Bis-O-(benzylidene)-D-glucitol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The following in-vitro studies have been conducted to assess the genetic toxicity of the substance (EC 251-136-4)

- In-vitro gene mutation in bacteria (Ames)

- In-vitro chromosome aberration in mammalian cells

- In-vitro gene mutation in mammalian cells (Mouse lymphoma assay) - conducted on structurally similar analogue substances.

In-vitro gene mutation in bacteria (Ames):

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.

- In-vitro chromosome aberration in mammalian cells:

This study was conducted to assess the potential chromosomal mutagenicity of a test material, on the metaphase chromosomes of the Chinese hamster lung (CHL) cell line.

Duplicate cultures of Chinese hamster lung (CHL) cells, treated with test material were evaluated for chromosome aberrations at a minimum of four dose levels together with vehicle and positive controls. Four treatment conditions were used: 4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 10% in standard co-factors with cell harvest at 20 hours and 30 hours after exposure.

Exposure in the absence of metabolic activation was continuous with cell harvest at 20 and 30 hours after culture initiation.

The dose range for metaphase analysis was selected from a series of at least four dose levels chosen on the basis of the results of a preliminary toxicity test. The test material was evaluated at doubling dose levels between 55.94 and 3580 µg/ml depending on the particular treatment regime used. The continuous exposures were more toxic than the pulse exposures.

All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line.

Both of the positive control treatments gave highly significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material demonstrated no statistically significant, dose-related increases in the frequency of cells with aberrations either with or without metabolic activation. The test material was shown to be toxic in CHL cells in vitro in the without metabolic treatment groups only.

Gell All D was shown to be non-clastosenic to CHL cells in vitro.

- In-vitro gene mutation in mammalian cells (Mouse lymphoma assay):

Source Substance; Gel All DX (EC-413 -110 -2):

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item (EC 413-110-2) on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. T

Results

The maximum dose level used in the Mutagenicity Test was limited by the formulation of the test item and the maximum achievable dose level was 1036.25 µg/ml. Precipitate of the test item was observed at and above 64.77 µg/ml in the Mutagenicity Test.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus.

The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Source Substance; Gel All MD (EC 402-950-5):

L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material (EC 402-950-5) at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls. The entire experiment was repeated to confirm the result of the first experiment. Four-hour exposures were used both with and without activation in Experiment 1. In Experiment 2, the exposure time without activation was increased to 24 hours.

The dose range of test material, plated for expression of mutant colonies, was selected based on the results and observations of a preliminary toxicity test and was 3.91 to 500 µg/ml in the absence of metabolic activation, and 3.91 to 62.5 µg/ml in the presence of metabolic activation for the first experiment. For the second experiment the dose range was 3.91 to 500 µg/ml both with and without activation.

Results: The maximum dose level used was limited by the presence and nature of the precipitate observed. In the preliminary toxicity test a precipitate of test material was observed at and above 15.63 µg/ml, increasing with intensity with increase in dose concentration until it aggregated at 1000 µg/ml. It was considered that the aggregation of the precipitate effectively reduced exposure to the cells and, therefore, this dose level was not selected for the mutagenicity tests. The vehicle (solvent) controls had acceptable mutant frequency values that that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce a statistically significant or dose-related increase in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion: The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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

Target gene:

histidine or tryptophan locus

Species / strain / cell type:

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

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.

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

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

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

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

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.

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

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 19 July 1996. Experimental completion date 18 October 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro chromosome aberration

Specific details on test material used for the study:

Sponsor's identification: Gell All D
Purity: 99.5 %
Date received: 01 July 1996
Description: White powder
Storage conditions: ambient <25 °C and 50% relative humidity

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Cell Line:
The Chinese hamster lung (CHL) cell line, isolated by Koyama et a/ (1970) and cloned by Ishidate and Sofuni (1985), was used. The CHL cell line has an average generation time of approximately 11 hours when grown in conditions described as follows:

Cell Culture:
Cells were grown in Eagles Minimal Essential medium with Earle's salts (MEM), supplemented with 10% foetal bovine serum and antibiotics, at 37°C with 5 % C02 in air.

Cytokinesis block (if used):

Mitosis was arrested by addition of demecolcine (0.1 /yg/ml) approximately two
hours before the required harvest time.

