Diethoxymethane

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Diethoxymethane

EC Number:

207-330-6

EC Name:

Diethoxymethane

Cas Number:

462-95-3

Molecular formula:

C5H12O2

IUPAC Name:

diethoxymethane

Test material form:

liquid

Details on test material:

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.

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.

Method

Target gene:

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

Species / strain

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.

Controls

Controls 1

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

Controls 2

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

Controls 3

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

Controls 4

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

Controls 5

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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).

Any other information on results incl. tables

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

 

 

 

 

 

 

Applicant's summary and conclusion

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.