Diethoxymethane

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 09 March 2018 Experimental completion date 29 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: Diethoxymethane
Synonym: Ethylal
CAS Number: 462-95-3
Physical State/Appearance: Clear colourless liquid
Storage Conditions: Approximately 4 °C, in the dark, under nitrogen
No correction for purity was required.

Target gene:

Histidine locus in the Salmonella strains and tryptophan locus in E.coli.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)

Test concentrations with justification for top dose:

Experiment 1 – Plate Incorporation Method
The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed.

Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 μg/plate.

Vehicle / solvent:

The test item was fully miscible in sterile distilled water at 50 mg/mL in solubility checks performed in-house. Sterile distilled water was therefore selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

2 μg/plate for WP2uvrA, 3 μg/plate for TA100, 5 μg/plate for TA1535

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

absence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

80 μg/plate for TA1537

Positive control substance:

9-aminoacridine

Remarks:

absence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 μg/plate for TA98

Positive control substance:

other: 4-Nitroquinoline-1-oxide

Remarks:

absence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, 10 μg/plate for WP2uvrA

Positive control substance:

other: 2-Aminoanthracene

Remarks:

presence of S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Remarks:

5 μg/plate for TA98

Positive control substance:

benzo(a)pyrene

Remarks:

presence of S9-mix

Details on test system and experimental conditions:

Test for Mutagenicity: Experiment 1 – Plate Incorporation Method

Without Metabolic Activation
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method

Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).


The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The solvent and negative controls were performed in triplicate.
The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation. The positive controls were performed in triplicate.

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. A fold increase greater than two times the concurrent solvent control for all strains (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 will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.

The vehicle (sterile distilled water) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1 (plate incorporation)
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (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).

Experiment 2 (pre-incubation)
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 μg/plate).
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (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).

Spontaneous Mutation Rates (ConcurrentNegativeControls)

Experiment1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
119		16		26		27		9
118	(127)	12	(15)	23	(30)	21	(25)	11	(10)
144		16		41		28		10
						28
						20	(25)†
						26

 

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
107		18		24		27		12
141	(131)	23	(18)	25	(31)	20	(25)	17	(13)
146		14		43		28		9

 

†             Experimental procedure repeated at a later date (with metabolic activation (S9 -mix)) due to contamination in the original test

 

Test Results: Experiment 1 – Without MetabolicActivation(Plate Incorporation)

 

Test Period		From: 13 March 2018					To: 16 March 2018
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Water)	150 150 120	(140) 17.3#	14 17 14	(15) 1.7	42 30 33	(35) 6.2	24 26 25	(25) 1.0	12 12 7	(10) 2.9
	1.5 µg	150 151 118	(140) 18.8	14 17 18	(16) 2.1	37 36 31	(35) 3.2	25 17 20	(21) 4.0	6 9 12	(9) 3.0
	5 µg	114 133 140	(129) 13.5	17 20 18	(18) 1.5	24 19 36	(26) 8.7	20 34 16	(23) 9.5	14 6 8	(9) 4.2
	15 µg	127 145 150	(141) 12.1	15 21 22	(19) 3.8	37 33 42	(37) 4.5	19 32 27	(26) 6.6	5 12 11	(9) 3.8
	50 µg	129 132 135	(132) 3.0	16 11 14	(14) 2.5	33 29 30	(31) 2.1	33 21 28	(27) 6.0	8 4 8	(7) 2.3
	150 µg	138 144 137	(140) 3.8	18 21 12	(17) 4.6	37 30 36	(34) 3.8	23 23 31	(26) 4.6	10 9 13	(11) 2.1
	500 µg	138 128 144	(137) 8.1	14 14 17	(15) 1.7	28 23 28	(26) 2.9	30 29 30	(30) 0.6	16 10 5	(10) 5.5
	1500 µg	129 148 155	(144) 13.5	13 10 14	(12) 2.1	34 34 36	(35) 1.2	15 12 23	(17) 5.7	9 6 12	(9) 3.0
	5000 µg	122 130 133	(128) 5.7	7 12 21	(13) 7.1	37 35 29	(34) 4.2	29 19 14	(21) 7.6	8 17 4	(10) 6.7
Positive controls S9-Mix (-)	Name DoseLevel No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		753 740 719	(737) 17.2	499 140 552	(397) 224.1	715 675 657	(682) 29.7	315 315 473	(368) 91.2	220 106 212	(179) 63.6

 

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

4NQO      4-Nitroquinoline-1-oxide

9AA        9-Aminoacridine

#            Standarddeviation

Test Results: Experiment 1 – With MetabolicActivation(Plate Incorporation)

 

