[No public or meaningful name is available]

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:

19-03-2020 to 28-04-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

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:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labor and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries (24 November 2000)

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

other: ICH S2(R1) guideline

Version / remarks:

June 2012 (ICH S2(R1) Federal Register

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

{2-[(2-methylundec-1-en-1-yl)oxy]ethyl}benzene

Cas Number:

2489743-82-8

Molecular formula:

C20H32O

IUPAC Name:

{2-[(2-methylundec-1-en-1-yl)oxy]ethyl}benzene

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: Dry area, protected from light, in a refrigerator (temp 2-8°C), store in a closed container with silica gel after first opening.
- Other: colourless liquid

Method

Target gene:

histidine or tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: Purchased from recognised supplier (dates within full study report) ; S9 Microsomal fraction: Lot No. 4189
- method of preparation of S9 mix: Documented in the full study report. Stored at -80ºC
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A Certificate of S9 QC and Production Certificate including Activity is presented in the full study report. Additionally, prior to testing each batch was subjected to in-house testing to assess batch-to-batch variation using promutagens: cyclophosphamide and benzo[a]pyrene (TA1535 and TA98, respectively) and was found to be acceptable. Furthermore, concurrent positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation method):
All strains (presence of S9-mix): 0, 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
All strains (absence of S9-mix): 0, 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
On the day of dosing and on the day of scoring, precipitate was seen at and above 500 μg (1.73 μmol) per plate for all five strains in the presence and absence of S9 mix.
There was no evidence of cytotoxicity as indicated by reductions in the growth of the background lawns or in the incidence of spontaneous revertant colonies.
Experiment 2 (pre-incubation method): Eight to ten test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item. The dose levels were selected based on the results of Experiment 1.
All strains (absence of S9-mix): 0, 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
All strains (presence of S9-mix): 0, 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
On the day of dosing and on the day of scoring, precipitate was seen at and above 500 μg (1.73 μmol) per plate for all five strains in the presence and absence of S9 mix.
Evidence of cytotoxicity as indicated by reductions in the growth of the background lawns or in the incidence of spontaneous revertant colonies was seen at 5000 μg (1.73 μmol) per plate for all five strains in the absence of S9 mix. There was no evidence of cytotoxicity for any of the five strains in the presence of S9 mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was miscible in DMSO at 100 mg/mL in solubility checks performed. DMSO was selected as the vehicle as a preferred solvent according to guideline(s).
- Other: Formulated concentrations would be adjusted/increased to allow for the stated water/impurity content, as applicable.. See 'Test Material Information' for further details, as applicable.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: Potassium dichromate

Remarks:

Without metabolic activation S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation S9 ; Each batch was subjected to in-house testing to assess batch-to-batch variation using promutagens: cyclophosphamide and benzo[a]pyrene (TA1535 and TA98, respectively) and was found to be acceptable.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation).
The test item was administered in solvent (DMSO) at a volume of 100 μL per plate for the plate incorporation method and a volume of 50 μL per plate when the liquid pre-incubation method was employed, within a maximum of two hours of formulation. For each test, a stock solution of test item in the solvent, DMSO, was prepared and then diluted in the same solvent to formulate the required test solutions. The solutions of test 1: A: 50 mg/mL (or 173 mmol/L) through H: 0.016 mg/mL (or 0.0555 mmol/L) ; test 2: I: 100 mg/mL (or 347 mmol/L) through P: 0.032 mg/mL (or 0.0111 mmol/L) are documented in the full study report.

