 2.3	25 19 31		(25) 6.0	13 18 32	(21) 9.8	9 7 6	(7) 1.5
	15 µg	87 85 102	(91) 9.3	14 9 7	(10) 3.6	21 15 21		(19) 3.5	29 25 26	(27) 2.1	10 7 8	(8) 1.5
	50 µg	91 86 81	(86) 5.0	7 7 13	(9) 3.5	15 26 20		(20) 5.5	18 22 20	(20) 2.0	7 3 6	(5) 2.1
	150 µg	99 89 87	(92) 6.4	8 13 8	(10) 2.9	19 17 20		(19) 1.5	25 18 29	(24) 5.6	8 3 10	(7) 3.6
	500 µg	85 92 75	(84) 8.5	8 13 10	(10) 2.5	17 30 23		(23) 6.5	15 35 25	(25) 10.0	7 5 5	(6) 1.2
	1500 µg	86 81 79	(82) 3.6	9 7 7	(8) 1.2	15 27 13		(18) 7.6	21 28 27	(25) 3.8	4 8 2	(5) 3.1
	5000 µg	80 70 76	(75) 5.0	8 14 13	(12) 3.2	24 12 21		(19) 6.2	26 22 30	(26) 4.0	7 2 7	(5) 2.9
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
		844 869 881	(865) 18.9	169 138 117	(141) 26.2	175 207 187		(190) 16.2	121 105 95	(107) 13.1	197 245 265	(236) 34.9

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

#            Standard deviation

Applicant's summary and conclusion

Conclusions:

PG-RAW-90-032 is not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay performed according to OECD 471 (1997).

Executive summary:

The mutagenic activity of PG-RAW-90-032 was evaluated in accordance with OECD 471 (1997) and according to GLP principles. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were 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 Experiment 1 (plate incorporation) was based on OECD TG 471 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 Experiment 1 and was 15 to 5000 µg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the potential toxicity of the test item following the change in test methodology.

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 and 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 OECD TG 471 recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (pre-incubation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix). 

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2.

There were no 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 (S9-mix) in Experiment 1 (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 (S9-mix) in Experiment 2 (pre‑incubation method). 

PG-RAW-90-032was considered to be non-mutagenic under the conditions of this test.