3-(4,4-dimethylcyclohex-1-en-1-yl)propanal

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2009

negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473, 2010

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10-07-2009 to 01-12-2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

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)

Remarks:

inspected: August 2008 ; signature: March 2009

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: prepared in house (dates within full study report) ; S9 Microsomal fraction: Lot No. PB/βNF S9 07/06/2009
- method of preparation of S9 mix: Documented in the full study report. Stored at -196ºC
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect mutagens used in the Ames Test. The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test. A Certificate of S9 Efficacy is not presented in the full study report. However, concurrent positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Test concentrations with justification for top dose:

Preliminary toxicity test (TA100 and E.coli strain WP2uvrA, only : with and without S9-mix) : 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Precipitate was observed and/or the test item was initially toxic to strains above 500 µg/plate
Experiment 1 (plate-incorporation method ; range-finding test – all strains : with and without S9-mix) : 0, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method – all strains : with and without S9-mix) : 0, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate
Up to seven test item dose levels were selected in Experiment 1 and 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item. The dose levels were selected based on the results of the preliminary toxicity test and then Experiment 1, prior to the final Experiment 2.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed. Dimethyl sulphoxide was selected as the vehicle.
- Other: Formulated concentrations were (if required) adjusted by an appropriate factor to allow for the stated purity of the test item. See ‘test material information’ for more details, as appropriate.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without metabolic activation S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With metabolic activation S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1. in medium; in agar (pre-incubation) ; Experiment 2. in medium; in agar (pre-incubation).
The choice of application was due to the test item to either have unknown volatility or was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls.

DURATION
- Exposure duration:
Experiment 1 (range-finding test – plate incorporation method) : Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test item formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. All of the plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Experiment 2 (pre-incubation method) : Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of the vehicle or test item formulation and incubated for 20 minutes at 37°C prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on to the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method. The procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test item both with and without S9-mix. Subsequently, the procedure for incubation and duration was the same as in Experiment 1. All of the plates were incubated at 37ºC for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

SELECTION AGENT (mutation assays): histidine-deficient agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

- Other: Preliminary toxicity test (TA100 and E.coli strain WP2uvrA, only) : In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test item. The nominal concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The assay was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA-), 0.1 mL of test item formulation, 0.5 mL of S9-mix or phosphate buffer and 2 mL of molten, trace histidine or tryptophan supplemented, top agar and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). Ten concentrations of the test item and a vehicle control (dimethyl sulphoxide) were tested. In addition, 0.1 mL of the maximum concentration of the test item and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile nutrient agar plate in order to assess the sterility of the test item. After approximately 48 hours incubation at 37ºC the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Rationale for test conditions:

In accordance with the relevant guidelines.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.

Statistics:

Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

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

S9-Mix (+)	Dose Level Per Plate	Number ofrevertants(mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	111 90 104	(102) 10.7#	10 12 8	(10) 2.0	35 18 21	(25) 9.1	19 27 22	(23) 4.0	12 7 10	(10) 2.5
	5 µg	100 109 84	(98) 12.7	11 9 11	(10) 1.2	20 27 20	(22) 4.0	14 27 26	(22) 7.2	13 14 7	(11) 3.8
	15 µg	77 98 89	(88) 10.5	10 11 10	(10) 0.6	27 30 32	(30) 2.5	23 18 23	(21) 2.9	15 10 7	(11) 4.0
	50 µg	95 100 102	(99) 3.6	11 9 10	(10) 1.0	27 21 35	(28) 7.0	25 22 23	(23) 1.5	8 12 14	(11) 3.1
	150 µg	102 95 93	(97) 4.7	10 9 12	(10) 1.5	29 25 20	(25) 4.5	20 25 19	(21) 3.2	10 7 10	(9) 1.7
	500 µg	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0
	1500 µg	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0
	5000 µg	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0
Positive controls S9-Mix (+)	Name Dose Level No. ofRevertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		3234 2888 3314	(3145) 226.4	123 109 164	(132) 28.6	361 308 323	(331) 27.3	203 245 167	(205) 39.0	210 219 228	(219) 9.0
S9-Mix (-)	Dose Level Per Plate	Number ofrevertants(mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	98 95 100	(98) 2.5#	14 19 14	(16) 2.9	22 20 21	(21) 1.0	21 21 16	(19) 2.9	12 16 15	(14) 2.1
	5 µg	101 106 79	(95) 14.4	13 15 18	(15) 2.5	25 15 22	(21) 5.1	20 21 12	(18) 4.9	11 12 11	(11) 0.6
	15 µg	82 91 96	(90) 7.1	12 19 11	(14) 4.4	26 20 21	(22) 3.2	16 18 16	(17) 1.2	12 15 15	(14) 1.7
	50 µg	100 97 96	(98) 2.1	16 18 13	(16) 2.5	26 27 24	(26) 1.5	20 14 16	(17) 3.1	11 9 12	(11) 1.5
	150 µg	92 93 77	(87) 9.0	14) 14 15	(14) 0.6	20 20 24	(21) 2.3	12 18 16	(15) 3.1	9 10 9	(9) 0.6
	500 µg	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0
	1500 µg	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0
	5000 µg	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0	0 *P 0 *P 0 *P	(0) 0.0
Positive controls S9-Mix (-)	Name Dose Level No. ofRevertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		289 302 339	(310) 25.9	100 109 103	(104) 4.6	147 178 170	(165) 16.1	107 114 114	(112) 4.0	307 565 420	(431) 129.3

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

4NQO   4-Nitroquinoline-1-oxide

9AA       9-Aminoacridine

BP          Benzo(a)pyrene

2AA       2-Aminoanthracene

N/T       Not tested at this dose level

P            Precipitate

*            Partial or complete absence of bacterial background lawn

#            Standard deviation

 

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

S9-Mix (+)	Dose Level Per Plate	Number ofrevertants(mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	86 89 80	(85) 4.6#	10 11 9	(10) 1.0	27 31 31	(30) 2.3	22 23 27	(24) 2.6	14 12 12	(13) 1.2
	0.5 µg	90 84 78	(84) 6.0	12 9 14	(12) 2.5	30 25 21	(25) 4.5	26 22 26	(25) 2.3	13 15 7	(12) 4.2
	1.5 µg	87 82 77	(82) 5.0	9 15 8	(11) 3.8	26 32 24	(27) 4.2	24 21 24	(23) 1.7	10 11 11	(11) 0.6
	5 µg	79 84 73	(79) 5.5	10 12 8	(10) 2.0	29 26 24	(26) 2.5	21 28 23	(24) 3.6	12 12 10	(11) 1.2
	15 µg	88 93 75	(85) 9.3	9 12 13	(11) 2.1	25 32 20	(26) 6.0	25 20 23	(23) 2.5	12 12 9	(11) 1.7
	50 µg	92 74 88	(85) 9.5	10 12 11	(11) 1.0	29 33 24	(29) 4.5	19 17 21	(19) 2.0	12 11 12	(12) 0.6
	150 µg	75 92 74	(80) 10.1	8 9 9	(9) 0.6	22 23 21	(22) 1.0	23 23 25	(24) 1.2	12 8 11	(10) 2.1
	500 µg	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0
Positive controls S9-Mix (+)	Name Dose Level No. ofRevertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		2479 2245 2153	(2292) 168.1	132 155 123	(137) 16.5	229 176 263	(223) 43.8	114 181 177	(157) 37.6	315 272 272	(286) 24.6
S9-Mix (-)	Dose Level Per Plate	Number ofrevertants(mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	96 108 106	(103) 6.4#	16 19 16	(17) 1.7	22 18 27	(22) 4.5	16 15 21	(17) 3.2	11 13 12	(12) 1.0
	0.5 µg	107 89 82	(93) 12.9	19 19 16	(18) 1.7	22 22 27	(24) 2.9	19 24 23	(22) 2.6	12 12 12	(12) 0.0
	1.5 µg	95 95 102	(97) 4.0	19 13 16	(16) 3.0	21 21 18	(21) 1.7	21 16 19	(19) 2.5	14 12 13	(13) 1.0
	5 µg	99 109 89	(80) 1.0	13 18 16	(16) 2.5	21 21 20	(21) 0.6	19 13 14	(15) 3.2	13 15 9	(12) 3.1
	15 µg	81 80 79	(97) 4.9	15 16 16	(16) 0.6	14 20 23	(19) 4.6	18 18 19	(18) 0.6	11 12 10	(11) 1.0
	50 µg	100 99 91	(97) 4.9	14 20 15	(16) 3.2	20 18 19	(19) 1.0	21 16 19	(19) 2.5	9 11 16	(12) 3.6
	150 µg	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0
	500 µg	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0	0 * 0 * 0 *	(0) 0.0
Positive controls S9-Mix (-)	Name Dose Level No. ofRevertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		486 474 476	(479) 6.4	200 179 221	(200) 21.0	168 143 142	(151) 14.7	156 136 90	(127) 33.8	366 449 493	(436) 64.5

