dec-1-en-4-yne

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, 2017

negative, in vitro chromosome aberration test (with and without S-9 activation), OECD TG 473, 2017

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:

03-05-2017 to 22-05-2017

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 2016 ; signature: October 2016

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:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: prepared in house (dates within full study report) ; S9 Microsomal fraction: Lot No. PB/βNF S9 17/02/2017
- method of preparation of S9 mix: Documented in the full study report. Stored at -196ºC
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A Certificate of S9 Efficacy is presented in the full study report. Additionally, 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 (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): 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
All strains (presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in DMSO at the same concentration in solubility checks performed. DMSO 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.

Key result

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

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

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

S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	130 102 112	(115) 14.2#	27 16 16	(20) 6.4	22 17 25	(21) 4.0	34 29 27	(30) 3.6	22 24 23	(23) 1.0
	1.5 µg	116 104 116	(112) 6.9	28 18 19	(22) 5.5	20 15 20	(18) 2.9	32 22 27	(27) 5.0	16 15 10	(14) 3.2
	5 µg	120 121 106	(116) 8.4	13 10 9	(11) 2.1	23 27 17	(22) 5.0	19 29 27	(25) 5.3	15 14 23	(17) 4.9
	15 µg	115 77 107	(100) 20.0	9 10 10	(10) 0.6	15 11 11	(12) 2.3	30 14 18	(21) 8.3	15 28 8	(17) 10.1
	50 µg	91 108 101	(100) 8.5	16 14 7	(12) 4.7	11 25 27	(21) 8.7	13 15 16	(15) 1.5	14 9 16	(13) 3.6
	150 µg	96 77 74	(82) 11.9	8 7 12	(9) 2.6	16 13 18	(16) 2.5	25 15 25	(22) 5.8	8 10 9	(9) 1.0
	500 µg	96 85 110	(97) 12.5	22 16 9	(16) 6.5	23 21 26	(23) 2.5	11 13 16	(13) 2.5	19 11 5	(12) 7.0
	1500 µg	103 89 111	(101) 11.1	7 9 8	(8) 1.0	19 25 27	(24) 4.2	29 10 9	(16) 11.3	10 10 4	(8) 3.5
	5000 µg	100 89 65	(85) 17.9	6 5 7	(6) 1.0	11 11 19	(14) 4.6	9 8 13	(10) 2.6	4 3 8	(5) 2.6
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
		560 1005 961	(842) 245.2	1366 1227 1375	(1323) 83.0	532 459 499	(497) 36.6	394 294 325	(338) 51.2	190 169 149	(169) 20.5
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	115 111 98	(108) 8.9#	19 12 16	(16) 3.5	26 13 31	(23) 9.3	21 18 18	(19) 1.7	7 15 16	(13) 4.9
	1.5 µg	114 116 76	(102) 22.5	13 8 18	(13) 5.0	28 15 26	(23) 7.0	34 24 19	(26) 7.6	8 9 12	(10) 2.1
	5 µg	104 124 119	(116) 10.4	8 9 12	(10) 2.1	30 33 32	(32) 1.5	26 20 19	(22) 3.8	14 14 12	(13) 1.2
	15 µg	120 98 109	(109) 11.0	10 8 8	(9) 1.2	31 31 24	(29) 4.0	17 19 18	(18) 1.0	10 10 10	(10) 0.0
	50 µg	120 95 112	(109) 12.8	8 9 12	(10) 2.1	24 38 24	(29) 8.1	31 21 24	(25) 5.1	8 14 11	(11) 3.0
	150 µg	120 116 113	(116) 3.5	11 9 10	(10) 1.0	34 39 29	(34) 5.0	23 25 24	(24) 1.0	10 9 14	(11) 2.6
	500 µg	98 123 102	(108) 13.4	8 7 10	(8) 1.5	33 18 32	(28) 8.4	28 18 30	(25) 6.4	7 7 14	(9) 4.0
	1500 µg	28 123 116	(89) 52.9	7 8 7	(7) 0.6	29 36 29	(31) 4.0	32 21 24	(26) 5.7	9 14 17	(13) 4.0
	5000 µg	108 112 116	(112) 4.0	10 8 10	(9) 1.2	31 36 25	(31) 5.5	24 25 21	(23) 2.1	11 16 12	(13) 2.6
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		994 636 997	(876) 207.6	231 151 163	(182) 43.1	1171 892 849	(971) 174.8	141 109 136	(129) 17.2	597 704 554	(618) 77.2