Test Period		From: 13 March 2018 From: 19 March 2018†					To: 16 March 2018 To: 22 March 2018†
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98†		TA1537
	Solvent Control (Water)	124 142 147	(138) 12.1#	14 15 16	(15) 1.0	36 36 29	(34) 4.0	24 22 35	(27) 7.0	9 9 8	(9) 0.6
	1.5 µg	127 133 118	(126) 7.5	14 21 17	(17) 3.5	35 40 33	(36) 3.6	25 22 27	(25) 2.5	9 10 9	(9) 0.6
	5 µg	145 129 143	(139) 8.7	16 18 8	(14) 5.3	28 44 37	(36) 8.0	41 33 37	(37) 4.0	12 13 10	(12) 1.5
	15 µg	102 130 133	(122) 17.1	20 12 24	(19) 6.1	36 45 36	(39) 5.2	38 30 34	(34) 4.0	11 10 12	(11) 1.0
	50 µg	146 129 120	(132) 13.2	16 24 12	(17) 6.1	36 34 40	(37) 3.1	32 37 38	(36) 3.2	6 5 10	(7) 2.6
	150 µg	144 143 145	(144) 1.0	10 12 13	(12) 1.5	33 37 28	(33) 4.5	33 22 43	(33) 10.5	6 6 7	(6) 0.6
	500 µg	136 137 145	(139) 4.9	10 8 14	(11) 3.1	33 42 38	(38) 4.5	26 28 31	(28) 2.5	6 10 11	(9) 2.6
	1500 µg	133 144 129	(135) 7.8	12 11 20	(14) 4.9	39 44 39	(41) 2.9	34 30 28	(31) 3.1	8 11 15	(11) 3.5
	5000 µg	123 109 136	(123) 13.5	9 17 22	(16) 6.6	28 47 44	(40) 10.2	20 28 38	(29) 9.0	8 4 9	(7) 2.6
Positive controls S9-Mix (+)	Name DoseLevel No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		2755 2870 2770	(2798) 62.5	341 381 336	(353) 24.7	328 326 319	(324) 4.7	195 184 171	(183) 12.0	493 440 464	(466) 26.5

 

 †            Experimental procedure repeated at a later date due to contamination in the original test

BP               Benzo(a)pyrene

2AA      2-Aminoanthracene

#            Standarddeviation

 

 

Test Results: Experiment 2 – Without MetabolicActivation(Pre- Incubation)

 

Test Period		From: 26 March 2018					To: 29 March 2018
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Water)	152 129 144	(142) 11.7#	9 13 13	(12) 2.3	38 27 35	(33) 5.7	24 27 15	(22) 6.2	12 9 16	(12) 3.5
	15 µg	130 157 132	(140) 15.0	10 6 15	(10) 4.5	31 28 17	(25) 7.4	18 30 18	(22) 6.9	11 11 10	(11) 0.6
	50 µg	142 150 132	(141) 9.0	16 11 11	(13) 2.9	29 30 24	(28) 3.2	18 27 19	(21) 4.9	7 6 8	(7) 1.0
	150 µg	137 129 153	(140) 12.2	11 9 7	(9) 2.0	28 29 35	(31) 3.8	22 19 28	(23) 4.6	16 10 10	(12) 3.5
	500 µg	122 133 130	(128) 5.7	12 14 16	(14) 2.0	33 36 26	(32) 5.1	16 16 15	(16) 0.6	8 8 5	(7) 1.7
	1500 µg	115 150 154	(140) 21.5	13 13 12	(13) 0.6	33 21 36	(30) 7.9	24 26 17	(22) 4.7	15 12 14	(14) 1.5
	5000 µg	144 150 130	(141) 10.3	13 13 13	(13) 0.0	38 41 38	(39) 1.7	17 20 20	(19) 1.7	13 11 13	(12) 1.2
Positive controls S9-Mix (-)	Name DoseLevel No. of Revertants	ENNG		ENNG		ENNG		4NQO		9AA
		3 µg		5 µg		2 µg		0.2 µg		80 µg
		800 780 710	(763) 47.3	500 507 544	(517) 23.6	790 789 723	(767) 38.4	340 368 422	(377) 41.7	571 921 739	(744) 175.0

 

 

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

4NQO                  4-Nitroquinoline-1-oxide

9AA          9-Aminoacridine

#                Standarddeviation

 

 

 

Test Results: Experiment 2 – With MetabolicActivation(Pre- Incubation)

 

Test Period		From: 26 March 2018					To: 29 March 2018
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains						Frameshift strains
		TA100		TA1535		WP2uvrA		TA98		TA1537
	Solvent Control (Water)	133 143 127	(134) 8.1#	14 17 19	(17) 2.5	40 28 41	(36) 7.2	32 29 29	(30) 1.7	14 8 13	(12) 3.2
	15 µg	149 147 145	(147) 2.0	8 10 11	(10) 1.5	38 39 50	(42) 6.7	21 23 25	(23) 2.0	11 13 13	(12) 1.2
	50 µg	128 130 142	(133) 7.6	12 12 12	(12) 0.0	30 40 45	(38) 7.6	29 24 28	(27) 2.6	15 12 13	(13) 1.5
	150 µg	132 143 152	(142) 10.0	16 10 13	(13) 3.0	32 38 37	(36) 3.2	29 35 24	(29) 5.5	12 14 11	(12) 1.5
	500 µg	135 139 151	(142) 8.3	9 20 11	(13) 5.9	47 33 45	(42) 7.6	43 34 28	(35) 7.5	13 10 13	(12) 1.7
	1500 µg	145 115 150	(137) 18.9	8 10 15	(11) 3.6	45 34 34	(38) 6.4	24 23 24	(24) 0.6	12 7 10	(10) 2.5
	5000 µg	150 136 129	(138) 10.7	12 13 13	(13) 0.6	41 37 47	(42) 5.0	26 34 29	(30) 4.0	7 14 9	(10) 3.6
Positive controls S9-Mix (+)	Name DoseLevel No. of Revertants	2AA		2AA		2AA		BP		2AA
		1 µg		2 µg		10 µg		5 µg		2 µg
		2152 2108 1971	(2077) 94.4	343 333 332	(336) 6.1	261 206 281	(249) 38.8	158 160 138	(152) 12.2	400 408 413	(407) 6.6