Conclusions:

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

Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test substance using the Ames direct plate incorporation and/or the pre incubation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). Formulated concentrations were adjusted by an appropriate factor to allow for the stated purity of the test item.The nominal dose range for the range-finding test was determined based on the results of a preliminary toxicity assay (conducted at 0 (control), 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) . The range-finding test (Experiment 1) was conducted at 0 (control), 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The test item nominal dose range was amended slightly to 0, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate with 20 minutes pre-incubation (Experiment 2), following the results of the range-finding test. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawns of the tester TA98, TA100, TA1535, TA1537 and WP2uvrA- strains in both the presence and absence of S9-mix at 500 µg/plate. Consequently, for the second mutation test the toxic limit was employed as the maximum dose concentration. Results from the second mutation test (pre-incubation method) confirmed the toxicity previously noted.The test item caused a visible reduction in the growth of the bacterial background lawns at 150 and 500 μg/plate in the absence and presence of S9, respectively. The test item was tested up to the maximum recommended dose level of 5000 μg/plate (range-finding test) and the toxic limit (main test). A precipitate (oily in appearance) was noted at and above 1500 μg/plate, although 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). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-11-2009 to 10-06-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

: Japanese Ministry of Economy, Trade and Industry (METI) Guidelines

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: 40 CFR 799.9537 TSCA in vitro mammalian chromosome aberration test.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: September 2009 ; signature: November 2009

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable (chromosome aberration test)

Species / strain / cell type:

lymphocytes: Human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Human lymphocytes from volunteers
- Suitability of cells: Cells used in accordance with OECD TG 473. Details on volunteers provided below.
- Normal cell cycle time (negative control): See below ; (17 hours = 1.0 normal cell cycle time ; ca. 24 hours = 1.5 times normal cell cycle time).

For lymphocytes:
- Sex, age and number of blood donors: For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (volunteers are typically: aged 18-35), sex not specified ; who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes for the donors used in this study: was considered to be approximately 17 hours. Therefore using this average it can be considered that the in-house exposure time for the experiments for 1.5 x AGT is 24 hours. Further details on the donors is available in the full study report.
- Whether whole blood or separated lymphocytes were used: Whole blood ; cells were cultured as follows : cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum, at 37ºC with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at approximately 90 μg/ml final concentration.
- Whether blood from different donors were pooled or not: None-pooled.
- Mitogen used for lymphocytes: (9 mg/mL) phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Cells were cultured as follows : cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum, at 37ºC with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at approximately 90 μg/ml final concentration. All incubations were carried out in a controlled environment, containing 5.0% CO2 humified air in the dark at 37.0°C.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: prepared in house (dates within full study report) ; S9 Microsomal fraction: Lot No. PB/βNF S9 13/09/2009 and 06/12/2009
- method of preparation of S9 mix: Documented in the full study report. Stored at -196ºC
- concentration or volume of S9 mix and S9 in the final culture medium: 2% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Before use, each batch of S9 was assayed for its ability to metabolise appropriate indirect mutagens used in the Ames Test. The S9-mix was prepared immediately before use using sterilised co-factors and maintained on ice for the duration of the test. A Certificate of S9 Efficacy is not presented in the full study report. However, concurrent positive control substances all produced a positive response (p ≤ 0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.

Test concentrations with justification for top dose:

The maximum dose level was 1660 µg/mL, or 10 mM concentration, the maximum recommended dose level. The test item was insoluble in culture medium (MEM) at 16.6 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 166 mg/mL in solubility checks. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 1660 μg/mL range (full results recorded in the full study report).
The purity of the test item was accounted for in the test item formulations, if required (see 'confidential details on test material').

I. Preliminary toxicity test: 0 (control) , 6.48, 12.97, 25.94, 51.88, 103.75, 207.5, 415, 830 and 1660 μg/mL
Within three exposure groups:
i) 4-hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.

