reaction mass of diethyl (E)-2-methylbut-2-enedioate and diethyl (Z)-2-methylbut-2-enedioate

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:

08 September 2016 - 16 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

diethyl 2-methylidenebutanedioate

EC Number:

607-321-0

Cas Number:

2409-52-1

Molecular formula:

C9H14O4

IUPAC Name:

diethyl 2-methylidenebutanedioate

Method

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:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I & Ia:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II:
TA 1537 and TA 100: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
All remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine / 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation test (experiment I & Ia), pre-incubation test (experiment II)

DURATION
- Preincubation period: 60 minutes
- Exposure duration: ≥ 48 hours

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants using a validated computer system

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

no statistical analysis

Results and discussion

Test results

Test results 1

Key result

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

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 2500 µg/plate (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 2500 µg/plate (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 2500 µg/plate (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at ≥ 1000 µg/plate (experiment I), at ≥ 2500 µg/plate (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate (experiment II)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in experiment I & Ia and at 5000 μg/plate in experiment II. No precipitation of the test item in the overlay agar on the incubated agar plates was observed.

Any other information on results incl. tables

Table 1: Summary of Experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100
Without Activation	DMSO		17 ± 4	13 ± 2	22 ± 9	150 ± 2
	Untreated		12 ± 2	11 ± 5	26 ± 2	148 ± 8
	PI 27319	3 µg	14 ± 7	9 ± 3	20 ± 5	139 ± 25
		10 µg	17 ± 5	14 ± 4	26 ± 3	135 ± 17
		33 µg	18 ± 3	15 ± 1	23 ± 4	136 ± 5
		100 µg	12 ± 3	12 ± 2	25 ± 8	149 ± 13
		333 µg	14 ± 5	10 ± 3	23 ± 8	132 ± 7
		1000 µg	15 ± 1	10 ± 6	24 ± 2	66 ± 9
		2500 µg	10 ± 4	7 ± 2	23 ± 6	65 ± 3
		5000 µg	10 ± 5	7 ± 2	18 ± 3	66 ± 16
	NaN3	10 µg	1400 ± 12			2126 ± 51
	4-NOPD	10 µg			430 ± 46
	4-NOPD	50 µg		85 ± 11
With Activation	DMSO		13 ± 2	12 ± 2	29 ± 9	108 ± 7
	Untreated		15 ± 5	14 ± 4	33 ± 9	146 ± 13
	PI 27319	3 µg	14 ± 7	14 ± 6	38 ± 2	102 ± 4
		10 µg	9 ± 2	12 ± 6	34 ± 4	102 ± 11
		33 µg	11 ± 3	12 ± 3	38 ± 6	100 ± 5
		100 µg	14 ± 3	14 ± 2	40 ± 7	93 ± 5
		333 µg	11 ± 2	14 ± 3	42 ± 6	105 ± 12
		1000 µg	14 ± 4	16 ± 3	32 ± 6	94 ± 24
		2500 µg	14 ± 3	12 ± 6	36 ± 4	93 ± 10
		5000 µg	11 ± 3	12 ± 3	32 ± 8	55 ± 18
	2-AA	2.5 µg	329 ± 27	308 ± 40	3613 ± 479	3697 ± 101

NaN3: sodium azide, 2 -AA: 2-aminoanthracene, 4 -NOPD: 4-nitro-o-phenylene-diamine

Table 2 Summary of Experiment Ia

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			WP2 uvrA
Without Activation	DMSO		37 ± 9
	Untreated		39 ± 9
	PI 27319	3 µg	41 ± 13
		10 µg	40 ± 8
		33 µg	42 ± 8
		100 µg	41 ± 6
		333 µg	36 ± 6
		1000 µg	32 ± 5
		2500 µg	27 ± 7^R
		5000 µg	24 ± 6^R
	NaN3	2.0 µL	1024 ± 27
With Activation	DMSO		41 ± 7
	Untreated		46 ± 15
	PI 27319	3 µg	48 ± 2
		10 µg	40 ± 4
		33 µg	45 ± 4
		100 µg	48 ± 4
		333 µg	44 ± 7
		1000 µg	42 ± 6
		2500 µg	33 ± 5
		5000 µg	28 ± 3
	2-AA	10.0 µg	452 ± 34

MSS: methyl methane silfonate, 2-AA: 2-aminoanthracene, R: Reduced background growth