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate

Test concentrations with justification for top dose:

Preliminary Toxicity Study: 0, 13.98 ,27.97, 55.94, 111.88, 223.75, 447.5, 895, 1790, 3580 µg/ml.

Main test:
Without S9: 55.94, 111.88, 223.75, 335.63, 447.5, 895, 1790 µg/ml.
With S9: 447.5 895, 1790, 3580 µg/ml.

The molecular weight of the test material was 358 and therefore the maximum
dose level was 3580 µg/ml which was calculated to be equivalent to 10 mM.

Vehicle / solvent:

Dimethyl sulfoxide (DMSO) was used as the vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

0.05 µg/ml

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

10 µg/ml

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Preparation of Test and Control Materials:
The test material was accurately weighed and prepared in dimethyl sulphoxide and appropriate dilutions made. Analysis for concentration, homogeneity and stability of the test material preparation was not a requirement of the test guidelines and therefore not performed.

Vehicle and positive controls were used in parallel with the test material. Solvent treatment groups were used as the vehicle controls.

Preliminary Toxicity Study:
A preliminary cytotoxicity test was performed on cell cultures using a 4-hour exposure time with metabolic activation followed by a 16-hour culture period
in treatment free media. Treatment without metabolic activation had a continuous treatment with cell harvest at 20 hours. Growth inhibition was estimated by counting the number of cells at the end of the culture period on an electronic cell counter (Coulter) and expressing the cell count as a percentage of the concurrent vehicle control value.

Microsomal Enzyme Fraction:
Lot No. Aro. S9/15/07/96 SPL, was prepared in house on 15/07/96. It was prepared from the livers of male Sprague-Dawley rats weighing ~ 200 g.
These had received a single ip injection of Aroclor 1254 at 500 mg/kg, five days before S9 preparation. The S9 was stored at -196°C in a liquid nitrogen freezer.

Culture Conditions:
Cultures were established approximately 24 hours prior to treatment, 0.3 x 10^6 cells and 0.2 x 10^6 cells were seeded per flasks for 20 and 30 hour cultures respectively, and exposed to doses of the test material, vehicle and positive controls, both with and without metabolic activation, in duplicate (A + B ). Cultures were maintained at 37°C in a humidified atmosphere of 5% C02 in air.

The treatment regimens are as follows:
Without Metabolic Activation:
i) 20 continuous hours exposure to the test material.
ii) 30 continuous hours exposure to the test material.

With Metabolic Activation
i) 4 hours exposure to the test material and S9 mix (0.5 ml per 4.5 ml culture medium, of 10% S9 in standard co-factors). A phosphate buffered saline
wash and then a further 16 hours in treatment-free media prior to cell harvest.
ii) 4 hours exposure to the test material and S9 mix (0.5 ml per 4.5 ml culture medium, of 10% S 9 in standard co-factors). A phosphate buffered saline
wash and then a further 26 hours in treatment-free media prior to cell harvest.

Cell Harvest:
Mitosis was arrested by addition of demecolcine (0.1 /yg/ml) approximately two hours before the required harvest time. After incubation with demecolcine, the
cells were trypsinised to detach them from the tissue culture flask and suspended in 5 ml of culture medium. A sample of the cell suspension from each harvest time was counted to measure growth inhibition at each concentration. The cells were centrifuged, the culture medium drawn off and discarded, and the cells resuspended in hypotonic KCI. After fifteen minutes ( including five minutes centrifugation) most of the hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the KCI suspension into 3 mis fresh methanol/glacial acetic acid (3 :1 v/v). The fixative was changed at least twice and the cells stored at 4 ° C for at least four hours to ensure complete fixation.

Preparation of Metaphase Spreads:
The cells were resuspended in several mis of fresh fixative if necessary before centrifugation and resuspension in fixative. Three or four drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining:
When the slides were dry they were stained in 2% Gurrs Giemsa for five minutes, rinsed, dried and coverslipped using Depex mountant.

Coding
After checking that the slide preparations were of good quality, the slides were coded using a computerised random number generator.

Evaluation criteria:

Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 23 to 27 chromosomes, any gaps,
breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity
testing. Aberrations recorded by the slide scorer were checked by a senior cytogeneticist. Cells with 28 to 31 chromosomes were scored as aneuploid cells. Cells with greater than 31 chromosomes were classified as polyploid cells with the % incidence of polyploid cells reported. Endoreduplicated cells are included as polyploid cells but can also be evaluated separately if necessary.

Key result

Species / strain:

other: Chinese hamster lung (CHL)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Cytotoxicity Study:
In the absence of S9 the test material showed some evidence of cell toxicity. The presence of a precipitate affected the electronic cell counter, giving falsely evaluated counts in some cases. Microscopic assessment of the slides prepared from the treatment cultures showed metaphases present up to 3580 µg/ml in the 20 hour treatment with activation and at up to 895 µg/ml
in the 20 hour treatment without activation. The mitotic index data confirm the absence of toxicity in the presence of activation and show that in
the absence of activation excessive toxicity was observed at and above 447.5 µg/ml. Less toxicity was observed at 895 µg/ml than expected and this
was considered to be probably a result of inconsistent exposure related to the precipitation of the test material.

Chromosome Aberration Study:
GelI All D showed the expected level of toxicity in the without activation treatment group and an absence of toxicity in the presence
of activation. The toxicity dose-response curve in the absence of activation was shown to be particularly steep by the absence of metaphase cells in the intermediate dose group of 335.63 µg/ml.

The vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range of 0 - 3 %.

All the positive control cultures gave highly significant increases in the frequency of cells with aberrations indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected.

The test material was seen to induce no statistically significant increases in the frequency of cells with aberrations in any treatment group.

The test material did not induce a significant increase in the numbers of polyploid cells at any dose level in any of the treatment cases.

Conclusions:

The test material, Gell All D, did not induce any statistically significant, dose-related increases in the frequency of cells with chromosome aberrations either in the presence or absence of a liver enzyme metabolising system or after various exposure times.
Gell All D is therefore considered to be non-clastogenic in CHL cells in vitro.

Executive summary:

This study was conducted according to Safepharm Definitive Method and was designed to assess the potential chromosomal mutagenicity of a test material, on the metaphase chromosomes of the Chinese hamster lung (CHL) cell line. The method follows that described in the OECD Guidelines for Testing of Chemicals (1981) No. 473 "Genetic Toxicology: Chromosome Aberration " Test.

Duplicate cultures of Chinese hamster lung (CHL) cells, treated with test material were evaluated for chromosome aberrations at a minimum of four dose levels together with vehicle and positive controls. Four treatment conditions were used: 4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 10% in standard co-factors with cell harvest at 20 hours and 30 hours after exposure.

Exposure in the absence of metabolic activation was continuous with cell harvest at 20 and 30 hours after culture initiation.

The dose range for metaphase analysis was selected from a series of at least four dose levels chosen on the basis of the results of a preliminary toxicity test. The test material was evaluated at doubling dose levels between 55.94 and 3580 µg/ml depending on the particular treatment regime used. The continuous exposures were more toxic than the pulse exposures.

All vehicle (solvent) controls gave frequencies of cells with aberrations within the range expected for the CHL cell line.

Both of the positive control treatments gave highly significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material demonstrated no statistically significant, dose-related increases in the frequency of cells with aberrations either with or without metabolic activation. The test material was shown to be toxic in CHL cells in vitro in the without metabolic treatment groups only. Gell All D was shown to be non-clastosenic to CHL cells in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 10 June 2013 and 20 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Cell culture:
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 μg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/ml) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO 2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20) and without serum (R0) are used during the course of the study.

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (master stocks of cells were tested and found to be free of mycoplasma).

- Periodically "cleansed" against high spontaneous background: yes
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/ml), Hypoxanthine (15 μg/ml), Methotrexate (0.3 μg/ml) and Glycine (22.5 μg/ml). For the following 24 hours the cells were cultured in THG medium
(i.e. THMG without Methotrexate) before being returned to R10 medium.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/beta-naphthoflavone induced rat liver (S9-mix)

Test concentrations with justification for top dose:

Preliminary Toxicity Test: 4.05 to 1036.25 µg/ml
Mutagenicity Test:
Experiment 1 and 2: 64.77, 129.53, 259.06, 518.13, 777.19, 1036.25 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle:
Following solubility checks performed in-house, the test item was accurately weighed and formulated in dimethyl sulfoxide (DMSO) prior to serial dilutions being prepared.
The molecular weight of the test item was 414.49 and the purity was> 99%. Due to formulation problems the maximum achievable dose was 1036.25 µg/ml. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate (EMS) was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) was used as the positive control in the presence of metabolic activation.