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 (DMSO)	99 84 71	(85) 14.0#	31 30 32	(31) 1.0	21 28 25	(25) 3.5	29 21 25	(25) 4.0	12 6 11	(10) 3.2
	1.5 µg	87 64 85	(79) 12.7	22 30 34	(29) 6.1	28 23 25	(25) 2.5	28 29 26	(28) 1.5	8 10 8	(9) 1.2
	5 µg	77 75 75	(76) 1.2	28 26 31	(28) 2.5	28 37 34	(33) 4.6	21 25 25	(24) 2.3	8 9 6	(8) 1.5
	15 µg	61 76 91	(76) 15.0	34 28 30	(31) 3.1	26 42 36	(35) 8.1	22 29 29	(27) 4.0	9 9 4	(7) 2.9
	50 µg	79 72 60	(70) 9.6	29 23 14	(22) 7.5	31 36 21	(29) 7.6	21 13 18	(17) 4.0	6 12 8	(9) 3.1
	150 µg	67 64 64	(65) 1.7	23 29 32	(28) 4.6	36 26 18	(27) 9.0	25 8 17	(17) 8.5	10 7 8	(8) 1.5
	500 µg	60 62 62	(61) 1.2	21 21 32	(25) 6.4	42 43 14	(33) 16.5	29 20 7	(19) 11.1	8 11 6	(8) 2.5
	1500 µg	70 69 73	(71) 2.1	23 31 23	(26) 4.6	26 20 22	(23) 3.1	17 15 21	(18) 3.1	6 S 4 S 4 S	(5) 1.2
	5000 µg	57 S 52 S 66 S	(58) 7.1	22 S 23 S 25 S	(23) 1.5	29 S 24 S 23 S	(25) 3.2	20 S 8 S 23 S	(17) 7.9	6 S 4 S 5 S	(5) 1.0
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
		988 845 884	(906) 73.9	790 722 657	(723) 66.5	386 392 400	(393) 7.0	302 280 223	(268) 40.8	314 369 304	(329) 35.0
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (DMSO)	71 79 94	(81) 11.7#	16 22 27	(22) 5.5	43 38 34	(38) 4.5	30 32 23	(28) 4.7	18 11 20	(16) 4.7
	5 µg	84 75 89	(83) 7.1	15 30 21	(22) 7.5	38 39 38	(38) 0.6	13 30 25	(23) 8.7	10 25 20	(18) 7.6
	15 µg	75 77 77	(76) 1.2	22 29 18	(23) 5.6	35 36 32	(34) 2.1	31 36 26	(31) 5.0	16 12 20	(16) 4.0
	50 µg	84 66 76	(75) 9.0	23 27 27	(26) 2.3	25 33 30	(29) 4.0	32 21 24	(26) 5.7	14 13 15	(14) 1.0
	150 µg	92 66 69	(76) 14.2	28 27 29	(28) 1.0	22 50 19	(30) 17.1	25 22 15	(21) 5.1	16 9 19	(15) 5.1
	500 µg	76 67 84	(76) 8.5	26 23 21	(23) 2.5	32 32 42	(35) 5.8	13 18 36	(22) 12.1	19 24 12	(18) 6.0
	1500 µg	72 68 76	(72) 4.0	29 19 21	(23) 5.3	29 41 41	(37) 6.9	39 15 16	(23) 13.6	14 21 14	(16) 4.0
	5000 µg	77 S 83 S 71 S	(77) 6.0	22 S 24 S 22 S	(23) 1.2	45 39 32	(39) 6.5	12 S 17 S 15 S	(15) 2.5	9 S 15 S 9 S	(11) 3.5
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
		1237 1193 885	(1105) 191.8	567 539 613	(573) 37.4	209 220 217	(215) 5.7	148 175 158	(160) 13.7	265 241 202	(236) 31.8

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 (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. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.In the first mutation test (absence of S9-mix), the test item induced substantial reductions in the frequency of revertant colonies (below a factor of 0.5 fold under the concurrent vehicle control) initially from 1500 µg/plate to Salmonella strains TA1535 and TA1537 and at 5000 µg/plate to E.coli strain WP2uvrA and Salmonella strain TA98. A small reduction in TA100 revertant colony frequency was also noted at 5000 µg/plate. In the presence of S9-mix, small reductions in TA1535 revertant colony were noted from 500 µg/plate. Consequently for the second mutation test, the same maximum dose level (5000 µg/plate) was employed. 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:

09-05-2017 to 21-08-2017

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 29/03/2018Type and composition of metabolic activation system: Rat liver S9
- source of S9: prepared in house (dates within full study report) ; S9 Microsomal fraction: Lot No. PB/βNF S9 31/03/2017
- method of preparation of S9 mix: Documented in the full study report. Stored at -196ºC
- concentration or volume of S9 mix and S9 in the final culture medium: 2% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test and a Certificate of S9 Efficacy is presented in the full study report. Additionally, concurrent positive control substances all produced a positive response (p ≤ 0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.

Test concentrations with justification for top dose:

The maximum dose level was 1362.4 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 1362.4 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), 5.32, 10.64, 21.29, 42.58, 85.15, 170.3, 340.6, 681.2 and 1362.4 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*, 7*, 14*, 28*, 42*, 49, 56, 84 μg/mL and MMC 0.4* μg/mL
4(20)-hour with S9: 0*, 14*, 28*, 42*, 56*, 70, 84, 98 μg/mL and CP 1* μg/mL
24-hour without S9: 0*, 28*, 42*, 56*, 84*, 98, 112, 168 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. The test item was insoluble in culture media at 13.62 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 136.24 mg/mL in solubility checks performed in-house. The maximum dose level (determined prior to the test based on molecular weight) was 1362.4 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 1362.4 μg/mL range (full results recorded in the full study report). The test item was formulated within two hours of it being applied to the test system.
- Other: Formulated concentrations were adjusted/increased to allow for the stated water/impurity content. See 'Test Material Information' for further details

Untreated negative controls:

other: Vehicle control served as the negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation, 0.4 μg/ml for 4(20)-hour and 0.1 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 as the negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation 1 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)
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.

Rationale for test conditions:

In accordance with the OECD TG 473 guidelines.

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)

Key result

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 170.3 µg/mL in all three 4(20)-hour and 24-hour (continuous) exposure groups.
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 42.58 μg/mL in the 4(20)-hour exposure groups and at and above 21.29μg/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 also observed at the end of the exposure period at and above 85.15 μg/mL in the 4(20)-hour exposure groups and at and above 170.3 μg/mL in the 24-hour continuous exposure group. This was considered to indicate overall toxicity to the cell population. A reduced pellet signifies that maximum exposure is occurring in the remaining cell population.
In the main test: Hemolysis was observed at and above 7 μg/mL and 14 μg/mL in the 4(20)-hour exposure groups in the absence and presence of S9, respectively and at and above 28 μg/mL in the 24-hour exposure group. A reduced cell pellet was also observed at the end of the exposure period at and above 49 μg/mL in the 4(20)-hour exposure in the absence of S9, at and above 70 μg/mL in the presence of S9 and at and above 98 μg/mL in the 24-hour exposure group.

