Ethyl enantate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is not genotoxic in bacteria or in mammalian cells.

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

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

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.

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.

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study using a read-across test substance

Justification for type of information:

Read-across from ethyl hexanoate to ethyl heptanoate is considered justified based on strong similarities with regard to chemical structure and metabolic pathways. A full read-across justification including comparison of toxicological profiles is included in section 13 of the IUCLID dossier.

Reason / purpose for cross-reference:

read-across source

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Experiment I without metabolic activation at 720 and 1440 µg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Read-across was done from ethyl hexanoate. Based on the result, and on the structural, chemical and toxicological similarities between ethyl hexanoate and ethyl heptanoate, ethyl heptanoate did not induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic.

Executive summary:

Read-across was done from ethyl hexanoate. In the current study the potential of ethyl hexanoate to induce gene mutations was assessed in an in vitro mammalian cell gene mutation test according to OECD 476 and GLP. The HPRT locus in the Chinese hamster cell line V79 was the target gene.

This in vitro test is an assay for the detection of forward gene mutations in mammalian cells. Gene mutations are discussed as an initial step in the carcinogenic process. The V79 cells are exposed to the test item both with and without exogenous metabolic activation. At a defined time interval after treatment the descendants of the treated original population are monitored for the loss of functional HPRT enzyme.

HPRT (hypoxanthine-guanine phosphoribosyl transferase) catalyzes the conversion of the nontoxic 6-TG (6-thioguanine) to its toxic ribophosphorylated derivative. Cells deficient in HPRT due to a forward mutation are resistant to 6-TG and are able to proliferate in the presence of 6-TG whereas the non-mutated cells die.

Phenotypic expression is achieved by allowing exponential growth of the cells for 7 - 9 days. The expression period is terminated by adding 6-TG to the culture medium.

The mutant frequency is determined by seeding known numbers of cells in medium containing the selective agent to detect mutant cells, and in medium without selective agent to determine the surviving cells. After a suitable period the colonies are counted. Mutant frequencies are calculated from the number of mutant colonies corrected for cell survival.

In order to establish a concentration response effect of the test item at least four concentration levels are tested. The highest concentration level should induce a reduced level of survival. To demonstrate the sensitivity of the test system reference mutagens are tested in parallel to the test item.

The assay was performed in two independent experiments. The treatment time was 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 24 hours without and 4 hours treatment with metabolic activation.

The maximum test item concentration (1440 μg/mL) was equal to a molar concentration of about 10 mM.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

The acceptability criteria were met. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus showed the sensitivity of the test system and the activity of the metabolic activation system.

Under the current experimental conditions, ethyl hexanoate did not induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic.

Based on the result, and on the structural, chemical and toxicological similarities between ethyl hexanoate and ethyl heptanoate, ethyl heptanoate is not expected to induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study using a read-across test substance

Justification for type of information:

Read-across from ethyl hexanoate to ethyl heptanoate is considered justified based on strong similarities with regard to chemical structure and metabolic pathways. A full read-across justification including comparison of toxicological profiles is included in section 13 of the IUCLID dossier.

Reason / purpose for cross-reference:

read-across source

Species / strain:

lymphocytes: Human

Remarks:

Experiment I

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

lymphocytes: Human

Remarks:

Experiment II

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

higher concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Read-across was done from ethyl hexanoate. Based on the result, and on the structural, chemical and toxicological similarities between ethyl hexanoate and ethyl heptanoate, ethyl heptanoate is considered to be unable to induce chromosome breaks and/or gain or loss in this test system and should be considered non-mutagenic.

Executive summary:

Read-across was done from ethyl hexanoate. In the current study the potential of ethyl hexanoate to induce micronuclei in human lymphocytes in vitro was assessed according to OECD 487.

The occurrence of micronuclei in interphase cells provides an indirect but easy and rapid measure of structural chromosomal damage and aneugenicity in cells that have undergone cell division during or after exposure to the test substance. Micronuclei arise from chromosomal fragments or whole chromosomes and are inducible by clastogens or agents affecting the spindle apparatus.

The induction of cytogenetic damage in human lymphocytes was assessed in two independent experiments and in each experimental group two parallel cultures were analyzed. Ethyl hexanoate was dissolved in DMSO. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix.

The cells were stimulated for 48 hour with phytohemeagglutinine (PHA) to activate the proliferation, before exposure. After exposure, the cells were washed and left to recover for 16 hours before the cytokinesis was blocked by cytochalasin B for 20 minutes. The cells were prepared 40 hours after start of treatment with ethyl hexanoate.

The highest applied concentration in this study was 1442 µg/mL. Dose selection of the cytogenetic experiment was performed considering the molecular weight of ethyl hexanoate and the OECD Guideline 487. The chosen treatment concentrations are in Experiment I (4 hours with and without S9 mix): 9.4, 16.4, 28.7, 50.2, 87.9, 154, 269, 471, 824 and 1442 µg/mL and in Experiment II (20 hours without S9 mix): 127, 191, 286, 428, 642, 962, 1442 µg/mL.

In Experiment I, phase separation of ethyl hexanoate in culture medium was observed at 824 μg/mL and above in the absence and presence of S9 mix at the end of treatment. In addition, phase separation occurred in Experiment II in the absence of S9 mix at 962 μg/mL and above at the end of treatment.

No relevant influence on osmolarity or pH was observed.

In each experimental group two parallel cultures were analyzed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation in Experiment I. In Experiment II, higher concentrations were not evaluable for cytogenetic damage due to strong cytotoxic effects.

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed.

In both experiments, either Demecolcin (100.0 ng/mL), MMC (1.5 μg/mL) or CPA (15.0 μg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, ethyl hexanoate did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, ethyl hexanoate is considered to be non-mutagenic in this in vitro micronucleus test.

Based on the result, and on the structural, chemical and toxicological similarities between ethyl hexanoate and ethyl heptanoate, ethyl heptanoate is considered to be unable to induce chromosome breaks and/or gain or loss in this test system and should be considered non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For this endpoint there are multiple studies available assessing the genotoxicity of ethyl heptanoate and the read-across substances ethyl octanoate and ethyl hexanoate.

For ethyl heptanoate there is an Ames test available according to OECD 471 using Salmonella typhimurium strains TA1535, TA1537, TA98, TA 100, and Escherichia coli strain WP2uvrA with and without liver microsomal activation. The concentrations were 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate in Experiment I and 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate in Experiment II. No precipitation of ethyl heptanoate occurred and no substantial increase in revertant colony numbers of any of the tester strains was observed following treatment at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Therefore, ethyl heptanoate did not induce gene mutations and is non-mutagenic.

Regarding the genotoxicity in mammalian cells, there are 3 recent studies on ethyl hexanoate and ethyl octanoate. For ethyl hexanoate an OECD 476 (in vitro mammalian cell gene mutation test) and an OECD 487 (in vitro mammalian cell micronucleus test) are available, for ethyl octanoate a study similar to OECD 473 is available.

To investigate the potential of ethyl hexanoate to induce gene mutations in vitro in mammalian cell, a gene mutation test according to OECD 476 was performed. Chinese hamster V79 cells were exposed to ethyl hexanoate with and without metabolic activation. The HPRT locus is the target gene and the loss of functional HPRT enzyme is monitored with the use of 6-TG (6-thioguanine) as cells deficient in HPRT are resistant to 6-TG and are able to proliferate in the presence of 6-TG whereas the non-mutated cells die. The mutant frequency is calculated from the number of mutant colonies corrected for cell survival.

Treatment time was 4 hours in the first experiment with and without metabolic activation and 24 hours in the second experiment without and 4 hours treatment with metabolic activation.

The maximum test item concentration (1440 μg/mL) was equal to a molar concentration of about 10 mM.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments, while the acceptability criteria were met. Therefore, ethyl hexanoate did not induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic.

Based on the result, and on the structural, chemical and toxicological similarities between ethyl hexanoate and ethyl heptanoate, ethyl heptanoate is expected to not induce gene mutations at the HPRT locus in V79 cells and therefore is considered to be non-mutagenic.

The potential of ethyl hexanoate to induce chromosomal aberrations in vitro was assessed according to OECD 487 in human lymphocytes. In Experiment I, the exposure period was 4 hours with and without S9 mix and in Experiment II, the exposure period was 20 hours without S9 mix. The test concentrations were in Experiment I: 9.4, 16.4, 28.7, 50.2, 87.9, 154, 269, 471, 824 and 1442 µg/mL and in Experiment II: 127, 191, 286, 428, 642, 962, 1442 µg/mL.

1000 binucleate cells per culture were scored for cytogenetic damage on coded slides, the CBPI was determined in 500 cells per culture. In both experiments no biologically relevant increase in the number of cells carrying micronuclei was observed, while the controls were valid. Ethyl hexanoate did not induce micronuclei in human lymphocytes.

Based on the result, and on the structural, chemical and toxicological similarities between ethyl hexanoate and ethyl heptanoate, ethyl heptanoate is considered to be unable to induce chromosome breaks and/or gain or loss in this test system and should be considered non-mutagenic.

In the study on ethyl octanoate which was performed similar to the OECD Guideline 473, Chinese hamster fibroblast cells were exposed to ethyl octanoate at different doses up to 2 mg/mL for 24 and 48 hours, without metabolic activation. Chromosome preparations were made and 100 well-spread metaphases were observed under the microscope. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, were recorded. Ethyl octanoate was found to be negative as the percentage of polyploidization effects was below 10% and no structural aberrations were observed after 48 hours.

Taking togehter all the information above, ethyl heptanoate is not genotoxic in bacteria or mammalian cells.

Justification for classification or non-classification

In the Regulation No 1274/2008 on classification, labelling and packaging, the criteria for mutagenicity are set in section 3.5. The data from the in vitro mutagenicity chromosome aberration tests, in vitro mammalian cell gene mutation test and a bacterial reverse mutation test indicate that the test item is not to be considered as genotoxic/mutagenic according to the Regulation EC No 1272/2008, as all results were negative.