Bis-O-(benzylidene)-D-glucitol

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:

Experimental start date: 8 March 2017. Experimental completion date: 23 March 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

1

Reference substance name:

Bis-O-(benzylidene)-D-glucitol

EC Number:

251-136-4

EC Name:

Bis-O-(benzylidene)-D-glucitol

Cas Number:

32647-67-9

Molecular formula:

C20H22O6

IUPAC Name:

1-{2,6-diphenyl-hexahydro-[1,3]dioxino[5,4-d][1,3]dioxin-4-yl}ethane-1,2-diol

Test material form:

solid: particulate/powder

Details on test material:

Identification: Geniset D
CAS number: 32647-67-9
Appearance/physical state: white powder
Batch: 5434
Purity: 99.4%
Expiry date: 31 October 2018
Storage conditions: room temperature in the dark

Specific details on test material used for the study:

Identification: Geniset D
CAS No: 32647-67-9
Physical state/Appearance: White powder
Batch: 5434
Purity: 99.4%
Expiry Date: 31 October 2018
Storage Conditions: Room temperature in the dark

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ β-naphthoflavone induced rat liver S9 were used as the metabolic activation system

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation method): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate

Experiment 2 (pre-incubation method): 15, 50, 150, 500, 150, 5000 μg/plate.
The dose range used for Experiment 2 was determined by the results of Experiment 1

Vehicle / solvent:

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

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

(untreated controls to assess the spontaneous revertant colony rate)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

2 μg/plate for WP2uvrA. 3 μg/plate for TA100. 5 μg/plate for TA1535

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

(untreated controls to assess the spontaneous revertant colony rate)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

80 μg/plate for TA1537

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Controls 3

Untreated negative controls:

yes

Remarks:

(untreated controls to assess the spontaneous revertant colony rate)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 μg/plate for TA98

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation

Controls 4

Untreated negative controls:

yes

Remarks:

(untreated controls to assess the spontaneous revertant colony rate)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

1 μg/plate for TA100. 2 μg/plate for TA1535 and TA1537. 10 μg/plate for WP2uvrA

Positive control substance:

other: 2-Aminoanthracene

Remarks:

With metabolic activation

Controls 5

Untreated negative controls:

yes

Remarks:

(untreated controls to assess the spontaneous revertant colony rate)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

5 μg/plate for TA98

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Test Item Preparation and Analysis:
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. No correction for purity was required. 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-4 microns.
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.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method:
Dose selection:
The test item was tested using the following method. 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, solvent 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 above 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 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). Manual counts were performed at 5000 μg/plate because of test item precipitation. Several further manual counts were also required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method:
As the result of Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.

Dose selection:
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 µg/plate.
Six test item concentrations were selected in Experiment 2 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 from plate incorporation to pre-incubation.

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, 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 above 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 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). Manual counts were performed at 5000 μg/plate because of test item precipitation.

Rationale for test conditions:

The purpose of the study was to evaluate Geniset D for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria.

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 (Mahon et 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.

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:

other: S. typhimurium TA 1535, TA 1537, TA 98 and 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 2

Key result

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.

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 lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. 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), in the second mutation test (pre-incubation method). A test item precipitate (particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

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 toxicologically 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). Small, statistically significant increases in TA1537 revertant colony frequency were observed in the presence of S9-mix at 15, 150 and 1500 μg/plate in the second mutation test. 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 colony 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 1.6 times the concurrent vehicle control.

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.

Any other information on results incl. tables

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in the Tables below for Experiments 1 and 2.

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

Test Period		From: 13 March 2017					To: 16 March 2017
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)	120 132 98	(117) 17.2#	29 23 29	(27) 3.5	20 29 15		(21) 7.1	21 12 21	(18) 5.2	13 18 10	(14) 4.0
	1.5 µg	124 104 134	(121) 15.3	21 19 18	(19) 1.5	23 15 23		(20) 4.6	22 14 14	(17) 4.6	21 5 16	(14) 8.2
	5 µg	110 99 113	(107) 7.4	25 23 24	(24) 1.0	19 14 16		(16) 2.5	16 21 16	(18) 2.9	11 15 13	(13) 2.0
	15 µg	104 99 107	(103) 4.0	21 21 29	(24) 4.6	16 16 17		(16) 0.6	12 23 19	(18) 5.6	9 10 11	(10) 1.0
	50 µg	107 101 116	(108) 7.5	27 30 25	(27) 2.5	22 15 22		(20) 4.0	15 14 22	(17) 4.4	18 11 15	(15) 3.5
	150 µg	113 113 84	(103) 16.7	27 26 26	(26) 0.6	19 17 21		(19) 2.0	20 17 22	(20) 2.5	10 4 13	(9) 4.6
	500 µg	92 89 94	(92) 2.5	27 16 29	(24) 7.0	21 22 28		(24) 3.8	24 26 11	(20) 8.1	16 15 13	(15) 1.5
	1500 µg	87 P 94 P 93 P	(91) 3.8	27 P 28 P 24 P	(26) 2.1	19 P 21 P 20 P		(20) 1.0	19 P 11 P 15 P	(15) 4.0	21 P 12 P 9 P	(14) 6.2
	5000 µg	94 P 73 P 62 P	(76) 16.3	15 P 13 P 15 P	(14) 1.2	12 P 14 P 23 P		(16) 5.9	16 P 13 P 13 P	(14) 1.7	8 P 8 P 12 P	(9) 2.3
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
		527 515 579	(540) 34.0	597 477 439	(504) 82.5	851 726 820		(799) 65.1	156 156 177	(163) 12.1	245 317 437	(333) 97.0

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

Test Period		From: 13 March 2017					To: 16 March 2017
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)	118 98 113	(110) 10.4#	16 25 15	(19) 5.5	32 32 30		(31) 1.2	26 29 19	(25) 5.1	13 15 10	(13) 2.5
	1.5 µg	105 85 112	(101) 14.0	17 25 20	(21) 4.0	32 26 30		(29) 3.1	12 30 22	(21) 9.0	7 16 14	(12) 4.7
	5 µg	117 100 113	(110) 8.9	16 21 18	(18) 2.5	32 21 26		(26) 5.5	39 19 24	(27) 10.4	16 8 10	(11) 4.2
	15 µg	101 118 110	(110) 8.5	29 24 26	(26) 2.5	29 28 16		(24) 7.2	26 23 29	(26) 3.0	12 8 8	(9) 2.3
	50 µg	120 118 87	(108) 18.5	28 22 19	(23) 4.6	15 25 30		(23) 7.6	27 20 31	(26) 5.6	5 8 11	(8) 3.0
	150 µg	104 123 105	(111) 10.7	25 18 25	(23) 4.0	26 20 27		(24) 3.8	23 28 32	(28) 4.5	16 10 18	(15) 4.2
	500 µg	108 95 97	(100) 7.0	24 23 16	(21) 4.4	32 25 23		(27) 4.7	21 17 23	(20) 3.1	6 14 16	(12) 5.3
	1500 µg	117 P 108 P 113 P	(113) 4.5	16 P 18 P 23 P	(19) 3.6	27 P 22 P 20 P		(23) 3.6	24 P 30 P 18 P	(24) 6.0	16 P 10 P 12 P	(13) 3.1
	5000 µg	103 P 91 P 80 P	(91) 11.5	15 P 22 P 17 P	(18) 3.6	19 P 17 P 18 P		(18) 1.0	20 P 19 P 24 P	(21) 2.6	10 P 9 P 6 P	(8) 2.1
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
		2522 1238 1297	(1686) 724.9	188 174 327	(230) 84.6	387 360 384		(377) 14.8	209 260 296	(255) 43.7	349 338 381	(356) 22.3

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

Test Period		From: 20 March 2017					To: 23 March 2017
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)	98 114 79	(97) 17.5#	9 16 16	(14) 4.0	33 28 38		(33) 5.0	20 17 17	(18) 1.7	16 16 11	(14) 2.9
	15 µg	87 87 80	(85) 4.0	11 9 14	(11) 2.5	24 28 23		(25) 2.6	15 16 17	(16) 1.0	10 8 12	(10) 2.0
	50 µg	91 81 105	(92) 12.1	9 16 11	(12) 3.6	23 27 39		(30) 8.3	19 21 18	(19) 1.5	7 15 15	(12) 4.6
	150 µg	97 90 97	(95) 4.0	19 14 14	(16) 2.9	31 20 24		(25) 5.6	14 14 14	(14) 0.0	23 17 4	(15) 9.7
	500 µg	103 102 78	(94) 14.2	16 14 16	(15) 1.2	35 41 21		(32) 10.3	16 23 20	(20) 3.5	16 17 5	(13) 6.7
	1500 µg	88 P 103 P 81 P	(91) 11.2	16 P 13 P 13 P	(14) 1.7	19 P 19 P 27 P		(22) 4.6	26 P 14 P 17 P	(19) 6.2	16 P 4 P 6 P	(9) 6.4
	5000 µg	65 P 74 P 66 P	(68) 4.9	14 P 14 P 12 P	(13) 1.2	32 P 29 P 16 P		(26) 8.5	9 P 16 P 13 P	(13) 3.5	8 P 14 P 7 P	(10) 3.8
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
		762 692 788	(747) 49.7	264 244 226	(245) 19.0	934 939 864		(912) 41.9	150 185 213	(183) 31.6	259 349 422	(343) 81.6

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

Test Period		From: 20 March 2017					To: 23 March 2017
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)	119 97 79	(98) 20.0#	7 16 7	(10) 5.2	37 36 34		(36) 1.5	31 24 23	(26) 4.4	9 10 6	(8) 2.1
	15 µg	85 103 85	(91) 10.4	11 8 9	(9) 1.5	30 31 27		(29) 2.1	25 23 28	(25) 2.5	14 15 10	** (13) 2.6
	50 µg	85 98 108	(97) 11.5	9 10 7	(9) 1.5	35 35 30		(33) 2.9	29 27 27	(28) 1.2	9 9 9	(9) 0.0
	150 µg	96 84 92	(91) 6.1	8 14 12	(11) 3.1	31 37 36		(35) 3.2	28 27 28	(28) 0.6	13 14 14	** (14) 0.6
	500 µg	110 82 104	(99) 14.7	12 9 9	(10) 1.7	29 28 24		(27) 2.6	26 22 29	(26) 3.5	8 7 10	(8) 1.5
	1500 µg	83 P 78 P 80 P	(80) 2.5	8 P 8 P 9 P	(8) 0.6	35 P 34 P 23 P		(31) 6.7	19 P 27 P 23 P	(23) 4.0	14 P 13 P 10 P	* (12) 2.1
	5000 µg	65 P 64 P 75 P	(68) 6.1	11 P 11 P 8 P	(10) 1.7	35 P 34 P 33 P		(34) 1.0	17 P 22 P 21 P	(20) 2.6	11 P 11 P 10 P	(11) 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
		1675 1530 1716	(1640) 97.7	259 245 214	(239) 23.0	218 259 242		(240) 20.6	152 179 178	(170) 15.3	309 380 395	(361) 45.9

Applicant's summary and conclusion

Conclusions:

Geniset D 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 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 was predetermined and was 1.5 to 5000 μg/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 were selected in Experiment 2 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 lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. 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), in the second mutation test (pre-incubation method). A test item precipitate (particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

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 toxicologically 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). Small, statistically significant increases in TA1537 revertant colony frequency were observed in the presence of S9-mix at 15, 150 and 1500 μg/plate in the second mutation test. 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 colony 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 1.6 times the concurrent vehicle control.

Conclusion

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