RANGE-FINDING/SCREENING STUDIES: The dose range for the Preliminary Toxicity Test was 0 to 1362.4 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 84 μg/mL in the 4(20)-hour without S9 and 98 μg/mL in the 4(20)-hour with S9 (2%) exposure groups and up to 168 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*, 7*, 14*, 28*, 42*, 49, 56, 84 μg/mL and MMC 0.4* μg/mL

4(20)-hour with S9: 0*, 14*, 28*, 42*, 56*, 70, 84, 98 μg/mL and CP 1* μg/mL

24-hour without S9: 0*, 28*, 42*, 56*, 84*, 98, 112, 168 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 49 μg/mL in the 4(20)-hour exposure group in the absence of S9, up to

56 μg/mL in the 4(20)-hour exposure group in the presence of S9 and up to 84 μg/mL 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 at and above 7 μg/mL and 14 μg/mL in the 4(20)-hour exposure groups in the absence and presence of S9, respectively and at and above 28 μg/mL in the 24-hour exposure group. A reduced cell pellet was also observed at the end of the exposure period at and above 49 μg/mL in the 4(20)-hour exposure in the absence of S9, at and above 70 μg/mL in the presence of S9 and at and above 98 μg/mL in the 24-hour exposure group. A reduced cell pellet was also observed at the end of the exposure period at and above 49 μg/mL in the 4(20)-hour exposure in the absence of S9, at and above 70 μg/mL in the presence of S9 and at and above 98 μg/mL in the 24-hour exposure group.

 

In the 4(20)-hour exposure group in the absence of S9, 30%, 63% and 81% mitotic inhibition was achieved at 28, 42 and 49 μg/mL, respectively. In the presence of S9, 44% mitotic inhibition was demonstrated at 56 μg/mL and the dose level above (70 μg/mL) had insufficient cell numbers for scoring. In the 24-hour exposure group 49% mitotic inhibition was achieved at 56 μg/mL and 71% at 84 μg/mL. The maximum dose level selected for metaphase analysis was based on toxicity and was 42 μg/mL in the 4(20)-hour exposure group in the absence of S9 where marginally greater than acceptable toxicity was achieved. In the presence of S9, the maximum dose level selected for scoring was 56 μg/mL which was the maximum dose level with sufficient cells for scoring. For the 24-hour exposure group near optimum toxicity was achieved at 56 μg/mL and this was the maximum dose level selected for scoring.

 

- 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:

In the (20)-hour exposure group in the presence of S9 and 24-hour in the absence of S9 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. However, near optimum toxicity was achieved and it was considered that the test item had been adequately tested in accordance with the testing guideline. 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.

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 5.32 to 1362.4 μg/mL where cytotoxicity was observed at the dose levels before the onset of test item precipitation in the presence and absence of metabolic activation. 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 without S9-Mix : 0, 7, 14, 28, 42, 49, 56 84 μg/mL, for (20)-hour with S9-Mix (2%): 0, 14, 28, 42, 56, 70, 84, 98 μg/mL and 24-hour continuous exposure without 0, 28, 42, 56, 84, 98, 112, 168 μg/mL. The test item was markedly toxic and exhibited a steep toxicity curve. 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 included optimum (55±5%) or induced near optimum or greater 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, 2017 : 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 (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. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.In the first mutation test (absence of S9-mix), the test item induced substantial reductions in the frequency of revertant colonies (below a factor of 0.5 fold under the concurrent vehicle control) initially from 1500 µg/plate to Salmonella strains TA1535 and TA1537 and at 5000 µg/plate to E.coli strain WP2uvrA and Salmonella strain TA98. A small reduction in TA100 revertant colony frequency was also noted at 5000 µg/plate. In the presence of S9-mix, small reductions in TA1535 revertant colony were noted from 500 µg/plate. Consequently for the second mutation test, the same maximum dose level (5000 µg/plate) was employed. 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, 2017 : 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 5.32 to 1362.4 μg/mL where cytotoxicity was observed at the dose levels before the onset of test item precipitation in the presence and absence of metabolic activation. 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 without S9-Mix : 0, 7, 14, 28, 42, 49, 56 84 μg/mL, for (20)-hour with S9-Mix (2%): 0, 14, 28, 42, 56, 70, 84, 98 μg/mL and 24-hour continuous exposure without 0, 28, 42, 56, 84, 98, 112, 168 μg/mL. The test item was markedly toxic and exhibited a steep toxicity curve. 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 included optimum (55±5%) or induced near optimum or greater 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