DURATION
- Exposure duration:
Experiment 1 (plate incorporation method) : Briefly, frozen aliquots of the bacterial strains (-70°C or lower) were incubated overnight (8 to 10 hours) at 37°C in an orbital incubator (120 rpm) in nutrient broth. For strains TA98, TA100 and uvrA/pKM101, the nutrient broth was supplemented with ampicillin at 25 μg/mL. Aliquots (100 μL each) of bacterial culture were mixed with 100 μL of solvent, positive control or test formulation dilution and 500 μL of sodium phosphate buffer or S9 mix. Finally, 2 mL of molten 0.6% agar maintained at approximately 50°C and supplemented with low concentrations of biotin and histidine (each at 0.05 mmol/L) for the S. typhimurium strains, and tryptophan (0.018 mmol/L) for the E. coli strain, was added. The mixture was immediately poured onto minimal glucose agar plates and incubated for 3 days at 37°C. There were three plates in all solvent control, test item and positive control groups. After incubation at 37°C for 3 days, all plates were examined both macroscopically and microscopically for evidence of cytotoxicity, precipitates and any other effect relevant to the interpretation of the test. Microscopic examination was used to check the condition of the histidine/tryptophan-requiring microcolonies that make up the background lawns. Revertant (his+, trp+) colony numbers were scored using a Sorcerer Colony Counter. Manual counts may be performed, where automated counting cannot be performed: e.g. colonies spreading, colonies on the edge of the plates and artefacts on the plates, thus distorting the actual plate count.
Experiment 2 (pre-incubation method) : Aliquots (100 μL each) of bacterial culture were mixed with 50 μL of solvent, positive control or test item formulation dilution and 500 μL of sodium phosphate buffer or S9 mix and then incubated for 60 minutes at 37°C in an orbital incubator at 120 rpm. After incubation, 2 mL of molten 0.6% agar containing biotin and histidine/tryptophan (as above) was added to the mixture and poured onto minimal glucose agar plates and incubated for a further 3 days at 37°C. There were three plates in all solvent control, test item and positive control groups. After incubation at 37°C for 3 days, all plates were examined both macroscopically and microscopically for evidence of cytotoxicity, precipitates and any other effect relevant to the interpretation of the test. Microscopic examination was used to check the condition of the histidine/tryptophan-requiring microcolonies that make up the background lawns. Revertant (his+, trp+) colony numbers were scored using a Sorcerer Colony Counter. Manual counts may be performed, where automated counting cannot be performed: e.g. colonies spreading, colonies on the edge of the plates and artefacts on the plates, thus distorting the actual plate count.

SELECTION AGENT (mutation assays): histidine or tryptophan -deficient agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Other: Viability determination: Confirmation that adequate numbers of viable bacteria were exposed to the test item was obtained from viability determinations for each strain performed concurrently with the mutagenicity test. For each tester strain, a sample of the stock overnight bacterial suspension was diluted to 10-7 in phosphate buffered saline. 200 μL of the x10^-7 dilution was spread onto each of two nutrient agar plates. The plates were then inverted and incubated at room temperature for 3 days, after which, numbers of colonies were counted and recorded. A mean viable count of at least 20 colonies per plate should be present (>109 colony forming units [cfu] per mL).

Rationale for test conditions:

In accordance with the OECD TG 471 guidelines.

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
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data
5. For TA1535 or TA1537, the mean number of revertant colonies of one or more doses of the test item, with or without metabolic activation was equal to or greater than 3 times the concurrent solvent control mean value (2-fold increases outside the historical control range may be assessed as equivocal); for any other strain, the mean number of revertant colonies was equal to or greater than 2 times the concurrent solvent control mean value in the presence of one or more doses of the test item, with or without metabolic activation.
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

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:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The current Positive HCD dataset is presented in the full study report.
- Negative (solvent/vehicle) historical control data: The current background spontaneous revertant counts in concurrent untreated controls and/or or vehicle controls ; historic negative controls are presented in the full study report.