 

 

BP         Benzo(a)pyrene

2AA    2-Aminoanthracene

#            Standarddeviation

 

 

 

 

 

 

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Diethoxymethane did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test Diethoxymethane was concluded as non-mutagenic.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxicity of the test item following the change in test methodology.

Results

The vehicle (sterile distilled water) 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 and 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 OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) was employed in the second mutation test (pre-incubation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).

Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).

Conclusion

Diethoxymethane was considered to be non-mutagenic under the conditions of this test.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 28 July 2017 Experimental completion date 17 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Identification: Ethylal
Chemical name: Diethoxymethane
CAS Number: 462-95-3
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Approximately 4 °C in the dark under nitrogen
Intended use/Application: Not supplied
No correction for purity was made.

Target gene:

not applicable

Species / strain / cell type:

primary culture, other: whole blood

Details on mammalian cell type (if applicable):

Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (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.

The details of the donors used are:
Preliminary Toxicity Test: male, aged 29 years
Main Experiment: female, aged 25 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

standard metabolizing system (S9)

Test concentrations with justification for top dose:

Preliminary Toxicity Test
The dose range of test item used was 0, 4.07, 8.14, 16.28, 32.56, 65.13, 130.25, 260.5, 521 and 1042 μg/mL.

Main Experiment
The dose range of test item used for all three exposures was 0, 65.13, 130.25, 260.5, 521, 781.5, 1042 μg/mL.

Vehicle / solvent:

The test item was miscible in aqueous media (MEM) at 10.42 mg/mL by mixing on a vortex for approximately 10 seconds in a solubility check performed in-house. The highest concentration of the test item tested in this study was 10.42 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 μg/mL for 4-hour exposure

Positive control substance:

mitomycin C

Remarks:

Absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium

True negative controls:

no

Positive controls:

yes

Remarks:

0.075 μg/mL for 24-hour continuous exposure

Positive control substance:

other: Demecolcine

Remarks:

Absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium

True negative controls:

no

Positive controls:

yes

Remarks:

5 μg/mL for 4-hour exposure

Positive control substance:

cyclophosphamide

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)
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 1.0 mL 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. 1.0 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 the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.
After 4 hours at approximately 37 ºC, 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 reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

4-Hour Exposure Without Metabolic Activation (S9)
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 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, 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, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The preliminary toxicity test was performed using the exposure conditions as described for the Main Experiment but using single cultures only, whereas the Main Experiment used replicate cultures.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 0, 4.07, 8.14, 16.28, 32.56, 65.13, 130.25, 260.5, 521 and 1042 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test.

Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used for all three exposures was 0, 65.13, 130.25, 260.5, 521, 781.5, 1042 μg/mL.

Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Preparation of Microscope Slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.

Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.

When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
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 judgement and/or further investigations.
Test items that induce micronuclei in the MNvit test may do so because they induce chromosome breakage, chromosome loss, or a combination of the two. Further analysis using anti-kinetechore antibodies, centromere specific in situ probes, or other methods can be used to determine whether the mechanism of micronucleus induction is due to clastogenic and/or aneugenic activity.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.

Species / strain:

primary culture, other: whole blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test (Table 1)
The dose range for the Preliminary Toxicity Test was 4.07 to 1042 μg/mL. The molecular weight of the test item was given as 104.15, therefore, the maximum dose level was 1042 μg/mL, which was calculated to be equivalent to the 10mM concentration (the maximum recommended dose level).
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at any dose level tested in any of the exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to the maximum concentration in all three exposure groups. The test item induced no evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level for the Main Experiment was, therefore, based on the maximum recommended dose level (1042 μg/mL) for all three exposure group.

Micronucleus Test – Main Experiment
As during the Preliminary Toxicity Test, there were binucleate cells suitable for scoring at the maximum dose level of test item, 1042 μg/mL, for all three exposure groups.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at any dose level tested in any of the exposure groups.
The CBPI data confirm the qualitative observations in that no dose-related inhibition of CBPI was observed Tables 2 to 6).
The maximum dose level selected for analysis of binucleate cells was the maximum recommended dose level (1042 μg/mL) for all three exposure groups.
The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, 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 binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

 

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

 

Group	Final concentration of test item Ethylal (µg/mL)
4-hour without S9	0*,65.13, 130.25, 260.5, 521*, 781.5*, 1042*,MMC0.2*
4-hour with S9 (2%)	0*, 65.13, 130.25, 260.5, 521*, 781.5*, 1042*,CP5*
24-hour without S9	0*, 65.13, 130.25, 260.5, 521*, 781.5*, 1042*,DC0.075*