Table 3 Summary of Experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ± SD)
			TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		12 ± 4	10 ± 3	25 ± 5	127 ± 19	46 ± 7
	Untreated		9 ± 4	14 ± 4	26 ± 6	164 ± 8	49 ± 9
	PI 27319	3 µg		11 ± 2		127 ± 20
		10 µg	15 ± 4	11 ± 4	27 ± 3	146 ± 10	48 ± 5
		33 µg	12 ± 7	11 ± 1	24 ± 4	127 ± 8	40 ± 9
		100 µg	11 ± 4	12 ± 3	28 ± 7	132 ± 14	40 ± 4
		333 µg	14 ± 2	9 ± 2	29 ± 8	121 ± 6	43 ± 5
		1000 µg	11 ± 6	9 ± 3	17 ± 5	103 ± 11	41 ± 5
		2500 µg	11 ± 1	3 ± 2	15 ± 1	20 ± 6	24 ± 6
		5000 µg	16 ± 1	0 ± 1	0 ± 0	0 ± 1	18 ± 7
	NaN3	10 µg	1481 ± 130			2526 ± 39
	4-NOPD	10 µg			453 ± 10
	4-NOPD	50 µg		74 ± 4
	MMS	2.0 µL					890 ± 50
With Activation	DMSO		13 ± 3	11 ± 5	37 ± 11	119 ± 8	61 ± 8
	Untreated		9 ± 3	8 ± 3	45 ± 9	177 ± 13	53 ± 10
	PI 27319	3 µg		12 ± 5		96 ± 7
		10 µg	14 ± 2	9 ± 5	33 ± 7	109 ± 15	60 ± 4
		33 µg	13 ± 5	13 ± 4	33 ± 8	107 ± 10	54 ± 2
		100 µg	13 ± 2	11 ± 5	31 ± 4	96 ± 13	53 ± 5
		333 µg	12 ± 3	8 ± 3	36 ± 4	114 ± 11	55 ± 8
		1000 µg	11 ± 4	7 ± 3	28 ± 2	81 ± 13	49 ± 1
		2500 µg	10 ± 3	8 ± 2	9 ± 4	52 ± 6	29 ± 4
		5000 µg	11 ± 1	0 ± 1	1 ± 1	2 ± 1	20 ± 5
	2-AA	2.5 µg	394 ± 6	100 ± 16	3884 ± 271	4828 ± 603
	2-AA	10.0 µg					456 ± 10

NaN3: sodium azide, 2-AA: 2-aminoanthracene,4 -NOPD: 4-nitro-o-phenylene-diamine

Historical Data

Strain		without S9 mix				with S9 mix
		Mean	SD	Min	Max	Mean	SD	Min	Max
TA 1535	Solvent control	11	2.15	7	23	12	2.14	7	21
	Untreated control	12	2.97	6	24	12	2.71	7	26
	Positive control	1090	123.80	334	1372	392	62.85	176	549
TA 1537	Solvent control	10	1.83	6	18	13	3.27	7	27
	Untreated control	10	2.29	6	20	14	3.72	7	25
	Positive control	83	12.28	55	131	175	44.44	82	327
TA 98	Solvent control	24	3.75	16	36	33	5.55	18	51
	Untreated control	26	4.72	15	43	36	5.83	17	56
	Positive control	344	51.13	211	599	3822	857.83	319	5048
TA 100	Solvent control	155	24.19	84	194	145	31.81	81	204
	Untreated control	174	21.92	90	206	170	23.62	93	212
	Positive control	1956	279.93	658	2528	3606	676.07	722	4940
WP2 uvrA	Solvent control	41	5.72	27	63	51	6.91	37	72
	Untreated control	42	6.01	31	63	53	7.05	38	88
	Positive control	732	161.66	322	1066	362	72.26	212	858

Mean = mean value of revertants/plate, SD = standard deviation, Min = minimal value, Max = maximal value

Applicant's summary and conclusion

Conclusions:

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

A bacterial reverse mutation assay (OECD 471) was performed to investigate the potential of the test item to induce gene mutations using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2uvrA. A plate incorporation test (experiment I and Ia: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate) and a pre-incubation test (experiment II: TA 1537 and TA 100: 3; 10; 33; 100; 333; 1000; 2500; and 5000μg/plate All remaining strains: 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate) was performed.

The assay was performed in three independent experiments with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

The test item precipitated in the overlay agar in the test tubes from 2500 to 5000 μg/plate in experiment I & Ia and at 5000 μg/plate in experiment II. No precipitation of the test item in the overlay agar on the incubated agar plates was observed.

In experiment Ia the plates incubated with the test item showed reduced background growth from 2500 to 5000 μg/plate in the absence of metabolic activation (in strain WP2uvrA).

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains except strain TA 1535.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The controls confirmed the validity of the study.