(5E)-3,5-dimethyloct-5-en-4-ol

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, 2018

negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473, 2018

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:

09-03-2018 to 04-04-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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

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

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2017 ; signature: November 2017

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine or tryptophan locus

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:

Rat liver S9

Test concentrations with justification for top dose:

Experiment 1 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate

Experiment 2 (pre-incubation method): Up to eight 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 (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 µg/plate.
All strains (presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL and partially miscible in dimethyl sulphoxide at the same concentration, but was fully miscible in acetone at 100 mg/mL in solubility checks performed. Acetone was selected as the vehicle.
- Other: Formulated concentrations were adjusted/increased to allow for the stated water/impurity content. See 'Test Material Information' for further details.

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. All of the plates were pre-incubated in sealed, small volume containers, by application of 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media. All of the plates were sealed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) during the incubation procedure (37 ± 3 ºC for approximately 48 to 72 hours) to minimize potential losses of the test item from the plates. After incubation, the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts 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. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media Subsequently, the procedure for incubation and duration was the same as in Experiment 1.

SELECTION AGENT (mutation assays): histidine-deficient agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

In accordance with the OECD TG 471 guidelines.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5. 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).
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

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.

Remarks on result:

other: all strains/cell types tested

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
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	104 120 125	(116) 11.0#	17 18 13	(16) 2.6	28 28 33	(30) 2.9	22 22 19	(21) 1.7	6 9 11	(9) 2.5
	1.5 µg	115 125 103	(114) 11.0	15 12 9	(12) 3.0	31 46 23	(33) 11.7	20 18 13	(17) 3.6	10 2 8	(7) 4.2
	5 µg	100 89 91	(93) 5.9	11 14 14	(13) 1.7	30 37 22	(30) 7.5	30 17 19	(22) 7.0	4 9 5	(6) 2.6
	15 µg	97 113 92	(101) 11.0	20 17 11	(16) 4.6	21 31 22	(25) 5.5	19 10 22	(17) 6.2	5 7 10	(7) 2.5
	50 µg	95 92 87	(91) 4.0	14 13 15	(14) 1.0	29 36 26	(30) 5.1	14 23 23	(20) 5.2	8 6 5	(6) 1.5
	150 µg	0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0
	500 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
	1500 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
	5000 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		304 308 309	(307) 2.6	129 126 188	(148) 35.0	323 244 218	(262) 54.7	147 133 153	(144) 10.3	212 211 213	(212) 1.0
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	131 108 119	(119) 11.5#	8 11 12	(10) 2.1	44 37 37	(39) 4.0	25 21 26	(24) 2.6	8 6 7	(7) 1.0
	1.5 µg	112 147 124	(128) 17.8	7 21 18	(15) 7.4	42 41 35	(39) 3.8	29 33 34	(32) 2.6	8 13 9	(10) 2.6
	5 µg	123 129 117	(123) 6.0	14 15 13	(14) 1.0	48 39 32	(40) 8.0	25 24 26	(25) 1.0	8 3 8	(6) 2.9
	15 µg	100 96 115	(104) 10.0	7 10 7	(8) 1.7	40 39 40	(40) 0.6	31 31 21	(28) 5.8	6 10 11	(9) 2.6
	50 µg	113 151 104	(123) 24.9	11 15 10	(12) 2.6	40 37 46	(41) 4.6	28 27 20	(25) 4.4	8 10 12	(10) 2.0
	150 µg	94 S 104 S 88 S	(95) 8.1	6 S 11 S 8 S	(8) 2.5	34 38 41	(38) 3.5	16 S 21 S 17 S	(18) 2.6	10 S 10 S 7 S	(9) 1.7
	500 µg	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0	15 S 19 S 17 S	(17) 2.0	0 V 0 V 0 V	(0) 0.0
	1500 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
	5000 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		1760 1703 1887	(1783) 94.2	332 295 320	(316) 18.9	182 191 197	(190) 7.5	122 103 123	(116) 11.3	268 272 317	(286) 27.2

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

S            Sparse bacterial background lawn

V           Very weak bacterial background lawn

T            Toxic, no 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 of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	138 140 144	(141) 3.1#	20 15 16	(17) 2.6	28 30 21	(26) 4.7	17 24 22	(21) 3.6	10 13 8	(10) 2.5
	0.05 µg	127 126 139	(131) 7.2	13 23 21	(19) 5.3	21 27 21	(23) 3.5	21 26 18	(22) 4.0	8 6 7	(7) 1.0
	0.15 µg	127 135 142	(135) 7.5	18 16 14	(16) 2.0	28 22 19	(23) 4.6	19 11 15	(15) 4.0	11 11 7	(10) 2.3
	0.5 µg	140 148 147	(145) 4.4	14 19 17	(17) 2.5	30 36 25	(30) 5.5	19 20 14	(18) 3.2	13 7 7	(9) 3.5
	1.5 µg	140 135 137	(137) 2.5	17 17 15	(16) 1.2	22 15 36	(24) 10.7	25 15 14	(18) 6.1	9 6 10	(8) 2.1
	5 µg	128 130 145	(134) 9.3	20 18 13	(17) 3.6	31 26 29	(29) 2.5	19 18 19	(19) 0.6	9 9 6	(8) 1.7
	15 µg	119 128 120	(122) 4.9	12 24 15	(17) 6.2	28 27 35	(30) 4.4	21 23 23	(22) 1.2	5 9 15	(10) 5.0
	50 µg	124 119 110	(118) 7.1	11 17 12	(13) 3.2	34 31 32	(32) 1.5	17 24 20	(20) 3.5	5 7 11	(8) 3.1
	150 µg	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	26 S 21 S 28 S	(25) 3.6	19 S 12 S 7 S	(13) 6.0	11 S 7 S 10 S	(9) 2.1
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		536 534 583	(551) 27.7	274 323 294	(297) 24.6	567 505 496	(523) 38.7	359 332 327	(339) 17.2	184 153 223	(187) 35.1
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Acetone)	144 142 143	(143) 1.0#	19 9 12	(13) 5.1	34 33 25	(31) 4.9	23 27 23	(24) 2.3	9 10 11	(10) 1.0
	0.15 µg	140 122 151	(138) 14.6	8 10 17	(12) 4.7	35 33 24	(31) 5.9	18 32 17	(22) 8.4	8 10 5	(8) 2.5
	0.5 µg	122 132 122	(125) 5.8	16 16 16	(16) 0.0	34 33 33	(33) 0.6	22 21 26	(23) 2.6	5 9 7	(7) 2.0
	1.5 µg	141 147 141	(143) 3.5	14 18 7	(13) 5.6	23 39 32	(31) 8.0	30 25 21	(25) 4.5	12 11 5	(9) 3.8
	5 µg	140 138 141	(140) 1.5	9 13 14	(12) 2.6	33 37 27	(32) 5.0	30 23 25	(26) 3.6	14 11 11	(12) 1.7
	15 µg	142 133 141	(139) 4.9	9 18 16	(14) 4.7	31 29 30	(30) 1.0	31 22 30	(28) 4.9	13 8 6	(9) 3.6
	50 µg	139 144 133	(139) 5.5	7 12 11	(10) 2.6	38 30 34	(34) 4.0	25 19 27	(24) 4.2	6 8 10	(8) 2.0
	150 µg	120 S 118 S 106 S	(115) 7.6	7 S 6 S 7 S	(7) 0.6	27 29 38	(31) 5.9	15 S 21 S 19 S	(18) 3.1	9 S 8 S 10 S	(9) 1.0
	500 µg	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		1674 1752 1869	(1765) 98.1	291 282 299	(291) 8.5	208 213 185	(202) 14.9	137 139 147	(141) 5.3	268 286 274	(276) 9.2

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 item using the Ames pre incubation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Eight test item dose levels were again 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 was amended following the results of Experiment 1 and ranged between 0.05 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The vehicle (acetone) 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 or the toxic limit of the test item depending on the strain type and presence of S9-mix.The test item caused a visible reduction in the growth of the bacterial background lawns of all of the strains dosed in the absence of S9-mix from 150 μg/plate in both the presence and absence of S9-mix. There were no biologically relevant 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). A minor statistical value was noted in Experiment 1 (TA98 at 1.5 μg/plate in the presence of S9-mix), however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.In Experiment 2, the toxic limit was employed as the maximum concentration in the second mutation test from Experiment 1. The test item induced an identical toxic response to the first experiment with weakened bacterial background lawns noted from initially 150 μg/plate in both the presence and absence of S9-mix. No precipitates were observed at any dose level in either the presence or absence of S9-mix. 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:

20-04-2018 to 11-07-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: 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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

: Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

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: July 2017 ; signature: November 2017

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):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) 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. Based on over 20 years in house data for 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 it is considered to be approximately 16 hours. Therefore using this average 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.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal fraction: Lot No. PB/βNF S9 29/03/2018

Test concentrations with justification for top dose:

The maximum dose level was 1560 µg/mL, calculated to be equivalent to 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 1560 μg/mL range (full results recorded in the full study report). Formulated concentrations were adjusted/increased to allow for the stated water/impurity content. See 'Test Material Information' for further details.