 

*   = Dose levels selected for analysis of micronucleus frequency in binucleate cells

MMC  = MitomycinC

CP = Cyclophosphamide

DC            =Demecolcine

 

 

Table 1: CBPI - Preliminary Toxicity test

4-hour exposure without S9						4-hour exposure with S9						24-hour exposure without S9
Dose Level μg/mL	Nucleate Cells/500 Cells			CBPI	% Cytostasis	Dose Level μg/mL	Nucleate Cells/500 Cells			CBPI	% Cytostasis	Dose Level μg/mL	Nucleate Cells/500 Cells			CBPI	% Cytostasis
	Mono	Bi	Multi				Mono	Bi	Multi				Mono	Bi	Multi
0	248	228	24	1.55	0	0	229	244	27	1.60	0	0	83	313	104	2.04	0
4.07	-	-	-	-	-	4.07	-	-	-	-	-	4.07	-	-	-	-	-
8.14	-	-	-	-	-	8.14	-	-	-	-	-	8.14	-	-	-	-	-
16.28	-	-	-	-	-	16.28	-	-	-	-	-	16.28	-	-	-	-	-
32.56	-	-	-	-	-	32.56	-	-	-	-	-	32.56	-	-	-	-	-
65.13	-	-	-	-	-	65.13	-	-	-	-	-	65.13	-	-	-	-	-
130.25	-	-	-	-	-	130.25	-	-	-	-	-	130.25	-	-	-	-	-
260.5	244	229	27	1.57	0‡	260.5	270	210	20	1.50	17	260.5	101	306	93	1.98	6
521	296	193	11	1.43	22	521	201	282	17	1.63	0‡	521	54	354	92	2.08	0‡
1042	228	255	17	1.58	0‡	1042	259	224	17	1.52	13	1042	66	345	89	2.05	0‡

-     = Not selected for scoring

‡    = Cytosis reported as 0 as the CBPI value is equal to or higher than the solvent control

 

Table 2: CBPI Data - Main Experiment - 4HOUR Exposure With and Without Metabolic Activation (S9)

4-Hour exposure without S9								4-hour exposure with S9
Dose level (µg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis	Dose level (µg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis
		M ono	Bi	Multi						M ono	Bi	Multi
0	A	125	316	59	1.87	1.89	0	0	A	227	241	32	1.61	1.65	0
	B	109	333	58	1.90				B	203	251	46	1.69
65.13	A	-	-	-	-	-	-	65.13	A	-	-	-	-	-	-
	B	-	-	-	-				B	-	-	-	-
130.25	A	-	-	-	-	-	-	130.25	A	-	-	-	-	-	-
	B	-	-	-	-				B	-	-	-	-
260.5	A	-	-	-	-	-	-	260.5	A	-	-	-	-	-	-
	B	-	-	-	-				B	-	-	-	-
521	A	150	306	44	1.79	1.80	10	521	A	231	244	25	1.59	1.59	10
	B	150	302	48	1.80				B	233	244	23	1.58
781.5	A	125	331	44	1.84	1.84	5	781.5	A	217	253	30	1.63	1.59	9
	B	113	353	34	1.84				B	258	209	33	1.55
1042	A	144	325	31	1.77	1.76	15	1042	A	265	209	26	1.52	1.58	12
	B	172	286	42	1.74				B	218	247	35	1.63
MMC 0.2	A	268	227	5	1.47	1.48	46	CP 5	A	356	142	2	1.29	1.31	53
	B	258	238	4	1.49				B	346	146	8	1.32

MMC    = Mitomycin C

CP      = Cyclophosphamide

-        = No selected for scoring

 

 

Table 3: CBPI Data - Main Experiment - 24-Hour Exposure Without Metabolic Activation (S9)

24-Hour exposure without S9
Dose level (µg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis
		Mono	Bi	Multi
0	A	82	383	65	1.97	1.96	0
	B	96	340	64	1.94
65.13	A	-	-	-	-	-	-
	B	-	-	-	-
130.25	A	-	-	-	-	-	-
	B	-	-	-	-
260.5	A	-	-	-	-	-	-
	B	-	-	-	-
521	A	91	346	63	1.94	1.96	0‡
	B	89	336	75	1.97
781.5	A	75	366	59	1.97	1.99	0‡
	B	74	345	81	2.01
1042	A	69	366	65	1.99	1.97	0‡
	B	88	346	57	1.94
DC 0.075	A	196	259	45	1.70	1.66	31
	B	237	218	45	1.62

DC  = Demecolcine

-     = Not selected for scoring

‡    = Cytosis reported as 0 as the CBPI value is equal to or higher than the solvent control

 

 

Table 4: CBPI and Micronucleus Data - Main Experiment - 4-Hour Exposure Without Metabolic Activation (S9)

