2-methylpentyl 2-hydroxybenzoate

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, 2020

negative, in vitro mammalian cell micronucleus assay (with and without S-9 activation), TK6 human lymphoblastoid cells, OECD TG 487, 2020

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:

14-02-2020 to 02-03-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: ICH S2(R1) guideline

Version / remarks:

June 2012 (ICH S2(R1) Federal Register

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine or tryptophan locus

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation method):
All strains (presence of S9-mix): 0, 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
All strains (absence of S9-mix): 0, 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate

Experiment 2 (pre-incubation method): Eight to ten test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item. The dose levels were selected based on the results of Experiment 1.
All strains (presence of S9-mix): 0, 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate
All strains (absence of S9-mix): 0, 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate

Top doses were justified based on cytotoxicity. For more information on cytotoxicity and/or precipitation and/or chosen dose levels refer to ‘any other information on materials and methods incl. tables’.

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: Potassium dichromate

Remarks:

Without metabolic activation S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation).
The test item was administered in solvent (DMSO) at a volume of 100 μL per plate for the plate incorporation method and a volume of 50 μL per plate when the liquid pre-incubation method was employed, within a maximum of two hours of formulation. For each test, a stock solution of test item in the solvent, DMSO, was prepared and then diluted in the same solvent to formulate the required test solutions. The solutions of test 1: A: 50 mg/mL (or 225 mmol/L) through H: 0.016 mg/mL (or 0.072 mmol/L) ; test 2: I: 100 mg/mL (or 450 mmol/L) through P: 0.032 mg/mL (or 0.0144 mmol/L) are documented in the full study report.

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

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

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Other: Viability determination: Confirmation that adequate numbers of viable bacteria were exposed to the test item was obtained from viability determinations for each strain performed concurrently with the mutagenicity test. For each tester strain, a sample of the stock overnight bacterial suspension was diluted to 10^-7 in phosphate buffered saline. 200 μL of the x10^-7 dilution was spread onto each of two nutrient agar plates. The plates were then inverted and incubated at room temperature for 3 days, after which, numbers of colonies were counted and recorded. A mean viable count of at least 20 colonies per plate should be present (>10^9 colony forming units [cfu] per mL).

Rationale for test conditions:

In accordance with the OECD TG 471 guidelines.

Evaluation criteria:

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

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium 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

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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 of revertants (mean) +/- SD
		TA98		TA100		TA1535		TA1537		WP2 uvrA/pKM101
	Solvent Control (DMSO)	28 34 32	(31) 3.1#	140 135 143	(139) 4.0	10 12 13	(12) 1.5	9 11 13	(11) 2.0	202 195 194	(197) 4.4
	1.6 µg	33 35 30	(33) 2.5 1.1~	129 143 137	(136) 7.0 1.0	15 16 12	(14) 2.1 1.2	10 8 10	(9) 1.2 0.8	196 193 189	(193) 3.5 1.0
	5 µg	31 32 27	(30) 2.6 1.0	147 153 152	(151) 3.2 1.1	15 11 11	(12) 2.3 1.0	8 9 9	(9) 0.6 0.8	196 187 181	(188) 7.5 1.0
	16 µg	35 37 32	(35) 2.5 1.1	149 138 145	(144) 5.6 1.0	10 9 12	(10) 1.5 0.8	7 11 8	(9) 2.1 0.8	195 201 190	(195) 5.5 1.0
	50 µg	32 34 35	(34) 1.5 1.1	143 148 155	(149) 6.0 1.1	9 8 12	(10) 2.1 0.8	8 7 10	(8) 1.5 0.7	178 174 175	(176) 2.1 0.9
	160 µg	30 33 36	(33) 3.0 1.1	144 137 128	(136) 8.0 1.0	10 8 10	(9) 1.2 0.8	9 6 8	(8) 1.5 0.7	167 171 163	(167) 4.0 0.8
	500 µg	20 s P 22 s P 26 s P	(23) 3.1 0.7	81 s P 75 s P 70 s P	(75) 5.5 0.5	10 s P 8 s P 9 s P	(9) 1.0 0.8	6 s P 7 s P 9 s P	(7) 1.5 0.6	143 P 138 P 144 P	(142) 3.2 0.7
	1600 µg	13 r P 11 r P 10 r P	(11) 1.5 0.4	2 r P 1 r P 0 r P	(1) 1.0 0.0	4 r P 5 r P 7 r P	(5) 1.5 0.4	2 r P 4 r P 3 r P	(3) 1.0 0.3	127 P 129 P 126 P	(127) 1.5 0.6
	5000 µg	7 v P 6 v P 8 v P	(7) 1.0 0.2	0 v P 0 v P 0 v P	(0) 0.0 0.0	7 v P 5 v P 3 v P	(5) 2.0 0.4	1 v P 2 v P 0 v P	(1) 1.0 0.1	79 s P 81 s P 88 s P	(83) 4.7 0.4
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		2AA		2AA
		2 µg		2 µg		2 µg		2 µg		20 µg
		1435 1680 1607	(1574) 125.8# 50.8~	1625 1747 1587	(1653) 83.6 11.9	154 164 179	(166) 12.6 13.8	133 152 143	(143) 9.5 13.0	1884 2093 2259	(2079) 187.9 10.6
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		TA98		TA100		TA1535		TA1537		WP2 uvrA/pKM101
	Solvent Control (DMSO)	31 27 34	(31) 3.5#	122 129 123	(125) 3.8	12 14 12	(13) 1.2	9 9 10	(9) 0.6	151 148 146	(148) 2.5
	1.6 µg	35 37 31	(34) 3.1 1.1~	128 134 117	(126) 8.6 1.0	16 13 15	(15) 1.5 1.2	8 11 12	(10) 2.1 1.1	145 149 151	(148) 3.1 1.0
	5 µg	29 26 30	(28) 2.1 0.9	118 116 123	(119) 3.6 1.0	14 12 13	(15) 1.0 1.0	9 8 8	(8) 0.6 0.9	154 142 149	(148) 6.0 1.0
	16 µg	27 29 30	(29) 1.5 0.9	128 132 137	(132) 4.5 1.1	15 16 11	(14) 2.6 1.1	11 10 8	(10) 1.5 1.1	150 139 138	(142) 6.7 1.0
	50 µg	35 37 31	(34) 3.1 1.1	123 115 118	(119) 4.0 1.0	13 12 12	(12) 0.6 0.9	11 9 8	(9) 1.5 1.0	149 143 131	(141) 9.2 1.0
	160 µg	28 26 28	(27) 1.2 0.9	108 116 109	(111) 4.4 0.9	11 13 10	(11) 1.5 0.8	6 7 8	(7) 1.0 0.8	134 139 136	(136) 2.5 0.9
	500 µg	21 s P 17 s P 19 s P	(19) 2.0 0.6	96 s P 89 s P 91 s P	(92) 3.6 0.7	12 s P 10 s P 9 s P	(10) 1.5 0.8	6 s P 4 s P 5 s P	(5) 1.0 0.6	138 P 130 P 133 P	(134) 4.0 0.9
	1600 µg	21 r P 12 r P 15 r P	(16) 4.6 0.5	94 r P 89 r P 88 r P	(90) 3.2 0.7	9 r P 9 r P 7 r P	(8) 1.2 0.6	5 r P 5 r P 6 r P	(5) 0.6 0.6	128 P 121 P 116 P	(122) 6.0 0.8
	5000 µg	9 v P 10 v P 12 v P	(10) 1.5 0.3	82 v P 72 v P 84 v P	(79) 6.4 0.6	8 v P 7 v P 4 v P	(6) 2.1 0.5	4 v P 4 v P 5 v P	(4) 0.6 0.4	102 s P 113 s P 112 s P	(109) 6.1 0.7
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	2-nitrofluorene		Na azide		Na azide		9AA		K2Cr2O7
		1 µg		0.5 µg		0.5 µg		50 µg		25 µg
		329 270 326	(308) 33.3# 9.9~	585 610 500	(565) 57.7 4.5	384 524 455	(454) 70.0 34.9	229 194 217	(213) 17.8 23.7	1090 983 975	(1016) 64.2 6.9

N/T: Not tested at this dose level

s: slightly reduced lawn

r: reduced lawn

v: very reduced lawn

n: no survival

f: pseudorevertants

P: Precipitate

#: Standard deviation

~: fold increase over solvent control

 

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

S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		TA98		TA100		TA1535		TA1537		WP2 uvrA/pKM101
	Solvent Control (DMSO)	31 36 27	(31) 4.5#	133 144 168	(148) 17.9	12 14 16	(14) 2.0	14 12 13	(13) 1.0	191 206 186	(194) 10.4
	1.6 µg	37 43 37	(39) 3.5 1.3~	147 145 126	(139) 11.6 0.9	17 11 11	(13) 3.5 0.9	12 13 12	(12) 0.6 0.9	154 197 183	(178) 21.9 0.9
	5 µg	34 29 27	(30) 3.6 1.0	133 171 134	(146) 21.7 1.0	14 13 9	(12) 2.6 0.9	11 14 13	(13) 1.5 1.0	214 171 199	(195) 21.8 1.0
	16 µg	37 42 36	(38) 3.2 1.2	160 144 150	(151) 8.1 1.0	14 11 12	(12) 1.5 0.9	11 13 13	(12) 1.2 0.9	208 193 204	(202) 7.8 1.0
	50 µg	34 31 32	(32) 1.5 1.0	119 137 126	(127) 9.1 0.9	11 12 15	(13) 2.1 0.9	12 12 14	(13) 1.2 1.0	152 179 199	(177) 23.6 0.9
	160 µg	27 26 32	(28) 3.2 0.9	145 122 135	(134) 11.5 0.9	11 12 11	(11) 0.6 0.8	10 11 8	(10) 1.5 0.8	142 231 188	(187) 44.5 1.0
	500 µg	17 s P 22 s P 22 s P	(20) 2.9 0.6	20 s P 21 s P 6 s P	(16) 8.4 0.1	7 s P 8 s P 7 s P	(7) 0.6 0.5	8 s P 6 s P 9 s P	(8) 1.5 0.6	169 P 194 P 157 P	(173) 18.9 0.9
	1600 µg	25 r P 20 r P 19 r P	(21) 3.2 0.7	0 r P 0 r P 0 r P	(0) 0.0 0.0	0 r P 0 r P 0 r P	(0) 0.0 0.0	2 r P 2 r P 2 r P	(2) 0.0 0.6	135 P 167 P 153 P	(152) 16.0 0.8
	5000 µg	12 v P 13 v P 12 v P	(12) 0.6 0.4	0 v P 0 v P 0 v P	(0) 0.0 0.0	0 v P 0 v P 0 v P	(0) 0.0 0.0	1 v P 0 v P 0 v P	(0) 0.6 0.0	111 s P 118 s P 104 s P	(111) 7.0 0.6
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		2AA		2AA
		2 µg		2 µg		2 µg		2 µg		20 µg
		2385 2115 2090	(2197) 163.6# 70.9~	2421 2359 2577	(2452) 112.3 16.6	185 209 166	(187) 21.5 13.4	146 184 134	(155) 26.1 11.9	1496 1224 1766	(1495) 271.0 7.7
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		TA98		TA100		TA1535		TA1537		WP2 uvrA/pKM101
	Solvent Control (DMSO)	26 36 30	(31) 5.0#	119 129 122	(120) 3.6	14 15 17	(15) 1.5	11 11 10	(11) 0.6	173 165 175	(171) 5.3
	1.6 µg	30 35 24	(30) 5.5 1.0~	116 117 120	(123) 9.5 1.0	14 12 12	(13) 1.2 0.9	10 9 12	(10) 1.5 0.9	179 194 183	(185) 7.8 1.1
	5 µg	21 26 25	(24) 2.6 0.8	116 138 121	(115) 8.1 1.0	16 13 16	(15) 1.7 1.0	10 10 6	(9) 2.3 0.8	170 169 157	(165) 7.2 1.0
	16 µg	30 26 24	(27) 3.1 0.9	118 107 128	(124) 11.9 1.0	10 11 8	(10) 1.5 0.7	7 8 9	(8) 1.0 0.7	162 140 157	(153) 11.5 0.9
	50 µg	27 22 24	(24) 2.5 0.8	113 126 109	(120) 4.0 1.0	12 11 11	(11) 0.6 0.7	4 6 3	(4) 1.5 0.4	144 142 147	(144) 2.5 0.8
	160 µg	29 22 26	(26) 3.5 0.8	118 129 129	(120) 2.1 1.0	12 10 11	(11) 1.0 0.7	8 6 6	(7) 1.2 0.6	150 133 128	(137) 11.5 0.8
	500 µg	16 s P 20 s P 25 s P	(20) 3.2 0.8	121 s P 134 s P 116 s P	(117) 4.6 1.0	10 s P 10 s P 6 s P	(9) 2.3 0.6	7 s P 6 s P 8 s P	(7) 1.0 0.6	154 P 132 P 111 P	(132) 21.5 0.8
	1600 µg	21 r P 27 r P 26 r P	(20) 3.1 0.6	117 r P 96 r P 123 r P	(125) 2.1 1.0	9 r P 10 r P 14 r P	(11) 2.6 0.7	8 r P 7 r P 6 r P	(7) 1.0 0.6	147 P 119 P 137 P	(134) 14.2 0.8
	5000 µg	17 v P 23 v P 19 v P	(20) 3.1 0.6	126 v P 113 v P 107 v P	(0) 0.0 0.0	6 v P 6 v P 7 v P	(6) 0.6 0.4	6 v P 3 v P 6 v P	(5) 1.7 0.5	130 s P 149 s P 144 s P	(141) 9.8 0.8
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	2-nitrofluorene		Na azide		Na azide		9AA		K2Cr2O7
		1 µg		0.5 µg		0.5 µg		50 µg		25 µg
		1617 1490 1318	(1475) 150.1# 47.6~	546 498 506	(517) 25.7 4.2	428 369 448	(428) 52.3 28.5	156 188 111	(152) 38.7 13.8	1182 1111 1089	(1127) 48.6 6.6

s: slightly reduced lawn

r: reduced lawn

v: very reduced lawn

n: no survival

f: pseudorevertants

P: Precipitate

#: Standard deviation

~: fold increase over solvent control

Conclusions:

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

Executive summary:

The study was performed to the requirements of OECD Guideline 471 under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2 uvrA/pKM101 as indicator organisms in both the presence and absence of S-9 mix. The test strains were treated with the test item using the Ames (i) plate incorporation method and then (ii) pre incubation method at up to nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). A preliminary solubility assessment was conducted. As solubility was satisfactory in dimethyl sulphoxide, it was used throughout this study as the solvent for the test item. The dose range for Experiment 1 (plate incorporation method) was predetermined and was 1.6 to 5000 µg/plate (or 0.0072 to 22.5 μmol per plate) for all five indicator strains in both the presence and absence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix). The experiment was repeated in Experiment 2 (preincubation method) on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Up to nine test item dose levels were selected in Experiment 2 in order to achieve a minimum of four non-toxic dose levels and the toxic limit of the test item. The dose range employed ranged between 1.6 and 5000 µg/plate (or 0.0072 to 22.5 μmol per plate), in the presence or absence of S9-mix. The test item showed evidence of cytotoxicity. The minimum dose level at which cytotoxicity was seen was 500 μg (2.25 μmol) per plate. The maximum dose level scored for revertant colonies was 5000 μg (22.5 μmol) per plate. Evidence of cytotoxicity as indicated by reductions in the growth of the background lawns or in the incidence of spontaneous revertant colonies was seen for the four S. typhimurium strains at a dose of ≥ 500 μg(2.25μmol) per plate in the presence and absence of S9 mix and at a dose of 5000μg(22.5μmol) per plate for the E.coli strain in the presence and absence of S9 mix, respectively. On the day of dosing, precipitate was seen at a dose of ≥ 160μg(0.72μmol) per plate for all five strains in the presence and absence of S9 mix. On the day of scoring, precipitate was seen at a dose of ≥ 500μg(2.25μmol) per plate for all five strains in the presence and absence of S9 mix. The minimum dose at which precipitate was seen on the test plates was 500 μg (2.25 μmol) per plate. The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In both experiments there were no significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix. It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2 uvrA/pKM101 in the presence and absence of S-9 mix.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24-02-2020 to 23-04-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human lymphoblastoid cells (TK6) ; from laboratory in-house cell stock originally obtained from European Collection of Authenticated Cell Cultures (ECACC; part of Public Health England, United Kingdom), were used in this study. The ECACC states that its stocks of the TK6 cell line are obtained from the American Type Culture Collection (ATCC).
- Suitability of cells: TK6 cell line that was not cytokinesis blocked, the specific guidance relating to established cell lines and studies performed in the absence of cytochalasin B, was followed (OECD TG 487)
- Normal cell cycle time (negative control): See below for further information ; TK6 cell stocks are checked for mycoplasma and confirmed to be mycoplasma free. The doubling time for TK6 cells is between 13 and 15 hours (Lorge et al. (2016), Standardised cell sources and recommendations for good cell culture practices in genotoxicity testing. Mutation Research ; 809 : 1-15).

For cell lines:
- Absence of Mycoplasma contamination: TK6 cell stocks are checked for mycoplasma and confirmed to be mycoplasma free.
- Number of passages if applicable: Experiment 01: passage number 24 and Experiment 02: passage number 13 ; Experiment 03: passage number 17 and Experiment 04: passage number 23 ; (cell initiation dates indicated in the full study report).
- Methods for maintenance in cell culture: Vial-stock TK6 cells was removed from ultra-cold storage, thawed and initiated in culture using RPMI 1640 medium containing 10% heat-inactivated horse serum, antibiotics and Pluronic F68. Cells were passaged into fresh medium every 1 to 4 days for a minimum of 6 days prior to use. The cell density of TK6 cultures was not permitted to exceed 1.0 x 10^6 cells per mL at any time. Incubation was at 37°C in a humidified atmosphere of 5% CO2 in air.
- Cell cycle length, doubling time or proliferation index : The doubling time for TK6 cells is between 13 and 15 hours from Lorge et al. (2016).
- Modal number of chromosomes: Based on literature sources: Lorge et al. (2016), TK6 cells are expected to have a modal chromosome number of 47. Reported as confirmed by the laboratory.
- Periodically checked for karyotype stability: Yes
- Periodically ‘cleansed’ of spontaneous mutants: Yes [cleansing of working-cell stocks prior to ultra-cold storage is routine handling practice, according to Lorge et al. (2016)]
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: RPMI 1640 medium containing 10% heat-inactivated horse serum, antibiotics and Pluronic F68. Cells were passaged into fresh medium every 1 to 4 days for a minimum of 6 days prior to use. The cell density of TK6 cultures was not permitted to exceed 1.0 x 10^6 cells per mL at any time. Incubation was at 37°C in a humidified atmosphere of 5% CO2 in air. Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored during the study. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on historical (and control) data these temporary deviations are not considered to affect the integrity of the study.

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

TK6 cell line that was not cytokinesis blocked, the specific guidance relating to established cell lines and studies performed in the absence of cytochalasin B, was followed (OECD TG 487)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: from Recognised supplier (origin and dates within full study report). S9 Microsomal fraction: Lot No. 4189
- method of preparation of S9 mix: Documented in the full study report. Stored at -80ºC. Following thawing protein content was adjusted to 30 mg/mL by dilution with RPMI 1640 before incorporation into the S9 mix. S9-mix was prepared immediately before use. Excess was discarded.
- concentration or volume of S9 mix and S9 in the final culture medium: 1.0% v/v S9 (with Nicotinamide adenine dinucleotide phosphate 0.25 mM and Glucose-6-phosphate 1.25 mM)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A Certificate of S9 QC and Production Certificate including Activity is presented in the full study report. Additionally, prior to testing each batch was subjected to in-house testing to assess batch-to-batch variation using bacterial promutagens: cyclophosphamide and benzo[a]pyrene (TA1535 and TA98, respectively) and was found to be acceptable. Furthermore, 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:

- Experiment 1:
3 hours exposure time
(+S9) : 0 (control), 4.567, 6.851, 10.28, 15.41, 23.12, 34.68, 52.02, 78.04, 117.1, 175.6, 263.4, 395.1, 592.6, 888.9, 1333, 2000 μg/mL and 4.0 μg/mL CP (PC)
(-S9) : 0 (control), 4.567, 6.851, 10.28, 15.41, 23.12, 34.68, 52.02, 78.04, 117.1, 175.6, 263.4, 395.1, 592.6, 888.9, 1333, 2000 μg/mL and 50 ng/mL MMC
24 hours (continuous) exposure time
(-S9) : 0 (control), 4.567, 6.851, 10.28, 15.41, 23.12, 34.68, 52.02, 78.04, 117.1, 175.6, 263.4, 395.1, 592.6, 888.9, 1333, 2000 μg/mL and 30 ng/mL MMC and 7.5 ng/mL Colch
- Experiment 2 :
3 hours exposure time
(+S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 4.0 μg/mL CP (PC).
(-S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 50 ng/mL MMC
24 hours (continuous) exposure time
(-S9) : 0 (control), 7.500, 15.00, 30.00, 43.81, 53.01, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3 μg/mL plus 30 ng/mL MMC and 7.5 ng/mL Colch
- Experiment 3 :
3 hours exposure time
(+S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 4.0 μg/mL CP (PC).
(-S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 50 ng/mL MMC
- Experiment 4 :
3 hours exposure time
(-S9) : 0 (control), 7.500, 15.00, 30.54, 32.07, 33.67, 35.35, 37.12, 39.98, 40.93, 42.97, 45.17, 47.38, 49.75, 52.24, 54.85, 57.59, 60.47, 63.49, 66.67, 70.00 μg/mL and and 50 ng/mL MMC
The final dose ranges from which micronuclei were analysed in valid tests (Experiments were repeated until acceptable cytotoxicity values were obtained, and then scored for micronuclei) were:
Experiment 3: 3h (+S9) treatment schedule: 30.00 to 70.56 μg/mL
Experiment 4: 3h (-S9) treatment schedule: 45.12 to 63.49 μg/mL
Experiment 2: 24h (-S9) continuous treatment schedule: 30.00 to 64.14 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was soluble in DMSO at all tested concentrations and up to 200.00 mg/mL, within solubility checks were performed in-house. Test item concentrations were used within 1 hour after preparation. The final concentration of DMSO in the culture medium was 1.0% (v/v).
- Other: Formulated concentrations were adjusted/increased to allow for the stated water/impurity content (if applicable). See 'Test Material Information' for further details (if applicable). A test item-DMSO formulation solubility test was performed in R10P (RPMI 1640 media containing 10% heat inactivated horse serum, antibiotics and Pluronic F68) to assess the suitability of the solvent and to help with determining suitable test concentrations for the micronucleus assay. In the solubility test, precipitate was visible at a concentration of 2000 μg/mL after addition to R10P media. Following overnight incubation (humidified atmosphere of 5% CO2 at a temperature of 37°C) of the tubes (containing R10P media with test concentrations or solvent control), precipitate was visible at a concentration of 2000 μg/mL. In order to meet guideline criteria, 2000 μg/mL was therefore selected as the top concentration for all three treatment schedules. On the day of testing a stock solution of test item in DMSO was prepared at 200.0 mg/mL and further diluted in DMSO to prepare the required test solutions. In Experiment 2, 3 and 4, narrower dose ranges and lower top concentrations were prepared and dosed. Further details on test item concentrations are provided above and below.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

mitomycin C

Remarks:

without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1 (single)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): Vial-stock TK6 cells was removed from ultra-cold storage, thawed and initiated in culture using RPMI 1640 medium containing 10% heat-inactivated horse serum, antibiotics and Pluronic F68. Cells were passaged into fresh medium every 1 to 4 days for a minimum of 6 days prior to use. The cell density of TK6 cultures was not permitted to exceed 1.0 x 10^6 cells per mL at any time. Incubation was at 37°C in a humidified atmosphere of 5% CO2 in air. TK6 cells were harvested by centrifugation and re-suspended at 1.5 x 10^5 cells per mL in tissue culture medium, then dispensed into treatment tubes (3 hour treatments) or culture flasks (continuous treatments) in 4.95 mL aliquots.
- Test substance added in medium : Based on findings in the preliminary assessment of solubility, the test item was prepared in DMSO at 200.0 mg/mL. It was then prepared in a dilution series, typically with a 1.5-fold dilution factor and finally diluted for treatment by adding 50 μL to 4.95 mL tissue culture medium containing 1.5 x 10^5 cells per mL. For tests performed in the presence of metabolic activation, S9 mix was incorporated into the culture medium. Formulated test item (10 μL) was added to each tube/flask and gently mixed to ensure homogeneity. Cultures following the 3 hour treatment schedules, either in the presence or absence of S9 mix, were incubated (humidified atmosphere of 5% CO2 at a temperature of 37°C) for 3 hours in culture medium (±S9 mix), after which cells were washed and the medium replaced with fresh culture medium and then dispensed into culture flasks. Cultures were then incubated until 1.5 to 2.0 population doublings were achieved in the concurrent solvent control. Cultures following the continuous exposure treatment schedule (without S9 mix) were incubated (humidified atmosphere of 5% CO2 at a temperature of 37°C) until 1.5 to 2.0 population doublings were achieved in the concurrent solvent control. Appropriate positive controls were also included in each assay.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: See above.
- Exposure duration/duration of treatment: Experiment 1 and 2: 3 hours exposure time and/or 24 hours exposure time ; Experiment 3 and 4: 3 hours exposure time
- Harvest time after the end of treatment (sampling/recovery times): See above. Until 1.5 to 2.0 population doublings were achieved in the concurrent solvent control

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Not applicable, see below.
- If cytokinesis blocked method was used for micronucleus assay: TK6 cell line that was not cytokinesis blocked, the specific guidance relating to established cell lines and studies performed in the absence of cytochalasin B, was followed (OECD TG 487)
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Following the treatment and recovery periods (where applicable), cells were counted using an automated cell counter. Cells in the solvent treated controls were required to have achieved between 1.5 and 2.0 population doublings. If cells in the solvent treated controls achieved a population doubling (PD) of <1.5, cultures were incubated further until sufficient proliferation had been achieved. From the six solvent treated cultures, a minimum of two were required to achieve a PD of >1.5. If a PD of >2.0 was observed, the experiment was repeated. Relative Population Doubling (RPD) was calculated for each culture. After RPDs were calculated from cell counts, these data were used to select the concentrations from which to prepare slides for micronucleus analysis. From the cytotoxicity profile (inferred from RPDs), a minimum of 3 and a maximum of 6 concentrations per test item per treatment schedule were selected for microscopic analysis (according to appropriate criteria as specified in the guideline OECD TG 487). Cultures treated with concentrations selected for slide preparation and analysis (including solvent treated and positive controls) were harvested by centrifugation and resuspended in fresh medium (RPMI supplemented with Pluronic F68 alone), with cell densities adjusted to 2.5 x 10^5 cells per mL. Subsequently, thin monolayer cell preparations were made using a cytology centrifuge. The monolayers were allowed to air dry before fixation with methanol. Following fixation, slides were stained with 0.12 mg/mL acridine orange in Dulbecco’s phosphate buffered saline (pH 7.4).
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): All slides, including those for solvent and positive controls, were coded prior to analysis by someone other than the scorer.
- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification): Micronucleus analysis was performed on 2000 mononuclear cells per culture per test item concentration and control sample (i.e. where possible 1000 mononuclear cells per culture). Micronucleus scoring was performed by manual counting under a fluorescence microscope with typical magnification of x400. The number of mononuclear and micronucleated mononuclear cells scored were recorded. Micronucleus frequencies were recorded per 1000 mononuclear cells per culture. Mononucleated cells were selected if they were not overlapping and cytoplasm was clearly visible around the nuclei. Micronuclei were selected if they were less than one-third of the diameter of the main nucleus and located within the intact cytoplasm, evenly stained, spherical with a well-defined outline and not connected to the main nucleus. The number of micronuclei analysed from 2000 mononuclear cells for each selected test item dose was compared with that from the concurrent solvent control. Pair-wise statistical analysis employing a one-sided Fisher’s Exact test were used to evaluate statistical significance (p < 0.05). A linear trend test was employed (Cochran Armitage) in order to confirm a dose-response (p < 0.05).
- 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): Not applicable.
- Determination of polyploidy: Not applicable.
- Determination of endoreplication: Not applicable.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Population Doubling (PD) and/or Relative Population Doubling (RPD)
- Any supplementary information relevant to cytotoxicity: Cells in the solvent treated controls were required to have achieved between 1.5 and 2.0 population doublings. If cells in the solvent treated controls achieved a population doubling (PD) of <1.5, cultures were incubated further until sufficient proliferation had been achieved. From the six solvent treated cultures, a minimum of two were required to achieve a PD of >1.5. If a PD of >2.0 was observed, the experiment was repeated. Relative Population Doubling (RPD) was calculated for each culture. After RPDs were calculated from cell counts, these data were used to select the concentrations from which to prepare slides for micronucleus analysis. From the cytotoxicity profile (inferred from RPDs), a minimum of 3 and a maximum of 6 concentrations per test item per treatment schedule were selected for microscopic analysis (according to appropriate criteria as specified in the guideline OECD TG 487). Micronucleus analysis was performed on 2000 mononuclear cells per culture per test item concentration and control sample (i.e. where possible 1000 mononuclear cells per culture).