I. Preliminary toxicity test: 0 (control), 6.1, 12.2, 24.4, 48.8, 97.5, 195, 390, 780 and 1560 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:
4(20)-hour without S9: 0*, 25, 50, 100*, 150*, 200*, 250, 300 μg/mL and MMC 0.2* μg/mL
4(20)-hour with S9: 0*, 25, 50, 100*, 150*, 200*, 250, 300 μg/mL and CP 3* μg/mL
24-hour without S9: 0*, 6.25, 12.5, 25*, 50*, 75*, 100, 150 μg/mL and MMC 0.1* 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: In a previously conducted OECD TG 471 study (cited in the full study report), the test item was immiscible in aqueous media at 50 mg/mL, partially miscible in dimethyl sulphoxide at the same concentration. A further solubility check was performed within this study. Dimethyl sulphoxide and acetone indicated that the test item was miscible at 156 mg/mL. Dimethyl sulphoxide was chosen due to better suitability when used with human lymphocytes. The maximum dose level (determined prior to the test based on molecular weight) was 1560 µg/mL, which was calculated to be equivalent to 10 mM, 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 1560 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, 0.2 μg/ml for 4(20)-hour and 0.1 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 athe negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation 3 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:

METHOD OF APPLICATION: Other:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile non-vented plastic flasks for each culture: 9.05 mL MEM, 10% (FBS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; 0.75 mL heparinized whole blood

DURATION
- Preincubation period: Not reported.
- Exposure duration:
The preliminary toxicity test was performed using both of the exposure conditions as described for both experiments (below) in the absence of metabolic activation only.
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. 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.

SPINDLE INHIBITOR (cytogenetic assays): demecolcine (Colcemid 0.1 μg/ml)

NUMBER OF REPLICATIONS: The study conducted two replicates (A and B) at each dose level and exposure duration groups.

NUMBER OF CELLS EVALUATED: 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.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
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.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes. Cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid 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.
- Other: Scoring: Where possible, 300 consecutive well-spread metaphases from each concentration (150 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 then the analysis was terminated after a total of 15 metaphases with aberrations (excluding gaps) were recorded. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

Evaluation criteria:

Positive response 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 response 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 analyze 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 will be 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)

Species / strain:

lymphocytes: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

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. Sterile non-vented plastic flasks for each culture, thereby preventing evaporation from medium.
- 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 390 µg/mL in the 4(20)-hour exposure groups and at and above 780 µg/mL in the continuous exposure group.
Main test: No observations of precipitate were noted at the end of exposure in the blood cultures of any of the exposure groups.
- Other confounding effects: In the preliminary test: Hemolysis was observed following exposure to the test item at and above 97.5 μg/mL in the 4(20)-hour exposure groups and at and above 48.8 μg/mL in the 24-hour continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. Additionally, a reduced cell pellet was observed at and above 390 μg/mL in the exposure groups in the absence of metabolic activation (S9) and at and above 780 μg/mL in the exposure group in the presence of S9. A reduced pellet signifies that maximum exposure is occurring in the remaining cell population.
In the main test: Hemolysis was observed following exposure to the test item at and above 50 μg/mL in the 4(20)-hour and 24-hour exposure group in the absence of metabolic activation (S9) and at and above 100 μg/mL in the 4(20)-hour exposure group in the presence of S9. Additionally, a reduced cell pellet was observed at 300 μg/mL in the in both of the 4(20)-hour exposure groups and at and above 100 μg/mL in the 24-hour exposure group.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 1560 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. Specifically 300 μg/mL in the 4(20)-hour exposure groups and up to 150 µg/mL in the 24-hour continuous exposure group.

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. (statistically significant p ≤ 0.01 or were 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:

4(20)-hour without S9: 0*, 25, 50, 100*, 150*, 200*, 250, 300 μg/mL and MMC 0.2* μg/mL

4(20)-hour with S9: 0*, 25, 50, 100*, 150*, 200*, 250, 300 μg/mL and CP 3* μg/mL

24-hour without S9: 0*, 6.25, 12.5, 25*, 50*, 75*, 100, 150 μg/mL and MMC 0.1* μg/mL

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

 

The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Cell Growth Inhibition Test and that there were metaphases suitable for scoring present up to 250 μg/mL in the 4(20)-hour exposure groups and up to the maximum concentration in the 24-hour continuous exposure group. No observations of precipitate were noted at the end of exposure in the blood cultures of any of the exposure groups. Hemolysis was observed following exposure to the test item at and above 50 μg/mL in the 4(20)-hour and 24-hour exposure group in the absence of metabolic activation (S9) and at and above 100 μg/mL in the 4(20)-hour exposure group in the presence of S9. Additionally, a reduced cell pellet was observed at 300 μg/mL in the in both of the 4(20)-hour exposure groups and at and above 100 μg/mL in the 24-hour exposure group.