Exposure Time +/-S9	Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
4Hr-S9	0	A	125	316	59	1.87	0	5	0	0	0.50	0.65
		B	109	333	58	1.90		7	1	0	0.80
	521	A	150	306	44	1.79	10	3	0	0	0.30	0.40
		B	150	302	48	1.80		5	0	0	0.50
	781.5	A	125	331	44	1.84	5	6	0	0	0.60	0.50
		B	113	353	34	1.84		4	0	0	0.40
	1042	A	144	325	31	1.77	15	4	1	0	0.50	0.45
		B	172	286	42	1.74		3	1	0	0.40
	MMC 0.2	A	268	227	5	1.47	46	62	5	0	6.70	9.7***
		B	258	238	4	1.49		116	8	3	12.70

MMC   = Mitomycin C

***    = P<0.001

 

Table 5: CBPI and Micronucleus Data - Experiment - 4-Hour Exposure With Metabolic Activation (S9)

Exposure Time +/-S9	Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
4Hr+S9	0	A	227	241	32	1.61	0	5	0	0	0.50	0.45
		B	203	251	46	1.69		4	0	0	0.40
	521	A	231	244	25	1.59	10	1	1	0	0.20	0.25
		B	233	244	23	1.58		3	0	0	0.30
	781.5	A	217	253	30	1.63	9	5	1	1	0.70	0.55
		B	258	209	33	1.55		4	0	0	0.40
	1042	A	265	209	26	1.52	12	4	0	0	0.40	0.50
		B	218	247	35	1.63		6	0	0	0.60
	CP 5	A	356	142	2	1.29	53	35	2	0	3.70	4.15***
		B	346	146	8	1.32		44	2	0	4.60

CP   = Cyclophosphamide

*** = P<0.001

 

Table 6: CBPI and Micronucleus Data - Main Experiment - 24-Hour Exposure Without Metabolic Activation (S9)

Exposure Time +/- S9	Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
24Hr-S9	0	A	82	383	65	1.97	0	6	0	1	0.70	0.55
		B	96	340	64	1.94		3	0	1	0.40
	521	A	91	346	63	1.94	0‡	5	0	0	0.50	0.65
		B	89	336	75	1.97		6	0	2	0.80
	781.5	A	75	366	59	1.97	0‡	7	0	0	0.70	0.90
		B	74	345	81	2.01		11	0	0	1.10
	1042	A	69	366	65	1.99	0‡	0	0	0	0.00	0.40
		B	88	346	57	1.94		2	0	6	0.80
	DC 0.075	A	196	259	45	1.70	31	40	9	5	5.40	5.75***
		B	237	218	45	1.62		36	19	6	6.10

DC   = Demecolcine

***  = P<0.001

‡    = Cytosis reported as 0 as the CBPI value is equal to or higher than the solvent control

Conclusions:

Under the conditions of this test, the test item, Ethylal, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

 Introduction

 

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

 

 Methods

 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2%

final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

 

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be the maximum recommended limit (1042 µg/mL). The dose levels selected for the Main Test were as follows:

 

Group	Final concentration of test item Ethylal (µg/mL)
4-hour without S9	0, 65.13, 130.25, 260.5, 521, 781.5, 1042
4-hour with S9 (2%)
24-hour without S9

 

Results

 

All vehicle (Eagle's minimal essential medium with HEPES buffer (MEM)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

 

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

 

The test item was non-toxic to human lymphocytes and did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the maximum recommended dose level.

 

Conclusion

 

Under the conditions of this study, the test item, Ethylal, was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 August 2018 - 29 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Clear colourless liquid

Target gene:

Thymidine kinase, TK +/-

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Lot No. PB/βNF S9 29/03/18 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.
20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test.

Test concentrations with justification for top dose:

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test at a concentration range of 4.07 to 1042 μg/mL. The maximum dose level used in the Mutagenicity Test was the 10 mM Maximum Recommended Dose level (MRD).
The dose levels plated for viability and expression of mutant colonies were as follows: 32.56, 65.13, 130.25, 260.5, 521, 1042 μg/mL

Vehicle / solvent:

R0 culture media

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

The cells were counted and processed to give 1 x 10e6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10e6 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at six dose levels of the test item 32.56 to 1042 μg/mL for all three exposure groups, vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 2.0 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).
The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item. Colonies were scored manually by eye using qualitative judgment. Large colonies were defined as those that covered approximately ¼ to ¾ of the surface of the well and were generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies were scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Rationale for test conditions:

The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance processes to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al., 1980; Jotz and Mitchell, 1981).

Ref:
Amacher, D.E., Paillet, S.C., Turner, G.N., Ray, V.A. and Salsburg, D.S. (1980) Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. 2. Test validation and interpretation, Mutation Res., 72, pp 447-474.
Clive, D., Flamm, W.G., Machesko, M.R. and Bernheim, N.J. (1972) A mutational assay system using the thymidine kinase locus in mouse lymphoma cells. Mutation Res., 16, pp 77-87.
Clive, D., Johnson, K.O., Spector, J.F.S., Batson, A.G. and Brown, M.M.M. (1979) Validation and characterisation of the L5178Y TK +/- mouse lymphoma mutagen assay system. Mutation Res. 59, pp. 61-108.
Fischer, G.A. (1958) Ann. NY Acad. Sci., 76, 673-680.
JOTZ, M.M. and MITCHELL, A.D. (1981) Effects of 20 Coded Chemicals on the forward mutation frequency at the thymidine kinase locus in L5178Y mouse lymphoma cells. In: Evaluation of Short-term Tests for Carcinogens [de Serres F J and Ashby J (Eds)], Elsevier/North Holland, New York, pp 580-593.