II. Main Test:
EXP1:
4(20)-hour without S9: 0*, 3.25, 6.5*, 13*, 26*, 52, 78 μg /mL and MMC 0.4* μg/mL
4(20)-hour with S9 (2%): 0*, 13, 26*, 52*, 78*, 104, 208 μg /mL and CP 5* μg/mL
EXP2:
24-hour without S9: 0*, 3.25, 6.5*, 13*, 26*, 39*, 52 μg /mL and , MMC 0.2* μg/mL
4(20)-hour with S9 (1%): 0*, 13, 26*, 39*, 52*, 65*, 78 μg/mL and CP 5*μg/mL
where:
* = dose levels selected for metaphase analysis
MMC= Mitomycin C
CP = Cyclophosphamide

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble in culture medium (MEM) at 16.6 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 166 mg/mL in solubility checks performed in-house. The maximum dose level was 1660 µg/mL, or 10 mM concentration, the maximum recommended dose level. There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991) within the 0 to 2063 μg/mL range (full results recorded in the full study report). The test item was formulated within two hours of it being applied to the test system.
- Other: Formulated concentrations were adjusted/increased to allow for the stated water/impurity content. See 'Test Material Information' for further details

Untreated negative controls:

other: Vehicle control served as the negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation, 0.4 μg/ml for 4(20)-hour and 0.2 μg/ml for 24 h exposure periods, respectively ; Full details on the positive controls is reported in the full study report.

Untreated negative controls:

other: Vehicle control served as the negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation 5 μg/ml for a 4(20)-hour exposure period ; Full details on the positive controls is reported in the full study report.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Duplicate. The study conducted two replicates (A and B) at each dose level and exposure duration groups.
- Number of independent experiments: Two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Not applicable.
- Test substance added in medium;

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Not reported.
- Exposure duration/duration of treatment: The preliminary toxicity test was performed using both of the exposure conditions as described for both experiments (below).
I. With Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.1 mL (100 μL) of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and of the Main Experiment 1. In Experiment 2, 1 mL of 10% S9-mix (i.e. 1% final concentration of S9 in standard cofactors), was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL. After 4 hours at approximately 37 ºC, 5 % CO2 in humidified air the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.
II. Without Metabolic Activation (S9) Treatment:
- After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 mL (100 μL) of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL. After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours at approximately 37 ºC in 5 % CO2 in humidified air.
In the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.
- Harvest time after the end of treatment (sampling/recovery times): Three exposure groups:
i) 4-hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4ºC for at least four hours to ensure complete fixation.
- If cytokinesis blocked method was used for micronucleus assay: Not applicable.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Following fixation above, when the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Scoring: Where possible, 200 consecutive well-spread metaphases from each concentration (100 per duplicate) were assessed for observations, if the cell had 44 to 48 chromosomes, any breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations according to the simplified system of Savage (1976), ISCN (1985). Where the analysis of the slide resulted in a large frequency of aberrant cells (30 to 50%) then the analysis was terminated after a total of 50 metaphases with aberrations were recorded. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): Not applicable.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): See above.
- Determination of polyploidy: Yes. Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) including endoreduplicated cells, reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range was reported in the full study report.
- Determination of endoreplication: See above.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI)
- Any supplementary information relevant to cytotoxicity: The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
See above “scoring”, furthermore the frequency of cells with aberrations excluding gaps and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

- OTHER: Not applicable.

Evaluation criteria:

From the OECD TG 473 criteria :
Positive criteria:
A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related.
Negative criteria:
A test item can be classified as non-genotoxic if:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment.
Statistical analysis is also performed (see: ‘Statistics’). Biological relevance of the results are to be considered first. Statistical methods are used to analyse the increases in aberration data as recommended in the OECD 473 guideline. However, statistical significance will not be the only determining factor for a positive response. A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations are assessed on a case by case basis.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. Analysis of data from in vitro cytogenetic assays. In Statistical Evaluation of mutagenicity test data: UKEMS sub-committee on guidelines for mutagenicity testing. Report Part III (Ed: Kirkland, D.J.), Cambridge University Press (1989)

