[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17/07/2019 to 12/08/2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

Version / remarks:

June 2012

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Test guideline 4

Qualifier:

according to guideline

Guideline:

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

Test guideline 5

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(4E)-2,4-dimethyl-5-(4-methylphenyl)pent-4-enal

Cas Number:

1226911-73-4

Molecular formula:

C14H18O

IUPAC Name:

(4E)-2,4-dimethyl-5-(4-methylphenyl)pent-4-enal

Test material form:

liquid

Details on test material:

- Physical state: Liquid
- Storage condition of test material: approximately 4ºC, in the dark and under nitrogen
- Other: colourless liquid

Method

Target gene:

Salmonella strains: histidine locus
E. coli strain: 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:

other: rfa- (increase permeability of bacterial cells to larger molecules); uvrB- (inactivation of the excision repair system) for all salmonella strains, and plasmid pKM101 R factor for TA98 and TA100 (to enhance chemical and UV induced mutagenesis)

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: uvrA- : DNA repair deficiency which enhances its sensitivity to some mutagenic compounds

Cytokinesis block (if used):

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 Moltox, Lot No. 4061 (Exp 1) and 4123 (Exp 2)
- method of preparation of S9 mix :
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.
- concentration: 10% of S9 in the S9-mix
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 Quality Control and Production Certificates are available in the study report.

Test concentrations with justification for top dose:

Experiment 1: 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 originally assayed in triplicate against each tester strain.

However, the test item induced excessive toxicity (resulting in an insufficient number of non-toxic dose concentrations) to all of the tester strains dosed in the absence of metabolic activation and, therefore, part of the experiment was repeated using an amended dose range of 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate.

Experiment 2:
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
All tester strains (absence of S9-mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 μg/plate.
All tester strains (presence of S9 mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl Sulfoxide (DMSO)
- Justification for choice of solvent/vehicle:
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.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene (2AA)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments
: 2

METHOD OF TREATMENT/ EXPOSURE:
The test item was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls. For volatile substances, the pre-incubation method may increase exposure of the bacteria to the test item in comparison to the standard plate incorporation method.

TREATMENT SCHEDULE:
- Preincubation period: 20 minutes
- Exposure duration/duration of treatment:
the plates were incubated in sealed anaerobic jars or bags at 37 ± 3 °C for between 48 and 72 hours

SELECTIVE AGENT: histidine, Tryptophan deficient agar

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction of the growth of the bacterial background lawn by microscopically assessment of the plate's thinning

METHODS FOR MEASUREMENTS OF GENOTOXICIY
: presence of revertant colonies using an automated colony counting system.

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 (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.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see tables 3 to 6

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium, other: TA98 and TA100 rfa, uvrB, pKM101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see tables 3 to 6

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium, other: TA1535 and TA1537, rfa, uvrB

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see tables 3 to 6

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: No test item precipitate was
observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Results for the negative controls (spontaneous mutation rates) were considered to be acceptable (see tables 1 and 2 below). 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.


Ames test:
- Signs of toxicity: in the experiment 1, the maximum dose level of the test item was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. However, the test item induced excessive toxicity to all of the tester strains dosed in the absence of metabolic activation (S9-mix) and, therefore, part of the experiment was repeated using an amended dose range of 0.015 to 50 μg/plate.
The test item induced a toxic response with weakened bacterial background lawns noted to all of the tester strains dosed in the absence of metabolic activation (S9-mix) at and above 15 μg/plate. In the presence of metabolic activation (S9-mix), weakened bacterial background lawns were initially noted from 150 μg/plate (all of the Salmonella strains) and 500 μg/plate (WP2uvrA).

In the experiment 2, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of metabolic activation (S9-mix) from 15 μg/plate (all of the Salmonella strains) and 50 μg/plate (WP2uvrA). In the presence of metabolic activation (S9-mix), weakened bacterial background lawns were noted from 150 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535,TA98 and WP2uvrA).

- Mean number of revertant colonies per plate and standard deviation: 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 both experiments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, as well as the number of data)
- Positive historical control data: the current positive HCD dataset is presented in the full study report
- Negative (solvent/vehicle) historical control data: The combined Vehicle and untreated control HCD dataset is presented in the full study report

Any other information on results incl. tables

Table 1: Spontaneous Mutation Rates (Concurrent Negative Controls) in Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type			Frameshift type
TA100	TA1535	WP2uvrA	TA98		TA1537
96	29	34	26		29
103          (104)	23            (23)	34            (35)	39	(34)	25            (25)
112	16	38	36		21
147	21	38	44		15
120         (135)†	15           (20)†	36           (38)†	31	(35)†	13           (13)†
138	24	40	30		10

Table 2: Spontaneous Mutation Rates (Concurrent Negative Controls) in Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type				Frameshift type
TA100	TA1535		WP2uvrA	TA98		TA1537
131	9		23	27		15
112          (120)	14	(14)	28            (26)	24	(23)	9	(11)
116	20		27	18		9

 

Table 3: Results of Experiment 1 - without metabolic activation (pre-incubation method)

