Ethyl octanoate

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:

15.03.2016 - 08.04.2016

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

Qualifier:

according to guideline

Guideline:

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

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:

Justification dose selection: Minor toxic effects were observed in the pre-experiment.
Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate
Experiment II: 33, 100, 333, 1000, 2500 and 5000 μg/plate for WP2 uvrA and 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate for all remaining strains

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

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

DATA EVALUATION
- Counting: Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB9 7BN, UK) with software program Ames Study Manager (v.1.21)

DETERMINATION OF CYTOTOXICITY
- Method: 8 concentrations were tested for toxicity and mutation induction with each 3 plates. Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used. Since minor toxic effects were observed 7 concentrations were tested and 5000 μg/plate was chosen as maximal concentration in Experiment II.

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 and TA 98

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

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg/plate without S9 in Experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate without S9 in Experiment I and at 100 - 5000 µg/plate without S9 and 2500 - 5000 µg/plate with S9 in Experiment II

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 and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with S9 in Experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In experiment II in the absence of S9 mix the induction factor was below the toxic border of 0.5 after treatment with the test item at a concentration of 1000 μg/plate. This effect was judged to be caused by statistical fluctuations of the rather low numbers of colonies in strain TA 1537 and does not represent a true toxic effect.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment 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.
The historical range of positive controls was exceeded in strains TA 1535 and TA 98 (Exp. I) with metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay.

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.	15±5	8±2	32±8	158±25	43±10
Untreated	n.a.	17±2	8±2	31±12	164±14	51±1
Test item	3	13±4	10±3	30±2	160 ± 7	40±10
	10	8±1	7±1	24±3	162±9	30±7
	33	14±2	8±3	27±4	148±32	38±3
	100	9±3	13±1	23±4	109±4	42±6
	333	9±4	11±1	29±1	92±7	44±1
	1000	11±3	12±3	23±8	83±10	44±6
	2500	8±2	11±4	23±5	83±20	40±6
	5000	11±4	13±3	22±3	68±21	49±2
NaN3	10	1229 ± 17			2297 ± 65
4-NOPD	10			387±66
4-NOPD	50		89±6
MMS	2.0 µL					1028 ± 41
With metabolic activation
DMSO	n.a.	15±3	12±6	24±5	137±18	47±7
Untreated	n.a.	12±3	14±5	37±10	161 ± 9	54±9
Test item	3	14±2	10±2	44±10	127±10	52±4
	10	11±1	10±2	33±4	119 ± 7	57±8
	33	9±2	12±3	37±7	112 ± 7	50±8
	100	17±0	15±6	34±6	112±11	53±11
	333	18±6	16±3	34±6	143 ± 7	45±3
	1000	14±2	14±4	40±14	163±13	48±7
	2500	14±1	11±4	32±4	131±14	44±7
	5000	12±3	10±0	30 ± 3	148 ± 23	37 ± 12
2-AA	2.5	554±67	120 ± 21	5323 ± 212	3057 ± 382
2-AA	10					440 ± 22

 

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.	11±3	10±3	19±3	167±8	45±3
Untreated	n.a.	11±5	9±4	24±4	178±20	46±3
Test item	3	12±2	10±3	19±2	169±16
	10	13±3	10±4	24±5	142±16
	33	9±1	9±1	22±7	118±8	51±4
	100	10±0	10±4	23±5	69±12	39±4
	333	10±2	8±2	16 ± 5	61 ± 5R	33±14
	1000	10±2	4 ± 0M R	17 ± 10R	46±10R	40±6
	2500	10±3	5 ± 1M R	17 ± 4R	65±21R	41±4
	5000	9±4	5 ± 1M R	9 ± 1M R	43 ± 1R	38 ± 8
NaN3	10	1194 ± 11			1996 ± 123
4-NOPD	10			397 ± 30
4-NOPD	50		102±4
MMS	2.0 µL					764±22
With metabolic activation
DMSO	n.a.	12±2	11±4	33±4	131±9	49±9
Untreated	n.a.	12±2	10±2	37±2	200±7	51±1
Test item	3	13±4	8±1	36±8	123±5
	10	10±2	8±2	28±2	126±14
	33	12±2	9±3	33±7	124±12	54±8
	100	11±2	13±3	40±1	134±10	52±7
	333	11±2	9±3	33±6	137±10	49±8
	1000	11±1	7±5	35±6	116±11	49±10
	2500	8±2	8±3	25±3	57±9	35±4
	5000	12±3	11 ± 2	26 ± 2	57 ± 9	16 ± 2
2-AA	2.5	401±33	89 ± 15	4498 ± 698	3034 ± 203
2-AA	10					523 ± 38

 

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. The test item was tested at 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate in Experiment I and at 33, 100, 333, 1000, 2500 and 5000 μg/plate in Experiment II for WP2 uvrA and at 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate in Experiment II for all remaining strains. 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 strains TA 1537, TA 98 and TA 100 in the second experiment without metabolic activation. Toxic effects, evident as a reduction in the number of revertants, were observed in strains TA 100 (from 100 to 5000 μg/plate without S9 mix and from 2500 to 5000 μg/plate with S9 mix), TA 1537 (at 1000 μg/plate without S9 mix) and WP2 uvrA (at 5000 μg/plate with S9 mix).

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.

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

Justification for type of information:

Read-across from ethyl hexanoate to ethyl octanoate 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

Test concentrations with justification for top dose:

45.0, 90.0, 180.0, 360.0, 720.0 and 1440.0 µg/mL

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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 octanoate, ethyl octanoate 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 octanoate, ethyl octanoate did not 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

Justification for type of information:

Read-across from ethyl hexanoate to ethyl octanoate 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

Test concentrations with justification for top dose:

- 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
- Experiment II: 20 hours without S9 mix; 127, 191, 286, 428, 642, 962, 1442 µg/mL

Species / strain:

lymphocytes: Human lymphocytes from healthy non-smoking donors not receiving medication

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 lymphocytes from healthy non-smoking donors not receiving medication

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 octanoate, ethyl octanoate did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes and 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 micronuclei in human lymphocytes in vitro was assessed according to OECD 487 and in compliance to GLP.

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 octanoate, ethyl octanoate did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic in this in vitro micronucleus test.

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 ehtyl octanoate and the read-across substance ethyl hexanoate.

Regarding the genotoxicity in bacteria there are 2 Ames tests available for the substance itself.

For ethyl octanoate there is a recent Ames test (2016) and an older study (1984). The first study was according to OECD 471 and used the Salmonella typhimurium strains TA1535, TA1537, TA98, TA 100, and Escherichia coli strain WP2uvrA with and without liver microsomal activation. The test concentrations were 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate for the Salmonella typhimurium strains and 33, 100, 333, 1000, 2500 and 5000 μg/plate for the Escherichia coli strain. No precipitation of the test item occurred and no substantial increase in revertant colony numbers of any of the tester strains was observed following treatment with ethyl octanoate at any dose level with or without metabolic activation (S9 mix).

In the older study (1984) no OECD guideline was followed, however, the study was according to the method of Ames, McCann & Yamasaki (1975), the test strains S. typhimurium TA92, TA1535, TA100, TA1537, TA94 and TA98 were included, and the genotoxicity was investigated with and without metabolic activation. Ethyl octanoate was found to be negative for all S. typhimurium strains, confirming the first study.

Based on the results, ethyl octanoate is not mutagenic in Salmonella typhimurium and Escherichia coli strains.

Regarding the genotoxicity in mammalian cells, read-across is done to ethyl hexanoate. For ethyl hexanoate a recent OECD 476 (in vitro mammalian cell gene mutation test) and recent OECD 487 (in vitro mammalian cell micronucleus test) are 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 hexanate 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 octanoate, ethyl octanoate did 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 octanoate, ethyl octanoate is considered to be unable to induce chromosome breaks and/or gain or loss in this test system and should be considered non-mutagenic.

An older study (1984) on ethyl octanoate confirms this latter observation. In that study (1984) study, similar to the OECD Guideline 473, Chinese hamster fibroblast cells were exposed to ethyl octanoate at different doses for 24 and 48 hours (maximum dose tested was 2.0 mg/mL), without metabolic activation. Chromosome analysis was done by observing 100 well-spread metaphases 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 and no structural aberrations were observed after 48 hours.

Taking togehter all the information above, ethyl octanoate 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 bacterial reverse mutation tests 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.