Any other information on results incl. tables

Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls

S9-Mix (+)	Dose Level Per Plate	Number ofrevertants(mean) +/- SD
		TA98		TA100		TA1535		TA1537		WP2 uvrA/pKM101
	Solvent Control (DMSO)	29 34 26	(30) 4.0#	138 137 140	(138) 1.5	11 12 10	(11) 1.0	10 11 13	(11) 1.5	204 181 225	(203) 22.0
	1.6 µg	30 38 24	(31) 7.0 1.0~	134 125 126	(128) 4.9 0.9	12 12 8	(11) 2.3 1.0	13 10 14	(12) 2.1 1.1	199 183 178	(187) 11.0 0.9
	5 µg	30 25 31	(29) 3.2 1.0	127 129 135	(130) 4.2 0.9	11 12 7	(10) 2.6 0.9	12 11 11	(11) 0.6 1.0	196 195 160	(184) 20.5 0.9
	16 µg	29 36 29	(31) 4.0 1.0	121 142 147	(137) 13.8 1.0	12 11 16	(13) 2.6 1.2	10 12 11	(11) 1.0 1.0	153 190 193	(179) 22.3 0.9
	50 µg	37 31 36	(35) 3.2 1.2	126 138 128	(131) 6.4 0.9	10 10 13	(11) 1.7 1.0	10 13 15	(13) 2.5 1.2	185 178 186	(183) 4.4 0.9
	160 µg	27 29 26	(27) 1.5 0.9	103 107 137	(116) 18.6 0.8	12 13 12	(12) 0.6 1.1	12 12 14	(13) 1.2 1.2	193 155 180	(176) 19.3 0.9
	500 µg	26 P 30 P 25 P	(27) 2.6 0.9	128 P 130 P 126 P	(128) 2.0 0.9	11 P 10 P 9 P	(10) 1.0 0.9	14 P 14 P 15 P	(14) 0.6 1.3	169 P 159 P 170 P	(166) 6.1 0.8
	1600 µg	27 P 30 P 22 P	(26) 4.0 0.9	130 P 121 P 111 P	(121) 6.1 0.9	11 P 13 P 13 P	(12) 1.7 1.1	12 P 13 P 9 P	(11) 2.1 1.0	147 P 142 P 173 P	(154) 16.6 0.8
	5000 µg	30 P 27 P 34 P	(30) 3.5 1.0	119 P 129 P 118 P	(122) 6.1 0.9	10 P 11 P 17 P	(13) 3.8 1.2	9 P 9 P 19 P	(12) 5.8 1.1	137 P 137 P 153 P	(142) 9.2 0.7
Positive controls S9-Mix (+)	Name Dose Level No. ofRevertants	2AA		2AA		2AA		2AA		2AA
		2 µg		2 µg		2 µg		2 µg		20 µg
		1919 1743 1762	(1808) 96.6# 60.3~	2913 2376 2505	(2598) 280.3 18.8	133 157 147	(146) 12.1 13.3	109 103 102	(163) 22.5 14.8	2057 2090 1864	(2004) 122.1 0.9
S9-Mix (-)	Dose Level Per Plate	Number ofrevertants(mean) +/- SD
		TA98		TA100		TA1535		TA1537		WP2 uvrA/pKM101
	Solvent Control (DMSO)	30 27 29	(29) 1.5#	122 122 127	(124) 2.9	14 11 13	(14) 1.7	10 11 12	(25) 14.0	168 164 166	(166) 2.0
	1.6 µg	26 26 32	(28) 3.5 1.0~	126 113 119	(119) 6.5 1.0	10 11 14	(14) 3.1	11 12 13	(18) 2.1	162 147 165	(158) 9.6 1.0
	5 µg	27 22 24	(24) 2.5 0.8	107 109 103	(106) 3.1 0.9	11 10 9	(15) 5.0	13 11 13	(13) 0.6	158 139 162	(153) 12.3 0.9
	16 µg	25 30 25	(27) 2.9 0.9	122 111 109	(114) 7.0 0.9	12 13 12	(15) 3.2	15 15 11	(19) 9.8	163 165 152	(160) 7.0 1.0
	50 µg	25 24 30	(26) 3.2 0.9	129 123 123	(125) 3.5 1.0	11 10 102	(16) 6.7	9 8 10	(12) 3.5	157 169 170	(165) 7.2 1.0
	160 µg	22 24 30	(25) 4.2 0.9	113 121 126	(120) 6.6 1.0	11 10 12	(11) 3.0	11 12 8	(21) 10.5	155 162 145	(154) 8.5 0.9
	500 µg	26 P 31 P 27 P	(28) 2.6 1.0	102 P 90 P 124 P	(105) 17.5 0.8	10 P 10 P 9 P	(10) 4.5	11 P 12 P 7 P	(20) 11.9	185 P 155 P 165 P	(168) 15.3 1.0
	1600 µg	31 P 30 P 30 P	(30) 0.6 1.0	119 P 126 P 114 P	(120) 6.0 1.0	12 P 11 P 12 P	(15) 3.0	11 P 10 P 10 P	(19) 8.4	163 P 139 P 142 P	(148) 13.1 0.9
	5000 µg	26 P 30 P 34 P	(30) 4.0 1.0	127 P 122 P 126 P	(125) 2.6 1.0	11 P 11 P 14 P	(12) 8.1	11 P 13 P 11 P	(16) 5.9	169 P 169 P 160 P	(166) 5.2 1.0
Positive controls S9-Mix (-)	Name Dose Level No. ofRevertants	2-nitrofluorene		Na azide		Na azide		9AA		K2Cr2O7
		1 µg		0.5 µg		0.5 µg		50 µg		25 µg
		451 411 456	(439) 24.7# 15.1~	595 634 632	(620) 22.0 5.0	420 463 395	(426) 34.4 32.8	140 93 137	(123) 26.3 11.2	1029 954 978	(987) 38.3 5.9