Evaluation criteria:

1. The majority of the plates, for both viability (%V) and TFT resistance, are analyzable.
2. The absolute viability (%V) at the time of mutant selection of the solvent controls is 65-120 %.
3. The total suspension growth of the solvent control following 4 hour exposure should be in the range of 8 - 32. Following 24 hour exposure the total suspension growth should be in the range of 32 - 180.
4. The in-house vehicle control mutant frequency is in the range of 50 – 170 x 10-6 cells. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution, or exposure to the metabolic activation system, may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10-6 mutant frequency per survivor are not acceptable and will be repeated.
5. Every test should also be evaluated as to whether the positive controls (EMS and CP) meet at least one of the following two acceptance criteria developed by the IWGT workgroup:
The positive control should demonstrate an absolute increase in total MF, that is, an increase above the spontaneous background MF [an induced MF (IMF)] of at least 300 x 10-6. At least 40% of the IMF should be reflected in the small colony MF.
The positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent untreated/solvent control (a small colony IMF of 150 x 10-6).
6. The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures
7. The upper test item concentrations will be 10mM, 2 mg/mL or 2μL/mL whichever is the lowest. Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point. In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival.

Statistics:

The experimental data was analyzed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear-trend events.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary test:
The dose range of the test item used in the preliminary toxicity test was 1.25 to 320 μg/mL.
There was no evidence dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in the 4-hour exposure groups either in the absence or presence of metabolic activation when groups compared to the concurrent vehicle control groups. There was a modest reduction in %RSG in the 24-hour exposure group. No precipitate was observed. Therefore, the dose levels selected for the main test was up to the maximum recommended dose level of 1042 μg/mL for all three exposure groups.

Main test:
There was no evidence of marked dose related toxicity following exposure to the test item in the 4-hour exposure groups in either the absence or presence of metabolic activation as indicated by the %RSG and RTG values. As was observed in the preliminary toxicity test there was a modest reduction in %RSG and RTG values in the 24-hour exposure group. Acceptable levels of toxicity were seen with the positive control substances. No precipitate was observed.
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional. (tables 1 - 10)

Table 1            Summary of Results

 Experiment 1

Treatment (µg/mL)	4-hours-S-9			Treatment (µg/mL)	4-hours+S-9
	%RSG	RTG	MF§		%RSG	RTG	MF§
0	100	1.00	134.37	0	100	1.00	133.60
32.56	104	1.10	121.22	32.56	95	1.01	104.40
65.13	111	1.17	132.84	65.13	97	0.94	143.60
130.25	101	0.95	134.50	130.25	88	0.86	133.35
260.5	100	1.03	133.06	260.5	92	1.05	124.08
521	99	1.03	153.20	521	96	0.89	131.38
1042	101	1.06	128.94	1042	83	0.76	164.02
MF threshold for a positive response=260.37				MF threshold for a positive response=256.60
EMS	66	0.45	1637.45	CP	62	0.38	831.08
400				1.5

 

Treatment (µg/mL)	24-hours-S-9
	%RSG	RTG	MF§
0	100	1.00	133.14
32.56	99	1.02	137.78
65.13	81	0.94	107.17
130.25	95	0.99	138.96
260.5	96	1.05	157.24
521	95	1.00	144.90
1042	66	0.83	158.52
MF threshold for a positive response=259.14
EMS	38	0.36	1707.56
150

Table 2          Cell and 96-Well Plate Counts: Mutagenicity Test (-S9) 4-Hour Exposure

  

Treatment (µg/mL)		Cell counts $			Viability § after day 2 2 cells/well				Resistant mutants § after day 2 2000 cells/well
		0h	24h	48h
0	A B	8.07 8.01	7.36 7.26	6.37 6.20	72 77	75 74	76 81	72 83	12 20	20 18	16 20	15 26
32.56	A B	7.66 7.27	7.20 7.75	6.81 6.96	78 80	80 73			17 16	16 21
65.13	A B	8.16 8.19	7.13 7.29	7.03 6.88	77 77	77 80			19 18	18 21
130.25	A B	7.39 7.42	7.70 7.02	6.58 7.06	72 78	75 73			16 17	20 17
260.5	A B	7.30 6.50	7.55 7.95	7.03 6.75	80 75	75 79			17 19	21 18
521	A B	7.90 7.34	7.28 7.36	6.87 6.29	76 79	76 78			18 23	20 24
1042	A B	7.57 6.55	7.22 8.16	7.58 6.19	78 73	79 81			18 22	16 18
Positive Control EMS (µg/mL)
400	A B	7.03 7.06	5.86 5.09	6.03 6.64	70 67	62 54			92 73	76 77

Table 3            Summary Analysis: Mutagenicity Test (-S9) 4-Hour Exposure

 