Key result

Species / strain:

lymphocytes: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test item was dosed into media
- Effects of osmolality: There was no significant change osmolality (did not increase by more than 50 mOsm) when the test item was dosed into media
- Evaporation from medium: Not reported.
- Water solubility: Not applicable.
- Precipitation: In the preliminary test: A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 6.48 μg/mL; in the 4(20)-hour without metabolic activation exposure group, and at and above 207.5 μg/mL with 4(20)-hour metabolic activation exposure group. Cloudy precipitate was also observed at and above 207.5 μg/mL 4(20)-hour without metabolic activation exposure group and 24 hour continuous exposure group, and at and above 415 μg/mL 4(20)-hour with metabolic activation exposure group. In all three exposure groups Greasy/oily precipitate was observed at and above 830 μg/mL.
Main test: No precipitate was observed at the end of exposure, in any of the three exposure groups.
- Other confounding effects: In the preliminary test: Haemolysis was observed in all blood cultures at and above 51.88 μg/mL in all three exposure groups. Haemolysis indicates that the test item is causing disruption of the erythrocyte membranes and not indicative of any genotoxic response to the lymphocytes.
Main test: in Experiment 1: haemolysis was observed at and above 52 μg/mL in the absence of S9, and at and above 78 μg/mL in the presence of S9, in Experiment 2 haemolysis was observed at or above 26 μg/mL in the presence of S9

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 1660 μg/mL. The maximum dose was the maximum recommended dose level. The selection of the maximum dose level was based on toxicity for the main test.

COMPARISON WITH HISTORICAL CONTROL DATA:
- All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. (Within the Historic Control Data range presented in the full study report).
- All the positive control items induced statistically significant increases in the frequency of cells with aberrations. (Within the Historic Control Data range presented in the full study report).

ADDITIONAL INFORMATION ON CYTOTOXICITY: See ‘other confounding effects’ listed above.

1. Chromosome Aberration Test – Main Test

The dose levels of the controls and the test item are given below:

EXP1:

4(20)-hour without S9: 0*, 3.25, 6.5*, 13*, 26*, 52, 78 μg /mL and MMC 0.4* μg/mL

4(20)-hour with S9: 0*, 13, 26*, 52*, 78*, 104, 208 μg /mL and CP 5* μg/mL

EXP2:

24-hour without S9: 0*, 3.25, 6.5*, 13*, 26*, 39*, 52 μg /mL and , MMC 0.2* μg/mL

4(20)-hour with S9: 0*, 13, 26*, 39*, 52*, 65*, 78 μg/mL and CP 5*μg/mL

where: * = dose levels selected for metaphase analysis ; MMC= Mitomycin C and CP = Cyclophosphamide

 

In Experiment 1: The qualitative assessment of the slides determined that the toxicity was marginally increased to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 26 μg/mL in the absence of metabolic activation (S9), and 78 μg/mL in the presence of metabolic activation (S9). No precipitate of the test material was observed at the end of exposure period in either exposure group. However, haemolysis was observed at and above 52 μg/mL in the absence of S9, and at and above 78 μg/mL in the presence of S9. A dose-related inhibition of mitotic index was observed, and that 61% mitotic inhibition was achieved at 26 μg/mL in the absence of S9. In the presence of S9 62% mitotic inhibition was achieved at 78 μg/mL. The optimum level of toxicity, 50% mitotic inhibition, was not achieved in either exposure group. The maximum dose level selected for metaphase analysis was limited by toxicity in both exposure groups to 26 and 78 μg/mL in the absence and presence of S9 respectively. It was considered that the toxicity observed at these doses was acceptable (as it exceeded the optimal level of toxicity).

 

- All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.

- The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

- The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

- The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

 

In Experiment 2: The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test material dose level of 52 μg/mL in the absence of S9. In the presence of S9 the maximum test material dose level with metaphases suitable for scoring was 78 μg/mL. No precipitate of the test material was observed at the end of exposure in either exposure group. Haemolysis was observed at and above 26 μg/mL in the presence of S9. A dose-related inhibition of mitotic index was observed, and that a maximum of 92% mitotic inhibition was achieved at 52 μg/mL in the absence of S9. In the presence of S9 a maximum of 77% mitotic inhibition was achieved at 78 μg/mL. The maximum dose level selected for metaphase analysis was based on toxicity as it was in Experiment 1. In the absence of S9 the maximum dose selected was 39 μg/mL with 60% mitotic inhibition, and in the presence of S9 it was 65 μg/mL with 71% mitotic inhibition.

 

- All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.

- The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

- The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation. However, a modest but statistically significant increase in cells with chromosome aberrations was observed at 26 μg/mL in the absence of S9. The increase was very modest, and predominantly due to chromatid breaks and whilst just outside the historical vehicle control range for this harvest point with no response being recorded at the next dose level it was, therefore, considered to have no toxicological significance.