 

The qualitative observations indicated a dose-related inhibition of mitotic index was observed in all exposure groups. In the 4(20)-hour exposure group in the absence of S9, 11% and 47% mitotic inhibition was achieved at 150 and 200 μg/mL, respectively. Above this dose level, there were too few metaphases present to analyse. Therefore, the maximum dose level selected for metaphase analysis was 150 μg/mL because this dose level approached the optimum range for toxicity as defined by the OECD 473 test guideline (55±5%) and the dose above was considered too toxic for analysis. In the presence of S9, 32% and 49% inhibition of mitotic index was observed at 150 and 200 μg/mL, respectively. Above this dose level, there were too few metaphases present to assess. Consequently, the maximum dose level selected for metaphase analysis was 200 μg/mL because this dose level approached the optimum range for toxicity as defined by the OECD 473 test guideline (55±5%) and the dose above was considered too toxic for analysis. In the 24-hour continuous exposure group, an inhibition of mitotic index of 25% and 42% was noted at 50 and 75 μg/mL. Above 75 μg/mL, there were too few metaphases for analysis. The maximum dose level selected for metaphase analysis was 75 μg/mL because this dose level approached the optimum range for toxicity as defined by the OECD 473 test guideline (55±5%) and the dose above was considered too toxic for analysis.

 

- 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. There was one indication of endoreduplication noted.

 

2. Discussion:

All three exposure groups approached but did not quite enter the optimum toxicity range as defined by the OECD 473 test guideline (55±5%); and the dose level above indicated that there were too few metaphases for analysis. The test item dose range was also quite narrow confirming that the small differences between the Cell Growth Inhibition Test (Preliminary Toxicity Test) and the Main Experiment contributed to the failure to achieve optimum toxicity. It should be noted that the vehicle controls gave frequencies of chromosomal aberrations that were within the laboratory Historical Control Data range and that the positive controls gave frequencies of chromosomal aberrations in excess of the concurrent solvent control values confirming that the test system was working within normal parameters. Therefore, the test item was considered to have been adequately tested.

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 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. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test (Cell Growth Inhibition Test) between 6.1 to 1560 μg/mL where the results indicated that the maximum concentration should be limited toxicity for all exposure groups for dose selection. The dose levels selected for the Main Test were as follows: 4(20)-hour with and without S9-Mix (2%) : 0, 25, 50, 100, 150, 200, 250, 300 μg/mL and 24-hour continuous exposure without S9: 0, 6.25, 12.5, 25, 50, 75, 100, 150 μg/mL. All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes and which were within the current historical control data range. 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 toxic to human lymphocytes and but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that approached 55±5% mitotic inhibition. 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, 2018 : 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 item using the Ames pre incubation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. Eight test item dose levels were again 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 was amended following the results of Experiment 1 and ranged between 0.05 and 500 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The vehicle (acetone) 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 or the toxic limit of the test item depending on the strain type and presence of S9-mix.The test item caused a visible reduction in the growth of the bacterial background lawns of all of the strains dosed in the absence of S9-mix from 150 μg/plate in both the presence and absence of S9-mix. There were no biologically relevant 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). A minor statistical value was noted in Experiment 1 (TA98 at 1.5 μg/plate in the presence of S9-mix), however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.In Experiment 2, the toxic limit was employed as the maximum concentration in the second mutation test from Experiment 1. The test item induced an identical toxic response to the first experiment with weakened bacterial background lawns noted from initially 150 μg/plate in both the presence and absence of S9-mix. No precipitates were observed at any dose level in either the presence or absence of S9-mix. 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, 2018 : The study was performed to the requirements of OECD TG 473 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. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; a 4-hour exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4-hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation. The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test (Cell Growth Inhibition Test) between 6.1 to 1560 μg/mL where the results indicated that the maximum concentration should be limited toxicity for all exposure groups for dose selection. The dose levels selected for the Main Test were as follows: 4(20)-hour with and without S9-Mix (2%) : 0, 25, 50, 100, 150, 200, 250, 300 μg/mL and 24-hour continuous exposure without S9: 0, 6.25, 12.5, 25, 50, 75, 100, 150 μg/mL. All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes and which were within the current historical control data range. 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 toxic to human lymphocytes and but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that approached 55±5% mitotic inhibition. 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