Treatment (µg/mL)	SG	%RSG	%V	RTG	MF§
0	11.49	100	79.06	1.00	134.37
32.56	12.87	104	83.01	1.10	121.22
65.13	12.54	111	83.01	1.17	132.84
130.25	12.55	101	74.81	0.95	134.50
260.5	13.35	100	81.66	1.03	133.06
521	12.04	99	81.66	1.03	153.20
1042	13.24	101	83.01	1.06	128.94
Positive Control EMS
Treatment (µg/mL)	SG	%RSG	%V	RTG	MF§
400	8.67	66	53.77	0.45	1637.45

 GEF =126, therefore MF threshold for a positive response = 260.37

Table 4            Large and Small Colonies Analysis: Mutagenicity Test (-S9) 4-Hour Exposure

 

Treatment (µg/mL)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A B	72 77	75 74	76 81	72 83	5 11	9 8	7 7	10 12	7 9	11 10	9 13	5 14
32.56	A B	78 80	80 73			9 10	6 10			8 6	10 11
65.13	A B	77 77	77 80			14 9	5 7			5 9	13 14
130.25	A B	72 78	75 73			9 7	8 10			7 10	12 7
260.5	A B	80 75	75 79			7 6	9 9			10 13	12 9
521	A B	76 79	76 78			9 9	9 10			9 14	11 14
1042	A B	78 73	79 81			8 8	7 11			10 14	9 7
400 EMS	A B	70 67	62 54			44 38	38 38			48 35	38 39

Mutation frequencies

Treatment (µg/mL)			Small colonies			Large Colonies			Proportion small colony mutants
	Viable		Mutants		MF§	Mutants		MF§
	Yv	Nv	Ym	Nm		Ym	Nm
0	158	768	699	768	59.5	690	768	67.7	0.47
32.56	73	384	349	384	57.6	349	384	57.6	0.50
65.13	73	384	349	384	57.6	343	384	68.0	0.46
130.25	86	384	350	384	62.0	348	384	65.8	0.49
260.5	75	384	353	384	51.5	340	384	74.5	0.41
521	75	384	347	384	62.0	336	384	81.8	0.44
1042	73	384	350	384	55.8	344	384	66.3	0.46
400 EMS	131	384	116	384	1113.1	118	384	1097.2	0.50

Table 5            Cell and 96-Well Plate Counts: Mutagenicity Test (+S9) 4-Hour Exposure

 

Treatment (µg/mL)		Cell counts $			Viability § after day 2 2 cells/well				Resistant mutants § after day 2 2000 cells/well
		0h	24h	48h
0	A B	6.89 7.64	7.78 7.64	8.96 7.78	72 80	69 80	78 82	76 80	17 20	21 17	15 23	17 20
32.56	A B	6.82 7.51	8.37 8.02	8.03 7.22	79 86	76 74			18 11	18 16
65.13	A B	7.37 6.32	7.90 9.12	7.33 8.29	78 80	75 72			19 19	21 19
130.25	A B	7.12 6.67	7.44 8.65	7.39 7.54	71 79	82 73			15 19	20 19
260.5	A B	7.57 6.90	8.15 7.41	8.07 7.19	82 76	85 81			20 20	17 22
521	A B	7.54 6.72	6.44 7.60	9.42 8.51	75 77	79 70			15 17	19 19
1042	A B	6.59 7.06	7.56 7.19	7.63 7.86	71 78	75 73			18 21	24 20
Positive Control CP (µg/mL)
1.5	A B	6.81 6.00	6.56 7.05	6.85 6.52	62 66	58 57			58 50	49 60

Table 6            Summary Analysis: Mutagenicity Test (+S9) 4-Hour Exposure

 

Treatment (µg/mL)	SG	%RSG	%V	RTG	MF§
0	16.13	100	81.33	1.00	133.60
32.56	15.62	95	85.83	1.01	104.40
65.13	16.62	97	79.06	0.94	143.60
130.25	15.01	88	79.06	0.86	133.35
260.5	14.8	92	92.81	1.05	124.08
521	15.73	96	76.59	0.89	131.38
1042	14.28	83	74.24	0.76	164.02
Positive Control CP
Treatment (µg/mL)	SG	%RSG	%V	RTG	MF§
1.5	11.37	62	50.09	0.38	831.08

GEF =126, therefore MF threshold for a positive response = 259.60

Table 7            Large and Small Colonies Analysis: Mutagenicity Test (+S9) 4-Hour Exposure

 

Treatment (µg/mL)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A B	72 80	69 80	78 82	76 80	9 12	8 9	8 12	8 11	8 8	13 8	7 11	9 9
32.56	A B	79 86	76 74			13 1	6 7			5 10	12 9
65.13	A B	78 80	75 72			9 10	9 7			10 9	12 12
130.25	A B	71 79	82 73			9 9	5 4			6 10	15 15
260.5	A B	82 76	85 81			12 7	9 9			8 13	8 13
521	A B	75 77	79 70			10 4	6 7			5 13	13 12
1042	A B	71 78	75 73			10 10	10 10			8 11	14 10
1.5 CP	A B	62 66	58 57			37 28	38 33			21 22	11 27