- The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

Conclusions:

Interpretation of results:
Negative
Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

The study was performed to the requirements of OECD TG 473, EU Method B.10, and Japan METI guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Within a Cell Growth Inhibition Test (Preliminary Toxicity Test) the test item was tested at: 0 (control) , 6.48, 12.97, 25.94, 51.88, 103.75, 207.5, 415, 830 and 1660 μg/mL within three exposure groups and within three exposure groups of (i) 4-hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure; (ii) 4-hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure and/or (iii) 24-hour continuous exposure to the test item without S9-mix. Following this a main test was conducted. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited on precipitate and cytotoxicity. The dose levels selected for the Main Tests were as follows: Experiment 1 : (i) 4(20)-hour without S9: 0*, 3.25, 6.5*, 13*, 26*, 52, 78 μg /mL; (ii) 4(20)-hour with S9 (2%): 0*, 13, 26*, 52*, 78*, 104, 208 μg /mL and within Experiment 2: (iii) 24-hour without S9: 0*, 3.25, 6.5*, 13*, 26*, 39*, 52 μg /mL and/or (iv) 4(20)-hour with S9 (1%): 0*, 13, 26*, 39*, 52*, 65*, 78 μg/mL. The symbol ‘star’ denotes dose levels selected for metaphase analysis. All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item was cytotoxic and did not induce any toxicologically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included dose levels that marginally exceeded 50% mitotic inhibition (optimum cytotoxicity). Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key Study : OECD TG 471, 2009 :  The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test substance using the Ames direct plate incorporation and/or the pre incubation method at up to seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). Formulated concentrations were adjusted by an appropriate factor to allow for the stated purity of the test item.The nominal dose range for the range-finding test was determined based on the results of a preliminary toxicity assay (conducted at 0 (control), 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) . The range-finding test (Experiment 1) was conducted at 0 (control), 5, 15, 50, 150, 500, 1500 and 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The test item nominal dose range was amended slightly to 0, 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate with 20 minutes pre-incubation (Experiment 2), following the results of the range-finding test. Additional dose levels and an expanded dose range were selected in both experiments in order to achieve both four non-toxic dose levels and the toxic limit of the test item. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test, the test item induced a visible reduction in the growth of the bacterial background lawns of the tester TA98, TA100, TA1535, TA1537 and WP2uvrA- strains in both the presence and absence of S9-mix at 500 µg/plate. Consequently, for the second mutation test the toxic limit was employed as the maximum dose concentration. Results from the second mutation test (pre-incubation method) confirmed the toxicity previously noted.The test item caused a visible reduction in the growth of the bacterial background lawns at 150 and 500 μg/plate in the absence and presence of S9, respectively. The test item was tested up to the maximum recommended dose level of 5000 μg/plate (range-finding test) and the toxic limit (main test). A precipitate (oily in appearance) was noted at and above 1500 μg/plate, although 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). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.

Key Study : OECD TG 473, 2010 : The study was performed to the requirements of OECD TG 473, EU Method B.10, and Japan METI guidelines under GLP conditions to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocyte cultured mammalian cells. Within a Cell Growth Inhibition Test (Preliminary Toxicity Test) the test item was tested at: 0 (control) , 6.48, 12.97, 25.94, 51.88, 103.75, 207.5, 415, 830 and 1660 μg/mL within three exposure groups and within three exposure groups of (i) 4-hours exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure; (ii) 4-hours exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure and/or (iii) 24-hour continuous exposure to the test item without S9-mix. Following this a main test was conducted. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited on precipitate and cytotoxicity. The dose levels selected for the Main Tests were as follows: Experiment 1 : (i) 4(20)-hour without S9: 0*, 3.25, 6.5*, 13*, 26*, 52, 78 μg /mL; (ii) 4(20)-hour with S9 (2%): 0*, 13, 26*, 52*, 78*, 104, 208 μg /mL and within Experiment 2: (iii) 24-hour without S9: 0*, 3.25, 6.5*, 13*, 26*, 39*, 52 μg /mL and/or (iv) 4(20)-hour with S9 (1%): 0*, 13, 26*, 39*, 52*, 65*, 78 μg/mL. The symbol ‘star’ denotes dose levels selected for metaphase analysis. All vehicle (DMSO) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9 mix were validated. The test item was cytotoxic and did not induce any toxicologically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included dose levels that marginally exceeded 50% mitotic inhibition (optimum cytotoxicity). Under the conditions of this study, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity