Ethyl enantate

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:

09.12.2015 - 12.01.2016

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Ethyl enantate

EC Number:

203-382-9

EC Name:

Ethyl enantate

Cas Number:

106-30-9

Molecular formula:

C9H18O2

IUPAC Name:

ethyl heptanoate

Method

Target gene:

- Salmonella typhimurium: histidine (his)
- Escherichia coli: tryptophan (trp)

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:

S9 Mix

Test concentrations with justification for top dose:

Pre-experiment: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate
Pre-incubation test: 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

Untreated negative controls:

yes

Remarks:

concurrent untreated

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD; 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation
DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours
DATA EVALUATION
- Counting: Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with software program Ames Study Manager (v.1.21)
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn
ACCEPTABILITY CRITERIA
- regular background growth in negative and solvent control
- spontaneous reversion rates in negative and solvent control are in the range of historical data
- positive control substances should produce an increase above 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
- a minimum of 5 analysable dose levels should be present with at least 3 dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.

Evaluation criteria:

Test item is considered mutagen if biologically relevant increase in the number of revertants exceeds the threshold (twice or thrice) of the colony count of the corresponding solvent control.
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:

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

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537 and TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 - 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

333 - 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

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

Key result

Species / strain:

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

Metabolic activation:

with

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

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2500 - 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Summary of individual results of Experiment I

Treatment	Concentration (µg/plate)	Revertant Colony counts (mean±SD)
		TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
Without metabolic activation
DMSO	n.a.	11±7	8±1	23±7	124±7	39±3
Untreated	n.a.	12±2	14±2	29±4	132±8	38±4
Test item	3	13±6	8±2	23±5	141±20	34±1
	10	9±4	11±3	23±8	116±7	32±10
	33	11±3	9±4	22±4	131±9	38±6
	100	13±3	8±2	23±9	117±11	39±3
	333	9±1	10±2	22±7	68±10	37±6
	1000	13±5	10±2	22±3	64±7	31±8
	2500	10±3	8±2R	22±5	57±10	31±6
	5000	8±2R	8±2R	18±4	63±8	37±9
NaN3	10	1062 ± 127			2100 ± 119
4-NOPD	10			361±4
4-NOPD	50		79±15
MMS	2.0 µL					823±12
With metabolic activation
DMSO	n.a.	13±3	14±2	29±10	106±15	41±7
Untreated	n.a.	13±6	16±6	38±4	145±4	53±7
Test item	3	13±3	13±1	34±9	128±20	44±3
	10	9±2	16±6	36±10	127±12	45±6
	33	11±5	15±3	37±3	122±14	51±3
	100	11±1	12±4	35±8	127±25	55±6
	333	10±3	14±3	31±6	102±5	45±5
	1000	11±6	18±3	27±6	121±13	37±5
	2500	8±3R	14±3R	36±7R	116±9R	20±3R
	5000	8±5R	15±5R	28±8R	89±13R	11±1R
2-AA	2.5	386±38	192 ± 18	3597 ± 408	3412 ± 295
2-AA	10					322 ± 13

 

R = reduced background growth

 

NaN3 = sodium azide; 2-AA = 2-aminoanthracene; 4-NOPD = 4-nitro-o-phenylene-diamine; MMS = methyl methane sulfonate

Summary of individual results of Experiment II

Treatment	Concentration (µg/plate)	Revertant Colony counts (mean±SD)
		TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
Without metabolic activation
DMSO	n.a.	13± 4	11 ± 2	29 ± 2	145 ± 6	38 ± 7
Untreated	n.a.	8 ± 3	9 ± 3	31 ± 10	184 ± 16	42 ± 7
Test item	10	13 ± 1	12 ± 3	22 ± 7	161 ± 8	36 ± 7
	33	12 ± 4	10 ± 5	27 ± 7	142 ± 2	38 ± 6
	100	11 ± 2	7 ± 1	18 ± 4	128 ± 6	37 ± 7
	333	10 ± 2	5 ± 1	22 ± 6	50 ± 19	36 ± 7
	1000	7 ± 2^M R	5 ± 2^M R	13 ± 5^M R	40 ± 11^M R	24 ± 4
	2500	6 ± 1^M R	4 ± 2^M R	12 ± 4^M R	41 ± 3^M R	27 ± 7^R
	5000	5 ± 2^M R	3 ± 2^M R	8 ± 1^M R	39 ± 8^M R	29 ± 8^R
NaN3	10	1088± 34			1705± 608
4-NOPD	10			359± 35
4-NOPD	50		83± 8
MMS	2.0 µL					694± 81
With metabolic activation
DMSO	n.a.	9 ± 5	11 ± 3	41 ± 0	150 ± 17	57 ± 8
Untreated	n.a.	10 ± 3	14 ± 6	37 ± 8	207 ± 3	56 ± 14
Test item	10	9 ± 4	13 ± 5	30 ± 7	142 ± 11	46 ± 4
	33	13 ± 3	15 ± 4	38 ± 3	155 ± 14	41 ± 13
	100	10 ± 2	13 ± 4	31 ± 9	155 ± 12	49 ± 11
	333	9 ± 3	16 ± 0	31 ± 5	130 ± 11	53 ± 11
	1000	10 ± 5	13 ± 3	33 ± 8	133 ± 13	39 ± 2
	2500	8 ± 2	12 ± 5	26 ± 4	121 ± 4	25 ± 5
	5000	6 ± 3	11 ± 2	26 ± 4	95 ± 11	10 ± 2
2-AA	2.5	312± 19	180± 10	4278± 332	4246± 17
2-AA	10					276± 34

M = manual count

R = reduced background growth

 

NaN3 = sodium azide; 2-AA = 2-aminoanthracene; 4-NOPD = 4-nitro-o-phenylene-diamine; MMS = methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the Salmonella typhimurium and Escherichia coli strains used. Therefore, the test item is considered to be non-mutagenic.

Executive summary:

In the current study the potential of the test item to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA1535, TA1537, TA98, TA 100, and Escherichia coli strain WP2uvrA was assessed. The study was perfomed according to OECD 471 and GLP.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. In the pre-experiment the test item was tested at 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate and in the pre-incubation test at 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate. No precipitation of the test item occurred up to the highest investigated dose.

The plates incubated with the test item showed reduced background growth in all strains used. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains, indicating the test item was cytotoxic. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment 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.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, during the described mutagenicity test and under the experimental conditions reported, 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.