ethyl tetrahydrofuran-2-carboxylate

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:

2013-12-11 to 2014-01-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study Positive control was slightly higher than the range of the historical control in strain TA 1535 wihout S9. This does not influence the validity of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 1997-07-21

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2013-04-11

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

ethyl tetrahydrofuran-2-carboxylate

EC Number:

807-745-8

Cas Number:

16874-34-3

Molecular formula:

C7H12O3

IUPAC Name:

ethyl tetrahydrofuran-2-carboxylate

Test material form:

other: liquid

Details on test material:

- Physical state: clear liquid, colourless
- Storage condition of test material: at room temperature

Method

Target gene:

TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
E. coli WP2 uvrA: trp-

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 were used as the metabolic activation system. Protein concentration of the S9 prepartion ws 37.8 mg/mL.

Test concentrations with justification for top dose:

Pre-experiment/Experiment 1 (with and without S9 mix): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment 2 (with and without S9 mix): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Positive control without metabolic activation: dissolved in deionised water; concentration: 10 μg/plate (strains TA 1535, TA 100)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Remarks:

Positive control without metabolic activation: dissolved in DMSO; concentration: 10 μg/plate (strain TA 98); 50 μg/plate (strain TA 1537)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Positive control without metabolic activation: dissolved in deionised water; concentration: 2 μL/plate (strain WP2 uvrA)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Positive control with metabolic activation: dissolved in DMSO; concentration: 2.5 μg/plate (strains TA 1535, TA 1537, TA 98, TA 100); 10.0 μg/plate in WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation (pre-experiment/Experiment 1) and preincubation (Experiment 2)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: at least 48 hours at 37°C

NUMBER OF REPLICATIONS: for each strain and dose level including the controls, three plates were used.

The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with the software program Ames Study Manager (v.1.21).

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. Whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: in the first experiment, the test item precipitated in the overlay agar of the test tubes at 2500 and 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate. No precipitation was noted in the second experiment. The undissolved particles had no influence on the data recording.

CYTOTOXICITY:
No reduced background growth was observed in any of the experimental parts with and without metabolic activation.
No toxic effects, evident as a reduction of the number of revertants (below an induction factor of 0.5), occurred up to the maximum concentration with and without metabolic activation.

MAIN EXPERIMENTS:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any concentration 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.
Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Please also refer to table 1 and 2 in the field "Any other information on results incl. tables" below.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

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	WP2 uvrA
Without Activation	DMSO		16 ± 2	10 ± 3	29 ± 7	87 ± 22	45 ± 14
	Untreated		17 ± 3	7 ± 3	35 ± 7	105 ± 2	47 ± 9
	PI 27221	3 µg	13 ± 1	8 ± 1	27 ± 2	85± 6	40 ± 8
		10 µg	17 ± 7	10 ± 6	28 ± 8	87 ± 10	35 ± 3
		33µg	17 ± 4	10 ± 2	25 ± 4	82 ± 6	47 ± 9
		100 µg	18 ± 3	11 ± 2	25 ± 4	82 ± 14	45 ± 0
		333 µg	18 ± 2	10 ± 3	29 ± 8	91 ± 4	39 ± 7
		1000 µg	16 ± 6	7 ± 3	23 ± 3	82 ± 16	44 ± 1
		2500 µg	16 ± 4	7 ± 4	23 ± 5	84 ± 12	35 ± 1
		5000 µg	19 ± 3^P	13 ± 3^P	26 ± 5^P	85 ± 13^P	40 ± 14^P
	NaN3 4-NOPD 4-NOPD MMS	10 µg	2755 ± 147			1950 ± 101
		10 µg			271 ± 25
		50 µg		65 ± 9
		2.0 µL					639 ± 153
With Activation	DMSO		13± 2	15± 2	26± 4	106± 8	37± 6
	Untreated		19 ± 2	16 ± 2	41 ± 1	115 ± 16	45 ± 12
	PI 27221	3 µg	14 ± 2	22 ± 4	42 ± 8	99 ± 10	47 ± 6
		10 µg	15 ± 6	13 ± 3	33 ± 8	100 ± 11	37 ± 6
		33 µg	10 ± 2	11 ± 6	33 ± 6	87 ± 17	35 ± 5
		100 µg	10 ± 8	19 ± 2	26 ± 8	88 ± 16	33 ± 13
		333 µg	19 ± 5	14 ± 1	40 ± 8	80 ± 12	44 ± 6
		1000 µg	16 ± 5	19 ± 8	45 ± 11	88 ± 5	43 ± 9
		2500 µg	18 ± 1	17 ± 2	37 ± 12	71 ± 7	38 ± 3
		5000 µg	14 ± 2^P	11 ± 3	28 ± 4^P	73 ± 4^P	30 ± 1^P
	2-AA	2.5 µg	361 ± 38	351 ± 17	2122 ± 51	2286 ± 193
	2-AA	10.0 µg					191 ± 4

 

Key to Positive Controls		Key to Postfix Codes
NaN3	sodium azide	P	Precipitate
2-AA	2-aminoanthracene
4-NOPD	4-nitro-o-phenylene-diamine
MMS	methyl methane sulfonate

 

Table 2: 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		14 ± 2	13 ± 3	23 ± 8	93 ± 19	51 ± 9
	Untreated		9 ± 3	12 ± 3	17 ± 4	123 ± 11	49 ± 6
	PI 27221	3 µg	14 ± 1	11 ± 3	29 ± 1	81± 10	37 ± 2
		10 µg	12 ± 1	11 ± 2	21 ± 5	97 ± 9	57 ± 8
		33µg	13 ± 1	11 ± 2	21 ± 2	78 ± 7	45 ± 6
		100 µg	16 ± 6	9 ± 3	24 ± 4	81 ± 5	47 ± 12
		333 µg	14 ± 6	9 ± 2	22 ± 2	86 ± 6	52 ± 7
		1000 µg	13 ± 2	5 ± 3	19 ± 8	88 ± 8	45 ± 12
		2500 µg	17 ± 4	10 ± 3	21 ± 1	102 ± 7	40 ± 9
		5000 µg	15 ± 3^P	11 ± 3	22 ± 6	90 ± 7	47 ± 3
	NaN3	10 µg	2604± 78			2171 ± 72
	4-NOPD	10 µg			310 ± 24
	4-NOPD	50 µg		79 ± 2
	MMS	2.0 µL					611 ± 17
With Activation	DMSO		15± 7	18± 3	32± 1	101± 21	61± 7
	Untreated		12 ± 2	17 ± 7	42 ± 4	112 ± 2	52 ± 2
	PI 27221	3 µg	11 ± 3	17 ± 2	32 ± 9	92 ± 2	52 ± 2
		10 µg	17 ± 3	17 ± 3	34 ± 1	100 ± 12	54 ± 6
		33 µg	10 ± 3	17 ± 4	31 ± 7	105 ± 9	64 ± 6
		100 µg	9 ± 1	21 ± 5	30 ± 8	107 ± 6	66 ± 5
		333 µg	11 ± 3	18 ± 5	38 ± 9	99 ± 10	47 ± 6
		1000 µg	13 ± 3	18 ± 4	36 ± 11	95 ± 18	60 ± 3
		2500 µg	12 ± 4	17 ± 6	43 ± 8	119 ± 4	51 ± 13
		5000 µg	10 ± 5	18 ± 3	42 ± 4	112 ± 6	53 ± 7
	2-AA	2.5 µg	399 ± 7	346 ± 27	2897 ± 151	2944 ± 443
	2-AA	10.0 µg					275 ± 11

 

 

Key to Positive Controls
NaN3	sodium azide
2-AA	2-aminoanthracene
4-NOPD	4-nitro-o-phenylene-diamine
MMS	methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
According to Directive 67/548 /EEC and its subsequent amendments and according to Regulation (EC) No 1272/2008 and subsequent regulations, the test substance should not be considered to have a mutagenic potential, and hence no classification or labelling is required.

Executive summary:

A bacterial reverse mutation assay was performed with the test item according to the OECD guideline 471 (1997) using S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 as well as E. coli WP2 uvr A. The test was carried out using the plate incorporation method and the preincubation method with plating in triplicate with and without metabolic activation. The test item was tested at the following concentrations in both experiments: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate. Additionally, a negative control and positive controls were run concurrently.

In the plate incorporation test, the test item precipitated in the overlay agar of the test tubes at 2500 and 5000 μg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate. No precipitation was noted in the preincubation test.

No reduced background growth was observed in any of the experimental parts with and without metabolic activation. No toxic effects, evident as a reduction of the number of revertants (below an induction factor of 0.5), occurred up to the maximum concentration with and without metabolic activation.

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.

In conclusion, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.