Test Period		From: 01 August 2019			To: 04 August 2019
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)	130          (133) 131          4.9# 139	17             (16) 15              1.0 16	44             (33) 27              9.3 29		24           (27) 30            3.1 26	13            (14) 14             0.6 14
	0.015 µg	141 134          (141) 148           7.0	13 16             (15) 15              1.5	29 40             (33) 29              6.4		25 30           (28) 29            2.6	13 13            (16) 23             5.8
	0.05 µg	146 127          (134) 130          10.2	14 17             (16) 18              2.1	25 17             (21) 22              4.0		29 24           (25) 22            3.6	9 10            (11) 15             3.2
	0.15 µg	126 164          (153) 168          23.2	15 18             (16) 16              1.5	31 34             (32) 32              1.5		27 33           (30) 31            3.1	19 13            (15) 13             3.5
	0.5 µg	147 154          (146) 138           8.0	8 22             (13) 10              7.6	37 33             (33) 30              3.5		29 28           (28) 26            1.5	16 22            (17) 12             5.0
	1.5 µg	162          (148) 135          13.5 148	12             (14) 17              2.5 14	48             (41) 28             11.5 48		35           (32) 27            4.6 35	18            (14) 11             3.6 13
	5 µg	160          (153) 143           8.9 156	14             (15) 22              6.1 10	34             (34) 31              3.0 37		36           (32) 35            6.7 24	12            (15) 10             6.4 22
	15 µg	143 S 158 S        (158) 172 S         14.5	11 S 17 S           (14) 15 S            3.1	31 S 37 S            (33) 31 S             3.5		38 S 39 S          (35) 29 S           5.5	16 S 14 S          (15) 14 S           1.2
	50 µg	0 V 0 V            (0) 0 V           0.0	17 S 11 S           (14) 13 S            3.1	10 V 12 V           (13) 16 V            3.1		26 S 33 S          (30) 32 S           3.8	0 T 0 T            (0) 0 T            0.0

 

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation

 

Table 4: Results of Experiment 1 - with metabolic activation (pre-incubation method)

Test Period		From: 25 July 2019			To: 28 July 2019
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)	95            (91) 86           4.5# 91	15             (13) 9               3.8 16	40             (41) 44              2.3 40		26           (31) 30            6.1 38	21            (22) 21             1.2 23
	1.5 µg	92 83            (87) 85            4.7	17 16             (14) 8               4.9	46 31             (36) 30              9.0		36 40           (35) 28            6.1	24 16            (19) 16             4.6
	5 µg	96 98            (95) 90            4.2	29 17             (20) 13              8.3	55 37             (48) 52              9.6		32 37           (32) 27            5.0	26 21            (24) 25             2.6
	15 µg	90 107          (101) 106           9.5	21 24             (20) 16              4.0	56 45             (48) 42              7.4		36 29           (32) 31            3.6	25 24            (25) 25             0.6
	50 µg	91 103           (94) 88            7.9	16 27             (19) 14              7.0	44 42             (43) 44              1.2		33 34           (31) 25            4.9	22 32            (26) 24             5.3
	150 µg	0 V            (0) 0 V           0.0 0 V	18 S           (18) 23 S            4.5 14 S	41             (37) 37              3.5 34		32 S          (29) 28 S           2.6 27 S	15 S          (19) 27 S           6.9 15 S
	500 µg	0 T            (0) 0 T            0.0 0 T	0 T             (0) 0 T             0.0 0 T	37 S            (40) 47 S             5.8 37 S		0 V           (0) 0 V           0.0 0 V	0 V            (0) 0 V            0.0 0 V
	1500 µg	0 T 0 T            (0) 0 T            0.0	0 T 0 T             (0) 0 T             0.0	35 S 45 S            (37) 32 S             6.8		0 V 0 V           (0) 0 V           0.0	0 T 0 T            (0) 0 T            0.0
	5000 µg	0 T 0 T            (0) 0 T            0.0	0 T 0 T             (0) 0 T             0.0	56 S 57 S            (55) 52 S             2.6		0 T 0 T            (0) 0 T            0.0	0 T 0 T            (0) 0 T            0.0
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
		1571        (1494) 1522         93.6 1390	241           (273) 260            40.1 318	138            (148) 155             9.1 152		121         (116) 101          13.6 127	165          (172) 170            7.6 180

BP Benzo(a)pyrene
2AA 2-Aminoanthracene
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation

 

Table 5: Results of Experiment 2 - without metabolic activation (pre-incubation method)

Test Period		From: 08 August 2019			To: 11 August 2019
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)	113          (113) 122          9.5# 103	11             (11) 9               2.0 13	23             (25) 23              4.0 30		26           (25) 17            8.0 33	16            (12) 10             3.5 10
	0.015 µg	113 110          (110) 106           3.5	13 8              (12) 16              4.0	35 17             (29) 35             10.4		19 17           (20) 25            4.2	12 6              (9) 9              3.0
	0.05 µg	129 119          (121) 115           7.2	12 11             (11) 10              1.0	29 23             (31) 41              9.2		27 31           (25) 17            7.2	4 7              (7) 11             3.5
	0.15 µg	100 123          (112) 113          11.5	11 6               (9) 11              2.9	38 24             (31) 30              7.0		25 29           (25) 20            4.5	11 6              (9) 11             2.9
	0.5 µg	108 98           (103) 102           5.0	5 15              (8) 5               5.8	23 23             (24) 26              1.7		13 13           (19) 32           11.0	18 20            (16) 11             4.7
	1.5 µg	114          (111) 109           2.5 111	10             (10) 13              2.5 8	30             (29) 31              3.2 25		25           (27) 16           12.1 40	12            (12) 9              3.5 16
	5 µg	111          (111) 101           9.5 120	14             (10) 8               3.5 8	24             (20) 21              4.0 16		20           (20) 21            1.0 19	14            (12) 11             1.7 11
	15 µg	86 S 86 S          (85) 83 S           1.7	11 S 9 S            (11) 13 S            2.0	31 25             (27) 26              3.2		15 S 33 S          (23) 21 S           9.2	7 S 9 S            (8) 8 S            1.0
	50 µg	89 S 70 S          (84) 93 S          12.3	10 S 6 S             (8) 9 S             2.1	15 S 29 S            (26) 35 S            10.3		0 V 0 V           (0) 0 V           0.0	0 V 0 V            (0) 0 V            0.0
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
		376          (344) 258          75.3 398	769          (1341) 2145          716.6 1110	517            (498) 499            19.5 478		156         (181) 186          23.4 202	211          (187) 201           33.3 149

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine
4NQO 4-Nitroquinoline-1-oxide
9AA 9-Aminoacridine
S Sparse bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation

 

Table 6: Results of Experiment 2 - with metabolic activation (pre-incubation method)

Test Period		From: 08 August 2019			To: 11 August 2019
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          (104) 101          6.7# 112	14             (13) 14              2.3 10	31             (41) 48              8.7 43		26           (27) 29            1.5 27	18            (14) 11             3.6 13
	0.15 µg	115 106          (104) 92           11.6	6 14             (10) 11              4.0	36 46             (42) 45              5.5		30 25           (25) 21            4.5	12 10            (11) 10             1.2
	0.5 µg	94 112          (101) 98            9.5	14 15             (13) 11              2.1	29 40             (39) 49             10.0		27 31           (26) 19            6.1	14 7             (10) 8              3.8
	1.5 µg	80 102          (105) 133          26.6	18 12             (16) 17              3.2	41 47             (48) 57              8.1		21 23           (22) 21            1.2	15 12            (13) 12             1.7
	5 µg	108 130          (117) 113          11.5	6 8               (9) 13              3.6	47 42             (46) 48              3.2		17 28           (27) 35            9.1	17 18            (18) 19             1.0
	15 µg	116          (118) 119           1.5 118	9               (7) 7               2.5 4	50             (45) 39              5.6 46		33           (29) 27            3.5 27	9             (12) 14             2.6 13
	50 µg	98           (110) 122          12.0 111	21             (13) 11              7.6 6	32             (39) 43              5.9 41		29           (25) 19            5.1 26	17            (12) 13             5.6 6
	150 µg	64 S 67 S          (66) 66 S           1.5	11 9              (13) 18              4.7	34 42             (37) 35              4.4		35 34           (31) 24            6.1	6 S 7 S            (7) 9 S            1.5
	500 µg	0 T 0 T            (0) 0 T            0.0	11 S 6 S             (7) 5 S             3.2	40 S 32 S            (35) 33 S             4.4		0 V 0 V           (0) 0 V           0.0	0 V 0 V            (0) 0 V            0.0
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
		1306        (1366) 1397         52.3 1396	202           (217) 226            13.3 224	110            (110) 110             0.0 110		154         (159) 151          11.4 172	131          (147) 145           17.6 166

BP Benzo(a)pyrene
2AA 2-Aminoanthracene
S Sparse bacterial background lawn
T Toxic, no bacterial background lawn
V Very weak bacterial background lawn
# Standard deviation

Applicant's summary and conclusion

Conclusions:

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:

ST 08 C 19 was tested to evaluate its potential mutagenicity in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA-. The study was performed according to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing and under GLP conditions. 

Bacteria were exposed to the test item diluted in DMSO vehicle using the pre-incubation method in two independent experiments 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).
Due to excessive toxicity observed without metabolic activation in the experiment 1, the concentration selected for the first experiment were in the range of 0.015 to 50 µg/plate without metabolic activation and 1.5 to 5000 µg/plate with metabolic activation. The concentration range for Experiment 2 was amended following the results of Experiment 1 and was 0.015 to 50 µg/plate without metabolic activation and 0.15 to 500 μg/plate with metabolic activation. Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve four non toxic dose levels and to achieve the toxic limit of the test item.

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. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. Cytotoxicity was observed in all tester strains with and without metbaolic activation in both experiments (visible reduction in the growth of the bacterial background lawns).

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 both experiments.

It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.