METHODS FOR MEASUREMENTS OF GENOTOXICIY :
Statistical analysis is performed and/or provided the acceptability criteria are met :
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
(a) at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent solvent control
(b) the increase was dose-related in at least one experimental condition when evaluated with an appropriate trend test
(c) any of the results were outside the historical solvent control range (Poisson-based 95% control limits)
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
(a) none of the test concentrations exhibited a statistically significant increase compared with the concurrent solvent control
(b) there was no concentration-related increase when evaluated using an appropriate trend test
(c) all results were inside the distribution of the historical solvent control data (Poisson-based 95% control limits)
Although most experiments are expected to yield clearly positive or negative results, in some cases the data preclude making a definitive judgement about the activity of the test item Such equivocal responses may occur regardless of the number of times the experiment is repeated. Expert judgement in accordance with the OECD TG 487 and related mutagenicity guidelines is therefore utilised in the assessment.

OTHER:
ACTIVATION: The S9-mix was prepared prior to the dosing of the test cultures. Metabolic activation was achieved by adding 1.0% v/v S9 in the exposure medium (with Nicotinamide adenine dinucleotide phosphate 0.25 mM and Glucose-6-phosphate 1.25 mM).

Rationale for test conditions:

In accordance with the OECD TG 487 guidelines.

Evaluation criteria:

Statistical analysis is performed and/or provided the acceptability criteria are met :
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
(a) at least one of the test concentrations exhibited a statistically significant increase compared with the concurrent solvent control
(b) the increase was dose-related in at least one experimental condition when evaluated with an appropriate trend test
(c) any of the results were outside the historical solvent control range (Poisson-based 95% control limits)
A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
(a) none of the test concentrations exhibited a statistically significant increase compared with the concurrent solvent control
(b) there was no concentration-related increase when evaluated using an appropriate trend test
(c) all results were inside the distribution of the historical solvent control data (Poisson-based 95% control limits)
Although most experiments are expected to yield clearly positive or negative results, in some cases the data preclude making a definitive judgement about the activity of the test item Such equivocal responses may occur regardless of the number of times the experiment is repeated. Expert judgement in accordance with the OECD TG 487 and related mutagenicity guidelines is therefore utilised in the assessment.

An in vitro micronucleus test is considered acceptable if it meets the following Acceptability Criteria:
(a) Solvent (DMSO) controls : the frequency of mononucleate cells with micronuclei for the solvent cultures must approximate those of the acceptable ranges from the Test Facility’s historical control database and/or published values.
(b) Positive controls : the positive control chemicals must induce a statistically significant (p <0.05) increase in the frequency of micronuclei in mononucleate cells compared with the concurrent solvent controls.

Statistics:

Statistical analysis was performed using software package(s). The number of micronuclei analysed from 2000 mononuclear cells for each selected test item dose was compared with that from the concurrent solvent control. Pair-wise statistical analysis employing a one-sided Fisher’s Exact test were used to evaluate statistical significance (p < 0.05). A linear trend test was employed (Cochran Armitage) in order to confirm a dose-response (p < 0.05).
See above for further information on the statistical analysis and acceptability criteria.

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no reported significant changes in pH, when the test item was dosed into media.
- Effects of osmolality: There was no reported significant changes in osmolality (did not increase by more than 50 mOsm) when the test item was dosed into media. Specifically: at the end of each treatment period the osmolality and pH of the solvent control and highest test concentration in treatment medium were determined for each treatment schedule. There were no relevant changes observed between the solvent controls and test concentrations evaluated.
- Evaporation from medium: Not reported.
- Water solubility: Not applicable.
- Precipitation and time of the determination:
3 hours exposure
Experiment 1: (+S9 and -S9): upon addition of the test item to the culture medium, precipitate was observed at concentrations of 888.9 μg/mL and above. At the end of the treatment period precipitate was observed at a concentration of 175.6 μg/mL and above. In Experiment 2 and 3 : (+S9 and -S9): no precipitate was reported, up to 125.0 μg/mL dose level. In Experiment 4: (+S9 and -S9): no precipitate was reported, up to 70.0 μg/mL dose level.
24 hours (continuous) exposure
Experiment 1: (-S9): Upon addition of the test item to the culture medium, precipitate was observed at concentrations of 888.9 μg/mL and above. At the end of the treatment period precipitate was observed at a concentration of 263.4 μg/mL and above. In Experiment 2: (-S9): no precipitate was reported, up to 103.3 μg/mL dose level.
- Definition of acceptable cells for analysis: The highest dose level examined for micronuclei were the cultures that produced 55 ± 5% cytotoxicity (or as technically possible). The lowest dose level had little or no cytotoxicity (little or no effect on RPD, or approximately the same as solvent control). Also cultures treated with an intermediate dose level were examined. See above and below for more information. For test items that do not yield apparent cytotoxicity, where solubility is a limiting factor, the lowest concentration at which minimal precipitate was visible in cultures was selected as the highest concentration for slide preparation and micronucleus analysis. For test items that do not yield apparent cytotoxicity or solubility issues, the three highest test concentrations were selected for further microscopic analysis.
- Other confounding effects: None reported.

RANGE-FINDING/SCREENING STUDIES (if applicable): Not applicable.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See tables.
For all test methods and criteria for data analysis and interpretation: See tables. Also see above “Evaluation” and “Statistics” fields.
- Concentration-response relationship where possible: See tables.
- Statistical analysis: See tables.
- Other: Concentrations tested were as follows
- Experiment 1:
3 hours exposure time
(+S9) : 0 (control), 4.567, 6.851, 10.28, 15.41, 23.12, 34.68, 52.02, 78.04, 117.1, 175.6, 263.4, 395.1, 592.6, 888.9, 1333, 2000 μg/mL and 4.0 μg/mL CP (PC)
(-S9) : 0 (control), 4.567, 6.851, 10.28, 15.41, 23.12, 34.68, 52.02, 78.04, 117.1, 175.6, 263.4, 395.1, 592.6, 888.9, 1333, 2000 μg/mL and 50 ng/mL MMC
24 hours (continuous) exposure time
(-S9) : 0 (control), 4.567, 6.851, 10.28, 15.41, 23.12, 34.68, 52.02, 78.04, 117.1, 175.6, 263.4, 395.1, 592.6, 888.9, 1333, 2000 μg/mL and 30 ng/mL MMC and 7.5 ng/mL Colch
- Experiment 2 :
3 hours exposure time
(+S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 4.0 μg/mL CP (PC).
(-S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 50 ng/mL MMC
24 hours (continuous) exposure time
(-S9) : 0 (control), 7.500, 15.00, 30.00, 43.81, 53.01, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3 μg/mL plus 30 ng/mL MMC and 7.5 ng/mL Colch
- Experiment 3 :
3 hours exposure time
(+S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 4.0 μg/mL CP (PC).
(-S9) : 0 (control), 7.500, 15.00, 30.00, 53.01, 58.31, 64.14, 70.56, 77.62, 85.38, 93.91, 103.3, 113.6, 125.0 μg/mL and 50 ng/mL MMC
- Experiment 4 :
3 hours exposure time
(-S9) : 0 (control), 7.500, 15.00, 30.54, 32.07, 33.67, 35.35, 37.12, 39.98, 40.93, 42.97, 45.17, 47.38, 49.75, 52.24, 54.85, 57.59, 60.47, 63.49, 66.67, 70.00 μg/mL and and 50 ng/mL MMC
The final dose ranges from which micronuclei were analysed in valid tests (Experiments were repeated until acceptable cytotoxicity values were obtained, and then scored for micronuclei) were:
Experiment 3: 3h (+S9) treatment schedule: 30.00 to 70.56 μg/mL
Experiment 4: 3h (-S9) treatment schedule: 45.12 to 63.49 μg/mL
Experiment 2: 24h (-S9) continuous treatment schedule: 30.00 to 64.14 μg/mL

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: See tables.
o In the case of the cytokinesis-block method: CBPI and/or ; distribution of mono-, bi- and multi-nucleated cells : see tables.
o Other observations when applicable (complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells): see tables.

- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate: see tables.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- All the positive control items produced a statistically significant increase in the number of binucleated cells with micronuclei (statistically significant p ≤ 0.01 or were within the 95% Historic Control Data limit range presented in the full study report).
- Negative (solvent/vehicle) historical control data: All vehicle (DMSO) controls had frequencies of mono- and binucleated cells with micronuclei within the range expected for normal human lymphocytes. (Within the 95% Historic Control Data limit range presented in the full study report).

The final dose ranges from which micronuclei were analysed in valid tests (Experiments were repeated until acceptable cytotoxicity values were obtained, and then scored for micronuclei) were:

Experiment 3: 3h (+S9) treatment schedule: 30.00 to 70.56 μg/mL

Experiment 4: 3h (-S9) treatment schedule: 45.12 to 63.49 μg/mL

Experiment 2: 24h (-S9) continuous treatment schedule: 30.00 to 64.14 μg/mL

 

Table 1. Experiment 1 : Cytotoxicity From 3 hour Exposure Testing in the Presence of S9 Mix and Absence of S9 Mix

Short Treatment (3 hour) +S9 mix Experiment 1 (cytotoxicity only, no scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %	MN per culture	MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0	ND	ND	ND	N
4.567	ND	ND	ND	ND	ND	N
6.851	ND	ND	ND	ND	ND	N
10.28	ND	ND	ND	ND	ND	N
15.41	ND	ND	ND	ND	ND	N
23.12	ND	ND	ND	ND	ND	N
34.68	-0.34	100.34	ND	ND	ND	N
52.02	9.21	90.79	ND	ND	ND	N
78.04	12.33	87.67	ND	ND	ND	N
117.1	100.0	-62.38	ND	ND	ND	N
175.6	100.0	-146.24	ND	ND	ND	Y
263.4	ND	ND	ND	ND	ND	Y
395.1	ND	ND	ND	ND	ND	Y
592.6	ND	ND	ND	ND	ND	Y
888.9	ND	ND	ND	ND	ND	Y
1333	ND	ND	ND	ND	ND	Y
2000	ND	ND	ND	ND	ND	Y
CPA 4 µg/mL	17.45	82.55	ND	ND	ND	N
Short Treatment (3 hour) -S9 mix Experiment 1 (cytotoxicity only, no scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %	MN per culture	MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0	ND	ND	ND	N
4.567	ND	ND	ND	ND	ND	N
6.851	ND	ND	ND	ND	ND	N
10.28	ND	ND	ND	ND	ND	N
15.41	ND	ND	ND	ND	ND	N
23.12	ND	ND	ND	ND	ND	N
34.68	12.21	87.79	ND	ND	ND	N
52.02	19.25	80.75	ND	ND	ND	N
78.04	76.15	23.85	ND	ND	ND	N
117.1	100.0	-166.63	ND	ND	ND	N
175.6	100.0	-182.87	ND	ND	ND	Y
263.4	ND	ND	ND	ND	ND	Y
395.1	ND	ND	ND	ND	ND	Y
592.6	ND	ND	ND	ND	ND	Y
888.9	ND	ND	ND	ND	ND	Y
1333	ND	ND	ND	ND	ND	Y
2000	ND	ND	ND	ND	ND	Y
MMC 50 ng/mL	23.31	76.69	ND	ND	ND	N

Where: Conc. = concentration; RPD = Relative population doubling; MN = micronuclei; MN per culture = micronuclei per 1000 mononuclear cells; MN per conc = combined micronuclei per culture i.e. MN per 2000 mononuclear cells; CPA = cyclophosphamide; ND = Not determined; Precipitate observed at the end of treatment period = N = no / Y = yes.

 

Table 2. Experiment 3: Cytotoxicity and/or Cytotoxicity and Slide-Scored Data From 3 hour Exposure Testing in the Presence of S9 Mix and Absence of S9 Mix

Short Treatment (3 hour) +S9 mix Experiment 3 (cytotoxicity and scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %	MN per culture		MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0	12/1000		24/2000	N/A	N
			12/1000
7.500	ND	ND	ND		ND	ND	N
15.00	12.27	87.73	ND		ND	ND	N
30.00 #	12.27	87.73	13/1000		26/2000	ns	N
			13/1000
53.01 #	18.70	81.30	7/1000		14/2000	ns	N
			7/1000
58.31 #	30.84	69.16	10/1000		19/2000	ns	N
			9/1000
64.14 #	29.81	70.19	12/1000		23/2000	ns	N
			11/1000
70.56 #	56.10	43.90	7/1000		15/2000	ns	N
			8/1000
77.62	55.19	44.81	ND		ND	ND	N
85.38	53.96	46.04	ND		ND	ND	N
93.91	100.0	-165.58	ND		ND	ND	N
103.3	100.0	-155.06	ND		ND	ND	N
113.6	100.0	-166.10	ND		ND	ND	N
125.0	100.0	-162.92	ND		ND	ND	N
CPA 4 µg/mL #	36.88	63.12	60/1000		112/2000	1.602x10^-15	N
			52/1000
Short Treatment (3 hour) -S9 mix Experiment 3 (cytotoxicity only, no scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %		MN per culture	MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0		ND	ND	ND	N
7.500	3.95	96.05		ND	ND	ND	N
15.00	9.47	90.53		ND	ND	ND	N
30.00	11.99	88.01		ND	ND	ND	N
53.01	100.0	-5.38		ND	ND	ND	N
58.31	52.24	47.76		ND	ND	ND	N
64.14	100.0	-73.46		ND	ND	ND	N
70.56	100.0	-92.20		ND	ND	ND	N
77.62	100.0	-137.76		ND	ND	ND	N
85.38	100.0	-123.08		ND	ND	ND	N
93.91	100.0	-188.71		ND	ND	ND	N
103.3	100.0	-167.72		ND	ND	ND	N
113.6	100.0	-194.89		ND	ND	ND	N
125.0	100.0	-171.57		ND	ND	ND	N
MMC 50 ng/mL	41.02	58.98		ND	ND	ND	N

Where: Conc. = concentration; RPD = Relative population doubling; MN = micronuclei; MN per culture = micronuclei per 1000 mononuclear cells; MN per conc = combined micronuclei per culture i.e. MN per 2000 mononuclear cells; MMC = Mitomycin C; CPA = cyclophosphamide; N/A = not applicable; ND = Not determined; Precipitate observed at the end of the treatment period = N = no, ns = not significant (by Fisher’s exact test); # = next to concentration text in table indicates concentrations and controls that were analysed for micronuclei; Cochran-Armitage trend test = 1.899x10^-01 = not significant.

