Ethylene diformate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Jul - 16 Aug 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of the Government of the United Kingdom

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Ethylene diformate

EC Number:

211-077-7

EC Name:

Ethylene diformate

Cas Number:

629-15-2

Molecular formula:

C4H6O4

IUPAC Name:

2-(formyloxy)ethyl formate

Details on test material:

- Name of test material (as cited in study report): Ethylene Glycol Diformate
- Physical state: pale yellow liquid
- Analytical purity: minimum 80% (w/w)
- Lot/batch No.: 13082
- Expiration date of the lot/batch: 28 June 2013
- Storage condition of test material: room temperature in the dark

Method

Target gene:

his operon (for S. typhimurium strains)
trp operon (for E. coli strain)

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Oxoid Limited, Lot numbers 1078364 (08/16) and 1014570 (03/2016)

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Phenobarbitone/beta-Naphthoflavone.

Test concentrations with justification for top dose:

Preliminarty toxicity test: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation
First and second experiment: 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was fully soluble in DMSO at 50 mg/mL in solubility tests and DMSO was therefore selected as the vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG; 2, 3 and 5 µg/plate, -S9, WP2uvrA, TA100 and TA1535, respectively); 9-Aminoacridine (9AA; 80 µg/plate, -S9, TA1537); 4-Nitroquinoline-1-oxide (4NQO; 0.2 µg/plate, -S9, TA98)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA; 1, 2 and 10 µg/plate, +S9, TA100, TA1535 and TA1537, WP2uvrA, respectively); Benzo(a)pyrene (BP; 5 µg/plate, +S9, TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation (first experiment), pre-incubation (second experiment)

DURATION
- Exposure duration: 48 h

DETERMINATION OF CYTOTOXICITY
- Method: inspection of the bacterial background lawn

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

Evaluation criteria:

A result is considered positive, if any, one, or all of the following conditions are met: A dose-related increase in mutant frequency over the dose range tested (De Serres F J and Shelby M D (1979), Recommendations on data production and analysis using the Salmonella/microsome mutagenicity assay, Environmental Mutagenesis, 1, 87-92). A reproducible increase at one or more concentrations. Biological relevance against historical control ranges. Statistical analysis of data as determined by UKEMS (Mahon G A T et al., (1989) Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing, (Kirkland D J Ed.), Cambridge University PressReport, 26-65). Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
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 judgement about test item activity. Results of this type will be reported as equivocal.

Statistics:

Mean values and standard deviations were calculated.

Results and discussion

Test results

Species / strain:

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

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 applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH value was measured in Experiment 1 and 2 (7.58 and 7.56) and thus represented no problem to the Ames Test.

RANGE-FINDING/SCREENING STUDIES: In the preliminary toxicity test, the test item was tested at concentrations of 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate with and without metabolic activation. The test was performed in the strains TA100 and WP2uvrA. After 48 h incubation at 37°C the plates were assessed for numbers of revertant colonies using a colony counter and examined for effects on the growth of the bacterial background lawn.The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Test Results of Experiment 1 (plate incorporation).

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	0	137 ± 7.4	15 ± 2.6	30 ± 5.1	23 ± 3.6	10 ± 1.7
–	50	146 ± 1.5	17 ± 5.6	28 ± 7.6	22 ±3.8	12 ± 2.6
–	150	133 ± 27.8	17 ± 2.1	28 ± 2.1	25 ± 3.2	13 ± 3.1
–	500	125 ± 9.3	16 ± 1.5	28 ± 7.0	24 ± 2.5	11 ± 2.5
–	1500	144 ± 29.2	14 ± 4.0	31 ±7.0	19 ± 2.5	13 ± 1.0
–	5000	125 ± 10.4	21 ± 1.4	26 ± 3.0	24 ± 8.0	14 ± 3.8
Positive controls. –S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (μg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	742 ± 11.1	480 ± 28.2	787 ± 25.1	192 ± 5.5	1186 ± 123.7
+	0	136 ±12.2	18 ± 5.2	44 ± 2.1	30 ± 5.3	9 ± 2.1
+	50	126 ± 15.1	18 ± 7.5	37 ± 2.6	36 ± 10.1	10 ± 2.1
+	150	128 ± 7.0	22 ± 4.4	37 ± 5.1	22 ± 3.2	9 ± 2.1
+	500	112 ± 15.5	20 ± 5.9	32 ± 5.6	29 ± 3.2	12 ± 1.5
+	1500	115 ± 10.6	16 ± 4.0	31 ± 6.4	22 ± 2.5	9 ± 5.7
+	5000	125 ± 25.1	20 ± 9.0	42 ± 4.6	26 ± 12.4	9 ± 1.5
Positive controls. + S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	1349 ± 91.7	225 ± 7.2	387 ± 16.0	314 ± 39.0	148 ± 11.8

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

9AA 9-Aminoacridine

Table 2: Test Results of Experiment 2 (pre-incubation). 

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	0	99 ± 13.1	23 ± 5.8	22 ± 3.6	26 ± 5.6	14 ± 2.0
–	50	86 ± 8.1	17 ± 2.6	23 ± 3.1	23 ± 3.6	9 ± 4.5
–	150	75 ± 4.0	14 ± 5.0	24 ± 2.5	19 ± 1.5	15 ± 8.6
–	500	76 ± 4.0	17 ± 2.1	25 ± 6.0	21 ± 3.8	10 ± 4.0
–	1500	83 ± 9.2	19 ± 4.2	24 ± 4.0	16 ± 2.5	9 ± 1.5
–	5000	81 ± 6.7	15 ± 4.6	16 ± 2.5	17 ± 7.5	9 ± 2.6
Positive controls  –S9	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Concentrations (μg/plate)	3	5	2	0.2	80
	Mean No. of colonies/plate (average of 3 ± SD)	453 ± 107.2	359 ± 77.4	673 ± 7.4	155 ± 3.5	1010 ± 197.7
		TA 100	TA1535	WP2uvrA	TA98	TA1537
+	0	102 ± 25.2	13 ± 3.8	39 ± 2.3	34 ± 2.9	12 ± 5.6
+	50	91 ± 7.2	15 ± 1.2	36 ± 1.2	30 ± 1.2	11 ± 2.0
+	150	83 ± 21.0	13 ± 1.5	38 ± 0.6	33 ± 4.5	11 ± 4.2
+	500	87 ± 19.3	14 ± 6.4	36 ± 2.6	21 ± 1.0	16 ± 8.9
+	1500	80 ± 8.3	13 ± 2.9	30 ± 6.2	27 ± 4.4	14 ± 1.0
+	5000	96 ± 13.7	15 ± 2.0	35 ± 5.1	29 ± 10.2	16 ± 3.1
Positive controls +S9	Name	2AA	2AA	2AA	BP	2AA
	Concentrations (μg/plate)	1	2	10	5	2
	Mean No. of colonies/plate (average of 3 ± SD)	1206 ± 80.5	237 ± 6.7	300 ± 29.3	236 ± 4.5	205 ± 12.5

ENNG = N-ethyl-N-nitro-N-nitrosoguanidine

4NQO = 4-Nitroquinoline-1-oxide

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

9AA 9-Aminoacridine

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative