Hexanoic acid, 2-ethyl-, C12-15-alkyl esters

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

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:

18 May 1992- 02 June 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar 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:

histodine locus

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: sensibility to crystalviolet, histidine necessity, sensibility to ultra-violet radiations, resistance to Atrpicitlin

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: sensibility to crystalviolet, histidine necessity, sensibility to ultra-violet radiations, resistance to Atrpicitlin

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: sensibility to crystalviolet, histidine necessity, sensibility to ultra-violet radiations

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: sensibility to crystalviolet, histidine necessity, sensibility to ultra-violet radiations

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

other: sensibility to crystalviolet, histidine necessity, sensibility to ultra-violet radiations

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Dosage
The following concentrations expressed in µg/plate were used;
10.000
1.000
100
10
1
o (negative control)

Vehicle / solvent:

The test material COSMACOL EOI, was dissolved in Dimethylsulphoxide (DMSO) at a concentration of 100 mg/ml.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene

Details on test system and experimental conditions:

TEST PROCEDURE

Plate test without metabolic activation

0.1 ml of the appropriate dilution of the test solution and 0.1 ml of a bacterial suspension containing approximately 108 -109 cfu/ml were added in this sequence to 2 ml of molten top agar.
The ingredients were thoroughly mixed and immediately poured into plates containing minimal medium.
At the same time, negative control and positive controls using the chosen concentration of the reference mutagens were performed.
The plates were incubated at 37°C for 48 72 hours. After incubation the revertant colonies obtained in the plates were counted. Triplicate plates were used for the test material and two for the positive controls.

Plate test with metabolic activation

0.1 ml of the appropriate dilution of the test solution, 0.1 ml of bacterial suspensions and 0.5 ml of the metabolic activation system were added in this sequence to 2 ml of molten top agar.
The ingredients were thoroughly mixed and immediately poured into plates containing minimal medium.
At the same time, negative control and positive controls using the chosen concentration of the reference mutagens were performed.
The plates were incubated at 37°C for 48 72 hours. After incubation the revertant colonies obtained in the plates were counted.
Triplicate plates were used for the test material and two for the positive controls.

Evaluation criteria:

ACCEPTANCE CRITERIA
At the end of the assay, a sterility check on S9Mix must show less two viable colonies per 0.5 ml.
At the end of the test, a sterility check of the test material must show less than two viable colonies per plate.
The positive controls must produce at least a threefold increase in the number of revertant colonies with regard to the mean value for the respective negative control.
The mean number of spontaneous revertant colonies in the negative controls must correspond to the following values:
STRAIN ACCEPTABLE RANGE
TA 1535 20 ± 15
TA 1537 20 ± 15
TA 1538 15 ± 10
TA 98 40 ± 25
TA 100 150 ± 90

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 1538

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

Species / strain:

S. typhimurium 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

Species / strain:

S. typhimurium TA 100

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

Additional information on results:

TEST MATERIAL MUTAGENICITY (Table n 1 and n 2 - see background attached info)
The test material induced the development of as many revertant colonies as those induced by the negative control, both in the absence and in presence of the rat liver metabolizing system, at the maximum dose tested (10 mg/plate).

CONTROLS
Preliminary tests
The genetic characteristics of the bacterial strains were found to be unaltered.

Test material toxicity
The test material did not induce toxic effects at a concentration oOf 10 mg/plate both in the absence and in presence of the metabolic activation system.

Negative control
The number of revert~~t colonies on the negative control plates fell within the normal range.

Positive control
All the positive controls induced a significant increase in the number of revertant colonies.

Sterility test
The sterility test performed on the test material and on S9Mix did not show any bacterial contamination.

Conclusions:

The test material at maximum concentration 10 mg/petri dish produced an increase in number of revertant colonies similar to the negative control so the test material is unable to induce mutation under there conditions.

Executive summary:

A test was performed to verify the mutagenic potential of the test material COSMACOL EOI on Salmonella typhimurium.

The test was carried out on 5 strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 , TA100).

The mutagenic activity of the material was assessed by comparing the number of revertant colonies induced with the positive control.

This activity was tested in the presence and absence of a metabolizing system and done directly in a petri dish. The test material was tested at a concentration of 100000 µg/ml, 10000 µg/ml , 1000 µg/ml, 100 µg/ml, 10 µg/ml corresponding to the dose 10000 µg/petri dish, 1000 µg/petri dish, 100 µg/petri dish, 10 µg/petri dish and 1 µg/petri dish.

The results of the study can be summarized as follows:

The test material at maximum concentration 10 mg/petri dish produced an increase in number of revertant colonies similar to the negative control so the test material is unable to induce mutation under there conditions.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 February 2018 - 16 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

23 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- Identification: C12-15 Alkyl Ethylhexanoate
- CAS Number: 90411-66-8
- EC Number: 291-443-0
- Appearance: Clear liquid
- Purity (%): 100%

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human lymphocytes prepared form the pooled blood of two female doners

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Range finder: 1.814, 3.023, 5.039, 8.398, 14, 23.33, 38.88, 64.8, 108, 300, & 500 µg/mL
Main test: 50, 200, 400, & 750 µg/mL
Top dose selected based upon results of range finder.

Vehicle / solvent:

TO BE CONFIRMED

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: vinblastine

Species / strain:

human lymphoblastoid cells (TK6)

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

Additional information on results:

Treatment of cells with C12-15 Alkyl Ethylhexanoate for 24+24 hours in the absence and presence of S-9 in the main test resulted in frequencies of MNBN cells that were not significantly higher (at the p≤0.05 level), compared to those observed in the concurrent vehicle controls, at any concentration analysed. The MNBN cell frequencies were within the 95th percentile of the observed historical control (normal) range at all concentrations analysed and there was no statistically significant linear trend.
Overall, it was conclude that C12-15 Alkyl Ethylhexanoate did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of solubility for 3+21 hours and 24+24 hours in the absence of S-9 and up to 2000 µg/mL (the maximum concentration for in vitro cytogenetics studies required by the regulatory test guidelines) for 3+21 hours in the presence of S-9 under the experimental conditions described.

Micronucleus Experiment – Results summary

Treatment	Concentration (mg/mL)	Cytotoxicity (%)^$	Mean MNBN Cell Frequency (%)	Historical Control Range (%)^#	Statistical Significance
24+24 hour -S-9	Vehicle^a	-	0.6	0.00 – 0.80	-
Main test	50	2	0.65		NS
	200	9	0.5		NS
	400	32	0.4		NS
	750	38	0.6		NS
	*MMC, 0.10	16	19.3		p≤0.001
	*VIN, 0.04	37	3.5		p≤0.001

a Vehicle control was Ethanol

* Positive control

# 95th percentile of the observed range

$ Based on replication index

NS Not significant

Data for 24+24 hour treatments -S-9, Range-Finder- Male donors

Treatment (µg/mL)	Replicate	Mono	Bi	Multi	Total	RI	Cytotoxicity Based on RI (%)
Vehicle	A	33	149	18	200	0.93	-
	B	23	164	13	200	0.95
	Total	56	313	31	400	0.94
UTC	A	18	147	35	200	1.09	-
1.814	A	22	155	23	200	1.01	0
3.023	A	18	160	22	200	1.02	0
5.039	A	18	165	17	200	1	0
8.398	A	13	177	10	200	0.99	0
14	A	22	163	15	200	0.97	0
23.33	A	18	172	10	200	0.96	0
38.88	A	25	157	18	200	0.97	0
64.8	A	29	159	12	200	0.92	2:00 PM
108	A	29	159	12	200	0.92	2:00 PM
180	A	25	168	7	200	0.91	3:00 PM
300	A	34	159	7	200	0.87	8:00 PM
500	A	61	137	2	200	0.71	25 P

UTC = Untreated control

P = Precipitation observed at treatment

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

Conclusions:

The test material was determined not to be mutagenic under the conditions of the test.

Executive summary:

In this guideline (OECD 487) study, conducted with GLP certification, EC 291-443-0 was considered not to be mutagenic under the conditions of the test.

Overall, it was conclude that the test material did not induce micronuclei in cultured human peripheral blood lymphocytes when tested up to the limit of solubility for 3+21 hours and 24+24 hours in the absence of S-9 and up to 2000µg/mL (the maximum concentration for in vitro cytogenetics studies required by the regulatory test guidelines) for 3+21 hours in the presence of S-9 under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The registered substance failed to induce mutagenic reactions in any of the in vitro mutagenicity tests conducted. The registered substance is therefore considered not to fulfill the criteria for classification as a germ cell mutagen under the Classificaiton, Labelling, and Packaging (CLP) regulation (1272/2008).