 

Table 3. Experiment 4: Cytotoxicity and Slide-Scored Data From 3 hour Exposure Testing in the Absence of S9 Mix

Short Treatment (3 hour) -S9 mix Experiment 4 (cytotoxicity and scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %	MN per culture	MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0	8/1000	18/2000	N/A	N
			10/1000
7.500	ND	ND	ND	ND	ND	N
15.00	ND	ND	ND	ND	ND	N
30.54	ND	ND	ND	ND	ND	N
32.07	ND	ND	ND	ND	ND	N
33.67	ND	ND	ND	ND	ND	N
35.35	ND	ND	ND	ND	ND	N
37.12	ND	ND	ND	ND	ND	N
39.98	ND	ND	ND	ND	ND	N
40.93	ND	ND	ND	ND	ND	N
42.97	6.90	93.10	ND	ND	ND	N
45.12 #	1.39	98.61	19/1000+	19/2000+	ns	N
47.38	16.71	83.29	ND	ND	ND	N
49.75	21.62	78.38	ND	ND	ND	N
52.24	16.84	83.16	ND	ND	ND	N
54.85	18.63	81.37	ND	ND	ND	N
57.79 #	23.21	76.79	9/1000	17/2000	ns	N
			8/1000
60.47 #	48.61	51.39	7/1000	15/2000	ns	N
			8/1000
63.49 #	55.84	44.16	7/1000	15/2000	ns	N
			8/1000
66.67 #	51.13	48.87	ND	ND	ND	N
70.00	100.0	-145.56	ND	ND	ND	N
MMC 50 ng/mL #	24.87	75.13	60/1000	124/2000	<2.2x10^-16	N
			64/1000

Where: Conc. = concentration; RPD = Relative population doubling; MN = micronuclei; MN per culture = micronuclei per 1000 mononuclear cells; MN per conc = combined micronuclei per culture i.e. MN per 2000 mononuclear cells; MMC = Mitomycin C; N/A = not applicable; ND = Not determined; Precipitate observed at the end of the treatment period = N = no, ns = not significant (by Fisher’s exact test); + = All 2000 cells scored from one culture as the duplicate culture was unsuitable for analysis when examined under the microscope; # = next to concentration text in table indicates concentrations and controls that were analysed for micronuclei; Cochran-Armitage trend test = 4.385x10^-01 = not significant.

 

Table 4. Experiment 1 & 2 Cytotoxicity and/or Experiment 2: Cytotoxicity and Slide-Scored Data From Continuous (24 hour) Exposure Testing in the Absence of S9 Mix

Continuous Treatment (24 hour) -S9 mix Experiment 1 (cytotoxicity only, no scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %	MN per culture	MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0	ND	ND	N/A	N
4.567	ND	ND	ND	ND	ND	N
6.851	ND	ND	ND	ND	ND	N
10.28	ND	ND	ND	ND	ND	N
15.41	ND	ND	ND	ND	ND	N
23.12	ND	ND	ND	ND	ND	N
34.68	8.33	91.67	ND	ND	ND	N
52.02	14.07	85.93	ND	ND	ND	N
78.04	91.35	8.65	ND	ND	ND	N
117.1	100.0	-162.17	ND	ND	ND	N
175.6	100.0	-21.17	ND	ND	ND	N
263.4	100.0	-234.34	ND	ND	ND	Y
395.1	ND	ND	ND	ND	ND	Y
592.6	ND	ND	ND	ND	ND	Y
888.9	ND	ND	ND	ND	ND	Y
1333	ND	ND	ND	ND	ND	Y
2000	ND	ND	ND	ND	ND	Y
MMC 30 ng/mL	45.51	54.49	ND	ND	ND	N
COL 7.5 ng/mL	39.57	60.43	ND	ND	ND	N
Continuous Treatment (24 hour) -S9 mix Experiment 2 (cytotoxicity and scoring)
Conc. (µg/mL)	Cytotoxicity %	RPD %	MN per culture	MN per conc.	p-value	Precipitate
Solvent (DMSO)	0	100.0	7/1000	19/2000	N/A	N
			12/1000
7.500	ND	ND	ND	ND	ND	N
15.00	ND	ND	ND	ND	ND	N
30.00 #	-3.46	103.46	7/1000	16/2000	ns	N
			9/1000
43.81	20.97	79.03	ND	ND	ND	N
48.81 #	20.51	79.49	7/1000	15/2000	ns	N
			8/1000
53.01 #	22.70	77.30	9/1000	16/2000	ns	N
			7/1000
58.31 #	31.69	68.31	7/1000	21/2000	ns	N
			14/1000
64.14 #	59.85	40.15	7/1000	15/2000	ns	N
			8/1000
70.56	92.41	7.59	ND	ND	ND	N
77.62	87.62	12.38	ND	ND	ND	N
85.38	100.0	-146.07	ND	ND	ND	N
93.91	100.0	-109.05	ND	ND	ND	N
103.3	100.0	-115.11	ND	ND	ND	N
MMC 30 ng/mL #	44.67	55.33	65/1000	113/2000	<2.2x10^-16	N
			48/1000
COL 7.5 ng/mL #	38.35	61.65	63/1000	122/2000	<2.2x10^-16	N
			59/1000

Where: Conc. = concentration; RPD = Relative population doubling; MN = micronuclei; MN per culture = micronuclei per 1000 mononuclear cells; MN per conc = combined micronuclei per culture i.e. MN per 2000 mononuclear cells; MMC = Mitomycin C; COL = Colchicine; N/A = not applicable; ND = Not determined; Precipitate observed at the end of the treatment period = N = no; ns = not significant (by Fisher’s exact test); # = next to concentration text in table indicates concentrations and controls that were analysed for micronuclei; Cochran-Armitage trend test = 9.073x10^-01 = not significant.

Conclusions:

Interpretation of results: negative with and without metabolic activation
Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic to TK6 human lymphoblastoid cells.

Executive summary:

The study was performed to the requirements of OECD TG 487 under GLP conditions to assess the test item ability to induce micronuclei in TK6 human lymphoblastoid cells using the in vitro micronucleus test method with manual scoring of microscope slides, either in the presence or absence of a metabolic activation system (1.0% v/v S9-mix). The test as applied to the test system under three treatment schedules 3 hours in both the absence and presence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix), and a continuous (24 hour) treatment in the absence of S9 mix. Cells were harvested for micronucleus analysis when cells in the solvent control treated cultures achieved between 1.5 and 2.0 normal cell cycles in the relevant exposure condition. In all treatments, the solvent used was dimethyl sulphoxide (DMSO). TK6 human lymphoblastoid cells were not cytokinesis blocked and cell division was determined using a measure of relative population doubling (RPD). Duplicate cultures were used for each test concentration and micronuclei were scored in a minimum of 1000 mononuclear cells per culture (where possible), using two cultures per treatment concentration (2000 mononuclear cells in total). Initially, a test item-DMSO formulation solubility test was performed in R10P (RPMI 1640 media containing 10% heat inactivated horse serum, antibiotics and Pluronic F68) to assess the suitability of the solvent and to help with determining suitable test concentrations for the micronucleus assay. In the solubility test, precipitate was visible at a concentration of 2000μg/mL after addition to R10P media. Following overnight incubation (humidified atmosphere of 5% CO2 at a temperature of 37°C) of the tubes (containing R10P media with test concentrations or solvent control), precipitate was visible at a concentration of 2000μg/mL.In order tomeet guideline criteria, 2000μg/mL was therefore selected as the top concentration for all three treatment schedules for Experiment 1. On the day of testing a stock solution of test item in DMSO was prepared at 200.0 mg/mL and further diluted in DMSO to prepare the required test solutions. Upon addition of test item to the culture medium in Experiment 1 precipitate was observed at concentrations of 888.9 μg/mL and above in all treatment schedules. At the end of the treatment period precipitate was observed at concentrations of 175.6 μg/mL and above in the three hour treatment schedules and 263.4 μg/mL and above in the continuous treatment schedule. In Experiment 2, 3 and 4, narrower dose ranges and lower top concentrations were prepared and dosed due to non achievement of required cytotoxicity (55% ±5%) dose levels. The final dose ranges from which micronuclei were analysed in valid tests (Experiments were repeated until acceptable cytotoxicity values were obtained, and then scored for micronuclei) were: Experiment 3: 3h (+S9) treatment schedule: 30.00 to 70.56 μg/mL, Experiment 4: 3h (-S9) treatment schedule: 45.12 to 63.49 μg/mL and Experiment 2: 24h (-S9) continuous treatment schedule: 30.00 to 64.14 μg/mL. In Experiment 3, cytotoxicity of 56.10% was observed at a concentration of 70.56 μg/mL in the 3 hour +S9 mix treatment. In Experiment 4 cytotoxicity of 55.84% was observed at a concentration of 63.49 μg/mL in the 3 hour -S9 mix treatment. In Experiment 2 cytotoxicity of 59.85% was observed at a concentration of 64.14 μg/mL in the continuous treatment. In further experiments, no precipitate was observed at any concentrations tested during any stage of the experiment. In final tests, where slides were selected for analysis, for all treatment schedules, data for background micronucleus induction in the solvent controls were consistent with the historical control database (based on 95% Poisson confidence limits) for TK6 cells. The concurrent positive controls produced statistically significant (p <0.05) increases in micronuclei compared with the concurrent negative controls and were within the expected range based on the historical control database. No statistically significant increases in micronucleus formation were observed at any test item concentration analysed, all micronucleus values fell within the 95% Poisson confidence limits of the solvent control and there were no concentration related increases when evaluated with a Cochran-Armitage trend tests. Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic to TK6 human lymphoblastoid cells, in vitro in the presence or absence of S9 mix.