N/T      Not tested at this dose level

P            Precipitate

#           Standard deviation

~            fold increase over solvent control

 

Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls

S9-Mix (+)	Dose Level Per Plate	Number ofrevertants(mean) +/- SD
		TA98		TA100		TA1535		TA1537		WP2 uvrA/pKM101
	Solvent Control (DMSO)	30 30 31	(30) 0.6#	136 155 143	(145) 9.6	12 11 11	(11) 0.6	11 11 12	(11) 0.6	185 223 198	(202) 19.3
	1.6 µg	29 29 29	(29) 0.0 1.0~	134 130 152	(139) 11.7 1.0	10 8 9	(9) 1.0 0.8	12 15 10	(12) 2.5 1.1	229 210 204	(214) 13.1 1.1
	5 µg	35 31 37	(34) 3.1 1.1	156 141 154	(150) 8.1 1.0	10 9 8	(9) 1.0 0.8	13 13 11	(12) 1.2 1.1	198 190 199	(196) 4.9 1.0
	16 µg	35 30 39	(35) 4.5 1.2	147 160 162	(156) 8.1 1.1	11 11 13	(12) 1.2 1.1	14 14 14	(14) 0.0 1.3	204 191 210	(202) 9.7 1.0
	50 µg	36 29 32	(32) 3.5 1.1	140 146 129	(138) 8.6 1.0	12 13 12	(12) 0.6 1.1	10 12 9	(10) 1.5 0.9	225 203 184	(204) 20.5 1.0
	160 µg	30 24 29	(28) 3.2 0.9	119 125 149	(131) 15.9 0.9	11 10 9	(10) 1.0 0.9	12 11 13	(12) 1.0 1.1	193 190 173	(185) 10.8 0.9
	500 µg	31 P 32 P 39 P	(34) 4.4 1.1	127 P 129 P 109 P	(122) 11.0 0.8	10 P 12 P 14 P	(12) 2.0 1.1	12 P 13 P 14 P	(13) 1.0 1.2	186 P 192 P 193 P	(190) 3.8 0.9
	1600 µg	32 P 42 P 39 P	(38) 5.1 1.3	117 P 127 P 123 P	(122) 5.0 0.8	13 P 12 P 17 P	(14) 2.6 1.3	11 P 14 P 13 P	(13) 1.5 1.2	219 P 191 P 170 P	(193) 24.6 1.0
	5000 µg	33 P 36 P 32 P	(34) 2.1 1.1	113 P 109 P 131 P	(118) 11.7 0.8	9 P 12 P 12 P	(11) 1.7 1.0	13 P 9 P 9 P	(10) 2.3 0.9	180 P 211 P 185 P	(192) 16.6 1.0
Positive controls S9-Mix (+)	Name Dose Level No. ofRevertants	2AA		2AA		2AA		2AA		2AA
		2 µg		2 µg		2 µg		2 µg		20 µg
		2083 2178 2289	(2183) 103.1# 72.8~	3017 2689 2564	(2757) 234.0 19.0	155 211 193	(186) 28.6 16.9	207 201 228	(212) 14.2 19.3	1932 1917 1976	(1942) 30.7 9.6
S9-Mix (-)	Dose Level Per Plate	Number ofrevertants(mean) +/- SD
		TA98		TA100		TA1535		TA1537		WP2 uvrA/pKM101
	Solvent Control (DMSO)	31 30 26	(29) 2.6#	126 122 139	(129) 8.9	12 12 13	(12) 0.6	12 12 12	(12) 0.0	165 166 166	(166) 0.6
	1.6 µg	29 21 30	(27) 4.9 0.9~	126 116 97	(113) 14.7 0.9	12 13 11	(12) 1.0 1.0	11 12 11	(11) 0.6 0.9	158 159 156	(158) 1.5 1.0
	5 µg	32 31 29	(31) 1.5 1.1	116 97 103	(105) 9.7 0.8	11 13 14	(13) 1.5 1.1	12 10 13	(12) 1.5 1.0	166 154 162	(161) 6.1 1.0
	16 µg	24 23 28	(25) 2.6 0.9	116 121 116	(118) 2.9 0.9	14 13 13	(13) 0.6 1.1	11 14 10	(12) 2.1 1.0	152 144 186	(161) 22.3 1.0
	50 µg	25 27 29	(27) 2.0 0.9	110 100 102	(104) 5.3 0.8	12 11 11	(11) 0.6 0.9	10 11 8	(10) 1.5 0.8	130 124 136	(130) 6.0 0.8
	160 µg	30 24 33	(29) 4.6 1.0	112 109 102	(108) 5.1 0.8	10 12 11	(11) 1.0 0.9	11 12 11	(11) 0.6 0.9	136 143 136	(138) 4.0 0.8
	500 µg	34 P 28 P 29 P	(30) 3.2 1.0	117 P 127 P 100 P	(115) 13.7 0.9	12 P 11 P 11 P	(11) 0.6 0.9	10 P 10 P 11 P	(10) 0.6 0.8	139 P 136 P 137 P	(137) 1.5 0.8
	1600 µg	31 P 30 P 26 P	(29) 2.6 1.0	122 P 112 P 104 P	(113) 9.0 0.9	12 P 12 P 12 P	(12) 0.0 1.0	10 P 9 P 17 P	(12) 4.4 1.0	135 P 130 P 137 P	(134) 3.6 0.8
	5000 µg	0 nP 0 nP 0 nP	(0.0) 0.0 0.0	0 nP 0 nP 0 nP	(0.0) 0.0 0.0	0 nP 0 nP 0 nP	(0.0) 0.0 0.0	0 nP 0 nP 0 nP	(0.0) 0.0 0.0	107 rP 106 rP 113 rP	(109) 3.8 0.7
Positive controls S9-Mix (-)	Name Dose Level No. ofRevertants	2-nitrofluorene		Na azide		Na azide		9AA		K2Cr2O7
		1 µg		0.5 µg		0.5 µg		50 µg		25 µg
		1511 1533 1321	(1455) 116.6# 50.2~	610 486 562	(553) 62.5 4.3	412 478 444	(445) 33.0 37.1	223 335 429	(332) 98.0 27.7	1240 1404 1437	(1360) 105.5 8.2

N/T      Not tested at this dose level

R            reduced survive

P            Precipitate

#           Standard deviation

~            fold increase over solvent control

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative
Under the conditions of this study, the test item was considered to be non-mutagenic in the presence and absence of S9 activation.

Executive summary:

The study was performed to the requirements of OECD Guideline 471 under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2 uvrA/pKM101 as indicator organisms in both the presence and absence of S-9 mix. The test strains were treated with the test item using the Ames (i) plate incorporation method and then (ii) pre incubation method 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). A preliminary solubility assessment was conducted. As solubility was satisfactory in dimethyl sulphoxide, it was used throughout this study as the solvent for the test item. The dose range for Experiment 1 (plate incorporation method) was predetermined and was 1.6 to 5000 µg/plate (or (0.00555 to 17.3 μmol) per plate for all five indicator strains in both the presence and absence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix). The experiment was repeated in Experiment 2 (preincubation method) on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Up to eight test item dose levels were selected in Experiment 2 in order to achieve a minimum of four non-toxic dose levels and the toxic limit of the test item. The dose range employed was consistent with the Experiment 1 and ranged between 1.6 and 5000 µg/plate, in the presence or absence of S9-mix. The test item showed evidence of cytotoxicity. The minimum dose level at which cytotoxicity was seen was 5000 μg (17.3 μmol) per plate. The maximum dose level scored for revertant colonies was 5000 μg (17.3 μmol) per plate. The minimum dose at which precipitate was seen was 500 μg (1.73 μmol) per plate. The maximum dose level of the test item in both experiments were selected as the maximum recommended dose level of 5000 μg/plate or the toxic limit of the test item depending on the strain type and presence of S9-mix. The vehicle (DMSO) 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 both experiments there were no significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix. It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2 uvrA/pKM101 in the presence and absence of S-9 mix.