Details on test system and experimental conditions:

DURATION
- Exposure duration:
Experiment 1: 4 hours with and without metabolic activation
Experiment 2: 4 hours with metabolic activation and 24 hours without metabolic activation
- Expression time: 2 days

NUMBER OF REPLICATIONS: Duplicate
NUMBER OF CELLS EVALUATED: 2000 cells/well

PRELIMINARY TOXICITY TEST:
A preliminary toxicity test was performed on cell cultures at 5 x 10E5 cells/ml, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10E5 cells/ml using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 4.05 to 1036.25 μg/ml for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 10E5 cells/ml.

The cultures were incubated at 37 °C with 5% CO 2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10E5 cells/ml. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity, and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth (%RSG) value.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) Maximum recommended dose level, 5000 μg/ml or 10 mM.
ii) The presence of excessive precipitate where no test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required).


MUTAGENICITY TEST:
EXPERIMENT 1:
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10E6 cells/ml in 10 ml aliquots in R10 medium in unvented 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentratio) at six dose levels of the test item (64.77 to 1036.25 μg/ml in both the absence and presence of metabolic activation), vehicle and positive controls. To each flask was added 2 ml of S9-mix if required, 0.2 ml of the treatment dilutions (0.2 ml for the positive control), and sufficient R0 medium to bring the total volume to 20 ml.

The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.

EXPERIMENT 2:
As in Experiment 1, an exponentially growing stock culture of cells was established. The cells were counted and processed to give 1 x 10E6 cells/ml in 10 ml cultures in R10 medium in unvented 25 cm2 tissue culture flasks for the 4-hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours therefore 0.3 x 10E6 cells/ml in 10 ml cultures were established in unvented 25 cm2 tissue culture flasks. The treatments were performed in duplicate (A + B), both with and without metabolic activation (1% S9 final concentration) at six dose levels of the test item (64.77 to 1036.25 μg/ml in both the absence and presence of metabolic activation), vehicle and positive controls. To each flask was added 2 ml of S9-mix if required, 0.2 ml of the treatment dilutions, (0.2 ml for the positive control) and sufficient R0 medium to give a final volue of 20 ml (R10 is used for the 24-hour exposure group).

The treatment vessels were incubated at 37°C with continuous shaking using an orbital shaker within an incubated hood for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.


MEASUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY:
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10E5 cells/ml. The cultures were incubated at 37°C with 5% CO 2 in air and subcultured every 24 hours for the expression period of two days, by counting and diluting to 2 x 10E5 cells/ml, unless the mean cell count was less than 3 x 10E5 cells/ml in which case all the cells were maintained.

On Day 2 of the experiment, the cells were counted, diluted to 10E4 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.

The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

PLATE SCORING:
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37 °C with 5% CO 2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 ml of MTT solution (2.5 mg/ml in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two to three hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

Evaluation criteria:

For a test item to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value. Following discussions at an International Workshop on Genotoxicity Test Procedures in Plymouth, UK, 2002 (Moore et al 2003) it was felt that the IMF must exceed some value based on the global background MF for each method (agar or microwell). This Global Evaluation Factor (GEF) value was set following a further meeting of the International Workshop in Aberdeen, Scotland, 2003 (Moore et al 2006) at 126 x 10-6 for the microwell method. Therefore, any test item dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the GEF of 126 x 10-6 and demonstrates a positive linear trend will be considered positive. However, if a test item produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely,
when a test item induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Statistics:

Small significant increases designated by the UKEMS statistical package will be reviewed using the above evaluation criteria, and may be disregarded at the Study Director's discretion.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

optimum levels of toxicity were achieved in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Preliminary toxicity test: Precipitate observed at and above 16.19 µg/ml.
Mutagenicity Tests: Precipitate observed at and above 64.77 µg/ml.