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, 2020 : The study was performed to the requirements of OECD Guideline 471 under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2 uvrA/pKM101 as indicator organisms in both the presence and absence of S-9 mix. The test strains were treated with the test item using the Ames (i) plate incorporation method and then (ii) pre incubation method at up to nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). A preliminary solubility assessment was conducted. As solubility was satisfactory in dimethyl sulphoxide, it was used throughout this study as the solvent for the test item. The dose range for Experiment 1 (plate incorporation method) was predetermined and was 1.6 to 5000 µg/plate (or 0.0072 to 22.5 μmol per plate) for all five indicator strains in both the presence and absence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix). The experiment was repeated in Experiment 2 (preincubation method) on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Up to nine test item dose levels were selected in Experiment 2 in order to achieve a minimum of four non-toxic dose levels and the toxic limit of the test item. The dose range employed ranged between 1.6 and 5000 µg/plate (or 0.0072 to 22.5 μmol per plate), in the presence or absence of S9-mix. The test item showed evidence of cytotoxicity. The minimum dose level at which cytotoxicity was seen was 500 μg (2.25 μmol) per plate. The maximum dose level scored for revertant colonies was 5000 μg (22.5 μmol) per plate. Evidence of cytotoxicity as indicated by reductions in the growth of the background lawns or in the incidence of spontaneous revertant colonies was seen for the four S. typhimurium strains at a dose of ≥ 500 μg(2.25μmol) per plate in the presence and absence of S9 mix and at a dose of 5000μg(22.5μmol) per plate for the E.coli strain in the presence and absence of S9 mix, respectively. On the day of dosing, precipitate was seen at a dose of ≥ 160μg(0.72μmol) per plate for all five strains in the presence and absence of S9 mix. On the day of scoring, precipitate was seen at a dose of ≥ 500μg(2.25μmol) per plate for all five strains in the presence and absence of S9 mix. The minimum dose at which precipitate was seen on the test plates was 500 μg (2.25 μmol) per plate. The vehicle (DMSO) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. In both experiments there were no significant increase in numbers of revertant (histidine or tryptophan-independent) colonies was seen with any of the five indicator strains either in the presence or absence of S9 mix. It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2 uvrA/pKM101 in the presence and absence of S-9 mix.

Key study : OECD TG 487, 2020 : The study was performed to the requirements of OECD TG 487 under GLP conditions to assess the test item ability to induce micronuclei in TK6 human lymphoblastoid cells using the in vitro micronucleus test method with manual scoring of microscope slides, either in the presence or absence of a metabolic activation system (1.0% v/v S9-mix). The test as applied to the test system under three treatment schedules 3 hours in both the absence and presence of an in vitro activation system based on S9 fraction obtained from Aroclor 1254-induced rat liver (S9 mix), and a continuous (24 hour) treatment in the absence of S9 mix. Cells were harvested for micronucleus analysis when cells in the solvent control treated cultures achieved between 1.5 and 2.0 normal cell cycles in the relevant exposure condition. In all treatments, the solvent used was dimethyl sulphoxide (DMSO). TK6 human lymphoblastoid cells were not cytokinesis blocked and cell division was determined using a measure of relative population doubling (RPD). Duplicate cultures were used for each test concentration and micronuclei were scored in a minimum of 1000 mononuclear cells per culture (where possible), using two cultures per treatment concentration (2000 mononuclear cells in total). Initially, a test item-DMSO formulation solubility test was performed in R10P (RPMI 1640 media containing 10% heat inactivated horse serum, antibiotics and Pluronic F68) to assess the suitability of the solvent and to help with determining suitable test concentrations for the micronucleus assay. In the solubility test, precipitate was visible at a concentration of 2000μg/mL after addition to R10P media. Following overnight incubation (humidified atmosphere of 5% CO2 at a temperature of 37°C) of the tubes (containing R10P media with test concentrations or solvent control), precipitate was visible at a concentration of 2000μg/mL.In order tomeet guideline criteria, 2000μg/mL was therefore selected as the top concentration for all three treatment schedules for Experiment 1. On the day of testing a stock solution of test item in DMSO was prepared at 200.0 mg/mL and further diluted in DMSO to prepare the required test solutions. Upon addition of test item to the culture medium in Experiment 1 precipitate was observed at concentrations of 888.9 μg/mL and above in all treatment schedules. At the end of the treatment period precipitate was observed at concentrations of 175.6 μg/mL and above in the three hour treatment schedules and 263.4 μg/mL and above in the continuous treatment schedule. In Experiment 2, 3 and 4, narrower dose ranges and lower top concentrations were prepared and dosed due to non achievement of required cytotoxicity (55% ±5%) dose levels. The final dose ranges from which micronuclei were analysed in valid tests (Experiments were repeated until acceptable cytotoxicity values were obtained, and then scored for micronuclei) were: Experiment 3: 3h (+S9) treatment schedule: 30.00 to 70.56 μg/mL, Experiment 4: 3h (-S9) treatment schedule: 45.12 to 63.49 μg/mL and Experiment 2: 24h (-S9) continuous treatment schedule: 30.00 to 64.14 μg/mL. In Experiment 3, cytotoxicity of 56.10% was observed at a concentration of 70.56 μg/mL in the 3 hour +S9 mix treatment. In Experiment 4 cytotoxicity of 55.84% was observed at a concentration of 63.49 μg/mL in the 3 hour -S9 mix treatment. In Experiment 2 cytotoxicity of 59.85% was observed at a concentration of 64.14 μg/mL in the continuous treatment. In further experiments, no precipitate was observed at any concentrations tested during any stage of the experiment. In final tests, where slides were selected for analysis, for all treatment schedules, data for background micronucleus induction in the solvent controls were consistent with the historical control database (based on 95% Poisson confidence limits) for TK6 cells. The concurrent positive controls produced statistically significant (p <0.05) increases in micronuclei compared with the concurrent negative controls and were within the expected range based on the historical control database. No statistically significant increases in micronucleus formation were observed at any test item concentration analysed, all micronucleus values fell within the 95% Poisson confidence limits of the solvent control and there were no concentration related increases when evaluated with a Cochran-Armitage trend tests. Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic to TK6 human lymphoblastoid cells, in vitro in the presence or absence of S9 mix.

Justification for classification or non-classification

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