Mutation frequencies

Treatment (µg/mL)			Small colonies			Large Colonies			Proportion small colony mutants
	Viable		Mutants		MF§	Mutants		MF§
	Yv	Nv	Ym	Nm		Ym	Nm
0	151	768	691	768	65.0	695	768	61.4	0.51
32.56	69	384	357	384	42.5	348	384	57.3	0.43
65.13	79	384	349	384	60.4	341	384	75.1	0.45
130.25	79	384	357	384	46.1	338	384	80.7	0.37
260.5	60	384	347	384	54.6	342	384	62.4	0.47
521	83	384	357	384	47.6	341	384	77.5	0.39
1042	87	384	344	384	74.1	341	384	80.0	0.48
1.5 CP	141	384	248	384	436.4	303	384	236.5	0.63

Table 8            Cell and 96-Well Plate Counts: Mutagenicity Test (-S9) 24-Hour Exposure

 

Treatment (µg/mL)		Cell counts $			Viability § after day 2 2 cells/well				Resistant mutants § after day 2 2000 cells/well
		0h	24h	48h
0	A B	9.64 9.62	6.68 6.96	7.79 8.73	81 80	76 78	83 82	81 79	24 21	21 23	19 17	19 19
32.56	A B	9.68 9.10	6.67 6.59	8.58 9.18	86 75	78 81			23 23	17 21
65.13	A B	8.60 8.96	7.28 6.14	7.62 8.81	84 81	80 82			15 19	16 21
130.25	A B	10.17 9.24	6.45 6.31	7.81 8.77	78 82	82 83			23 21	24 20
260.5	A B	9.02 8.84	6.67 7.38	8.18 9.71	85 76	82 78			21 26	26 22
521	A B	9.31 9.93	6.74 6.05	7.74 9.11	77 84	79 86			26 25	17 24
1042	A B	8.02 8.72	6.79 5.77	8.29 7.45	77 85	80 86			24 26	25 26
Positive Control EMS (µg/mL)
150	A B	6.39 7.21	5.68 5.51	7.83 7.56	66 73	61 69			83 89	79 84

Table 9            Summary Analysis: Mutagenicity Test (-S9) 24-Hour Exposure

 

Treatment (µg/mL)	SG	%RSG	%V	RTG	MF§
0	90.41	100	89.59	1.00	133.14
32.56	92.14	99	89.59	1.02	137.78
65.13	80.66	81	95.38	0.94	107.17
130.25	85.55	95	93.66	0.99	138.96
260.5	93.52	96	90.38	1.05	157.24
521	86.38	95	94.51	1.00	144.90
1042	68.95	66	96.26	0.83	158.52
Positive Control EMS (µg/mL)
Treatment (µg/mL)	SG	%RSG	%V	RTG	MF§
150	48.79	38	60.29	0.36	1707.56

 GEF =126, therefore MF threshold for a positive response = 259.14

Table 10          Large and Small Colonies Analysis: Mutagenicity Test (-S9) 24-Hour Exposure

 

Treatment   (µg/mL)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A B	81 80	76 78	83 82	81 79	9 16	12 7	8 5	7 11	15 5	9 16	11 12	12 8
32.56	A B	86 75	78 81			10 11	10 12			13 12	7 9
65.13	A B	84 81	80 82			6 8	10 12			9 11	6 9
130.25	A B	78 82	82 83			15 12	9 11			8 9	15 9
260.5	A B	85 76	82 78			13 13	11 16			8 13	15 6
521	A B	77 84	79 86			13 13	8 10			13 12	9 14
1042	A B	77 85	80 86			16 12	9 11			8 14	16 15
150 EMS	A B	66 73	61 69			47 48	43 48			36 41	36 36

Mutation frequencies

Treatment (µg/mL)			Small colonies			Large Colonies			Proportion small colony mutants
	Viable		Mutants		MF§	Mutants		MF§
	Yv	Nv	Ym	Nm		Ym	Nm
0	128	768	693	768	57.4	680	768	67.9	0.46
32.56	64	384	341	384	66.3	343	384	63.0	0.51
65.13	57	384	348	384	51.6	349	384	50.1	0.51
130.25	59	384	337	384	69.7	343	384	60.3	0.53
260.5	63	384	331	384	82.2	342	384	64.1	0.56
521	58	384	340	384	64.4	336	384	70.6	0.48
1042	56	384	336	384	69.4	331	384	77.1	0.48
150 EMS	115	384	198	384	549.4	235	384	407.3	0.56

 

KEY TO TABLES 1 TO 10

 

$	=	Cell counts (x10e5cells/mL). Set up on previous day to 2 x 10e5cells/mL unless otherwise stated in parenthesis.
%RSG	=	Relative Suspension Growth
RTG	=	Relative Total Growth
%V	=	Viability Day 2
§ or #	=	Positive wells per tray, 96 wells plated unless otherwise stated in parenthesis
A,B	=	Replicate cultures
CP	=	Cyclophosphamide
EMS	=	Ethylmethanesulphonate
MF§	=	5-TFT resistant mutants/10e6 viable cells 2 days after exposure
Nv	=	Number of wells scored, viability plates
Yv	=	Number of wells without colonies, viability plates
Ym	=	Number of wells without colonies, mutation plates
Nm	=	Number of wells scored, mutation plates

Conclusions:

The test item, Ethylal did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in this assay.