COMPARISON WITH HISTORICAL CONTROL DATA:
None of the vehicle control mutatant frequency values were outside the acceptable range of 50 to 170 x 10-6 viable cells.
Positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

Preliminary Toxicity Test:

The results for the Relative Suspension Growth (%RSG) were as follows:

Dose Level (µg/ml)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
4.05	70	94	99
8.1	58	97	101
16.19	64	94	133
32.88	85	105	94
64.77	87	75	82
129.53	89	93	92
259.06	77	98	81
518.13	67	99	74
1036.25	36	96	74

In the 4-hour and 24 hour exposure groups in the absence of metabolic activation there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 4 hour exposure in the presence of metabolic activation there was no evidence of marked test item induced toxicity. Overall, precipitate of the test item was observed at and above 16.19 µg/ml. Based on the %RSG values observed, the maximum dose level in the subsequent Mutagenicity Test would be set at 1036.25 µg/ml.

Mutagenicity Test

A summary of the results from the test is presented in Table 1 (attached background material).

Experiment 1

The results of the microtitre plate counts and their analysis are presented in Tables 2 to 7 (attached background material).

There was evidence of marked dose-related toxicity following exposure to the test item in both the absence of metabolic activation, as indicated by the RTG and %RSG values (Tables 3 and 6). There was no evidence of a reduction in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred. Based on the RTG and %RSG values, it was considered that optimum levels of toxicity were achieved in the absence of metabolic activation. Whilst optimum levels of toxicity were not achieved in the presence of metabolic activation, the test item was exposed to the cultures at the maximum achievable concentration due to formulation issues above this concentration. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels (Tables 3 and 6). It should also be noted that the GEF was not exceeded at any of the dose levels. Precipitate of the test item was observed at and above 64.77 µg/ml in both exposure groups.

The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in Tables 8 to 13 (attached background material).

There was evidence of marked toxicity following exposure to the test item in the absence of metabolic activation, as indicated by the %RSG and RTG values (Table 9). Moderate levels of test item toxicity were observed in the presence of metabolic activation as indicated by the %RSG and RTG values (Table 9). Based on the %RSG and/or RTG values it was considered that optimum levels of toxicity were achieved in the absence of metabolic activation. Whilst optimum levels of toxicity were not achieved in the presence of metabolic activation, the test item was exposed to the cultures to the maximum achievable concentration due to formulation issues above this concentration. There was no evidence of reductions in viability (%V) in either the absence or presence of metabolic activation; therefore indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had no significant effect on the toxicity of the test item.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 170 x 10^-6 viable cells. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test item did not induce a statistically significant dose related (linear-trend) response in either the absence or presence of metabolic activation (Table 9 and 12). Precipitate of the test item was observed at and above 64.77 µg/ml.

The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals NO.476 "In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9 final concentration). In Experiment 2, the cells were treated with the test item at six dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9 final concentration) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test and for Experiment 1 and 2 was 64.77 to 1036.25 µg/ml in both the absence and presence of metabolic activation.

Results

The maximum dose level used in the Mutagenicity Test was limited by the formulation of the test item and the maximum achievable dose level was 1036.25 µg/ml. Precipitate of the test item was observed at and above 64.77 µg/ml in the Mutagenicity Test.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus.

The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please see Section 13 for "Read-across justification to support the REACH registration of Bis-O-(benzylidene)-D-glucitol (EC 251-136-4) at 10-100 tpa" for full details.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is proposed that the structural similarity and properties of the target substance and the structural analogue (sources substance) are sufficiently close for there to be a reasonable expectation of similar effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance:
IUPAC name: 1,3:2,4-bis-O-((3,4-dimethylphenyl)methylene) D-Glucitol
EC number: 413-110-2
CAS number: 135861-56-2


Target Substance: Bis-O-(benzylidene)-D-glucitol
IUPAC name: 1,3:2,4-bis-O-dibenzylidene-D-glucitol
EC number: 251-136-4
CAS number: 32647-67-9


3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity of the source substances and target substance, similarity of physic-chemical properties and similarity in experimental (eco)toxicological test data (and toxicokinetic behaviour assessment) it is concluded that target substance and the structural analogue (source substance) are sufficiently close for there to be a reasonable expectation of similar effects, for the endpoints where results have been read-across.

4. DATA MATRIX
Please see Section 13 for "Read-across justification to support the REACH registration of Bis-O-(benzylidene)-D-glucitol (EC 251-136-4) at 10-100 tpa" for full details.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

optimum levels of toxicity were achieved in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item (source substance EC 413-110-2) on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals NO.476"In Vitro Mammalian Cell Gene Mutation Tests", Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and be acceptable to the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9 final concentration). In Experiment 2, the cells were treated with the test item at six dose levels using a 4-hour exposure group in the presence of metabolic activation(1% S9final concentration) and a 24-hour exposure group in the absence of metabolic activation.

The dose range of test item was selected following the results of a preliminary toxicity test and for Experiment 1 and 2 was 64.77 to 1036.25 µg/ml in both the absence and presence of metabolic activation.

Results

The maximum dose level used in the Mutagenicity Test was limited by the formulation of the test item and the maximum achievable dose level was 1036.25 µg/ml. Precipitate of the test item was observed at and above 64.77 µg/ml in the Mutagenicity Test.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus.

The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test item did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment.

Conclusion

The test item was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

It is proposed that this result can be used in the assessment of the target substance.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study conducted between 21st March 2005 and 5th May 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase, TK +/- locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr J Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr D Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Cell Culture: Cells were routinely cultured in RPMI 1640 medium
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone beta-naphthaflavone induced rat liver, S9 mix

Test concentrations with justification for top dose:

preliminary study: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 µg/ml
Experiment 1: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 µg/ml
Experiment 2: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected as the vehicle because it provided a suspension that was considered to allow accurate dosing of the cultures.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

EMS used in the absence of metabolic activation, CP used in the presence of of metabolic activation Migrated to IUCLID6: was used in the absence of metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration:
Experiment 1: 4 hour exposure with and without metabolic activation.
Experiment 2: Exposure time without metabolic activation increased to 24 hours.
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 14 days


SELECTION AGENT (mutation assays): 5TFT

NUMBER OF REPLICATIONS: duplicate


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency and relative total growth

OTHER EXAMINATIONS:
- Other: colony size

Evaluation criteria:

The normal range for mutant frequency per survivor is 50-200 x 10E-6 for the TK+/- locus in L5l78Y cells at this laboratory. Vehicle controls results should ideally be within this range, although minor errors in cell counting and dilution or exposure to the metabolic activation system may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 250 x 10E-6 mutant frequency per survivor are not normally acceptable and will be repeated.

Positive control chemicals should induce at least three to five fold increases in mutant frequency greater than the corresponding vehicle control.

Any test material dose level that has a mutation frequency value that is greater than the corresponding vehicle control by the Global Evaluation Factor (GEF) of 126 x 10-6 will be considered positive. However, if a test material produces a modest increase in mutant frequency, which only marginally exceeds the GEF value and is not reproducible or part of a dose-related response, then it may be considered to have no toxicological significance. Conversely, when a test material induces modest reproducible increases in the mutation frequencies that do not exceed the GEF value then scientific judgement will be applied. If the reproducible responses are significantly dose-related and include increases in the absolute numbers of mutant colonies then they may be considered to be toxicologically significant.

Statistics:

The UKEMS statistical package was used to review the results.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no marked change in pH when the test material was dosed into media
- Effects of osmolality: the osmolality did not increase by more than 50 mOsm.
- Precipitation: A precipitate ofthe test material was observed at and above 15.63 µg/ml, and with heavy precipitate aggregation at 1000 µg/ml the maximum exposure was considered to be achieved at 500 µg/ml. In the subsequent mutagenicity experiments the maximum dose was limited to 500 µg/ml by the presence and nature ofthe precipitate.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test: In the 4-hour exposures, both in the absence and presence of metabolic activation (S9) there was an apparent reduction in the Relative Suspension Growth (%RSG) of cells treated with test material when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of S9 there was also an apparent reduction of %RSG values of cells treated with test material. However, based on the observations in the subsequent mutagenicity test the toxicity was most likely due to cells being washed of with the precipitate rather than true toxicity.
In the subsequent mutagenicity experiments the maximum dose was limited to 500 µg/ml by the presence and nature of the precipitate in the preliminary experiment.

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle (solvent) controls had acceptable mutant frequency values that that were within the normal range for the L5178Y cell line at the TK +I-Iocus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity ofthe metabolising system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the preliminary toxicity test a precipitate oftest material was observed at and above 15.63 µg/ml, increasing with intensity with increase in dose concentration until it aggregated at 1000 µg/ml. It was considered that the aggregation of the precipitate effectively reduced exposure to the cells and, therefore, this dose level was not selected for the mutagenicity tests.

During the course of the study the test material was tested to the limits of toxicity in the presence of metabolic activation, and in the absence of metabolic activation to a dose level where maximum exposure of the cells was achieved.

Conclusions:

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Introduction: The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/- locus of the L5178Y mouse lymphoma cell line.

Methods: L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls. The entire experiment was repeated to confirm the result of the first experiment. Four-hour exposures were used both with and without activation in Experiment 1. In Experiment 2, the exposure time without activation was increased to 24 hours.

The dose range of test material, plated for expression of mutant colonies, was selected based on the results and observations of a preliminary toxicity test and was 3.91 to 500 µg/ml in the absence of metabolic activation, and 3.91 to 62.5 µg/ml in the presence of metabolic activation for the first experiment. For the second experiment the dose range was 3.91 to 500 µg/ml both with and without activation.

Results: The maximum dose level used was limited by the presence and nature of the precipitate observed. In the preliminary toxicity test a precipitate of test material was observed at and above 15.63 µg/ml, increasing with intensity with increase in dose concentration until it aggregated at 1000 µg/ml. It was considered that the aggregation of the precipitate effectively reduced exposure to the cells and, therefore, this dose level was not selected for the mutagenicity tests. The vehicle (solvent) controls had acceptable mutant frequency values that that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce a statistically significant or dose-related increase in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion: The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

Please see Section 13 for "Read-across justification to support the REACH registration of Bis-O-(benzylidene)-D-glucitol (EC 251-136-4) at 10-100 tpa" for full details.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is proposed that the structural similarity and properties of the target substance and the structural analogue (sources substance) are sufficiently close for there to be a reasonable expectation of similar effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance:
IUPAC name: 1-(2,6-bis(4-tolyl)-1,3-dioxano(5,4-d)-1,3-dioxan-4-yl)ethane-1,2-diol
EC number: 402-950-5
CAS number: 87826-41-3

Target Substance: Bis-O-(benzylidene)-D-glucitol
IUPAC name: 1,3:2,4-bis-O-dibenzylidene-D-glucitol
EC number: 251-136-4
CAS number: 32647-67-9


3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity of the source substances and target substance, similarity of physic-chemical properties and similarity in experimental (eco)toxicological test data (and toxicokinetic behaviour assessment) it is concluded that target substance and the structural analogue (source substance) are sufficiently close for there to be a reasonable expectation of similar effects, for the endpoints where results have been read-across.

4. DATA MATRIX
Please see Section 13 for "Read-across justification to support the REACH registration of Bis-O-(benzylidene)-D-glucitol (EC 251-136-4) at 10-100 tpa" for full details.

Reason / purpose for cross-reference:

read-across source

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

Executive summary:

Methods:L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material (source substance EC 402-950-5) at up to eight dose levels, in duplicate, together with vehicle (solvent) and positive controls. The entire experiment was repeated to confirm the result of the first experiment. Four-hour exposures were used both with and without activation in Experiment 1. In Experiment 2, the exposure time without activation was increased to 24 hours.

The dose range of test material, plated for expression of mutant colonies, was selected based on the results and observations of a preliminary toxicity test and was 3.91 to 500 µg/ml in the absence of metabolic activation, and 3.91 to 62.5 µg/ml in the presence of metabolic activation for the first experiment. For the second experiment the dose range was 3.91 to 500 µg/ml both with and without activation.

Results:The maximum dose level used was limited by the presence and nature of the precipitate observed. In the preliminary toxicity test a precipitate of test material was observed at and above 15.63 µg/ml, increasing with intensity with increase in dose concentration until it aggregated at 1000 µg/ml. It was considered that the aggregation of the precipitate effectively reduced exposure to the cells and, therefore, this dose level was not selected for the mutagenicity tests. The vehicle (solvent) controls had acceptable mutant frequency values that that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce a statistically significant or dose-related increase in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion:The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

It is proposed that this result can be used in the assessment of the target substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance was found to be:

- non-mutagenic in a bacterial reverse mutation assay (Ames test).

- non-clastogenic in in-vitro chromosome aberration study (in CHL cells).

- non-mutgenic to L5178Y cells in a mouse lymphoma assay (based on results read-across from two structural analogues).

Based on the above, the substance has been assessed as not requiring classification as a germ cell mutagen.