Allyl hexanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro (Ames test): negative (±S9 mix) in vitro (Micronucleus assay): negative in vitro (Mammalian cell gene mutation assay): negative (±S9 mix)

Link to relevant study records

Reference

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-28 till 2009-12-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline conform GLP study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals, December 13, 2007, Draft Proposal for a new Guideline No. 487 “In vitro Mammalian Cell Micronucleus Test” (3rd version).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed by Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz (2009-03-30)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

not applicable

Species / strain / cell type:

lymphocytes: from human donors

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from a 27 year-old male donor for the first experiment and from a 29 year-old female donor for Experiment II. Blood samples were drawn by venous puncture and collected in heparinised tubes.
- Type and identity of media: The culture medium was DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1) already supplemented with 15 mM HEPES. The antibiotic solution contained 10,000 U/mL penicillin and 10,000 µg/mL streptomycin. Additionally, the medium was supplemented with the mitogen Phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), 10 % FBS (fetal bovine serum), the anticoagulant heparin (25,000 U.S.P.-U/mL) and L-glutamin 200 mM. All incubations were done at 37 °C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).
- Properly maintained: yes
Treatments were commence at around 44 - 48 hours after culture initiation when the cells are actively proliferating.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Pre-Experiment:
- without metabolic activation, 4 hours treatment: 10.2, 17.8, 31.2, 54.7, 95.7, 167.4, 292.9, 512.7, 897.1 and 1570.0 µg/mL
- with metabolic activation, 4 hours treatment: 10.2, 17.8, 31.2, 54.7, 95.7, 167.4, 292.9, 512.7, 897.1 and 1570.0 µg/mL
Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Dose selection of Experiment II was based on the results obtained in Experiment I.
Experiment II:
- without metabolic activation, 20 hours treatment: 31.2, 54.7, 95.7, 167.4, 292.9, 512.7, 897.1 and 1570.0 µg/mL
- with metabolic activation, 4 hours treatment: 95.7, 167.4, 292.9, 512.7, 897.1 and 1570.0 µg/mL
For more detail see table under "any other information on material and methods incl. tables".

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: On the day of the experiment (immediately before treatment), the test item was dissolved in DMSO (E. MERCK, 64293 Darmstadt, Germany; purity 99.9 %). The final concentration of DMSO in the culture medium was 0.5 % (v/v).
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Demecolcin; 4.0 µg/mL (Exp.I) and 0.05 µg/mL (Exp.II), dissolved in deionised water

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Migrated to IUCLID6: 1.0 µg/mL (Exp.I) and 0.3 µg/mL (Exp.II), dissolved in deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Migrated to IUCLID6: 12.5 µg/mL (Exp. I) and 10.0 µg/mL (Exp. II), dissolved in 0.9% saline

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (+16 hours recovery period) or 20 hours; Cells were either treated for 4 hours in the absence of S9 mix (Exp.I) and in the presence of S9 mix (Exp.I and II) or for 20 hours without S9 mix (Exp.II).
The culture medium was replaced with serum-free medium (Experiment I without S9 mix; Experiment I and II in with S9 mix) or complete medium with 10 % FBS (v/v) (Experiment II without S9 mix), containing the test item.
- Recovery period: After treatment for 4 hours, the cells were re-suspended in "saline G" and incubatet for further 16 hours.
- Cytokinesis blocker: After washing, the cells were re-suspended in complete culture medium containing Cytochalasin B (4 µg/mL) and cultured another approximately 20 hours until preparation.
- Fixation time (start of exposure up to fixation or harvest of cells): The cultures were harvested by centrifugation 40 hours after beginning of treatment. Thereafter the cells were fixed and slides were prepared.

STAIN (for cytogenetic assays): The cells were stained with Giemsa.

NUMBER OF REPLICATIONS: In each experimental group two parallel cultures were analysed.

NUMBER OF CELLS EVALUATED: At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

DETERMINATION OF CYTOTOXICITY
- Method: other: cytokinesis block proliferation index
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment. Cytotoxicity is characterised by the percentages of reduction in the cytokinesis block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture in duplicate. The experimental conditions in this pre-experimental phase were identical to those required and described for the mutagenicity assay.
The pre-experiment was performed with 10 concentrations of the test item and the negative, solvent and positive controls. All cell cultures were set up in duplicate. Exposure time was 4 hours (with and without S9 mix) and the cells were prepared 40 hours after start of the exposure.

OTHER EXAMINATIONS:
The frequency of micronucleated cells was reported as % micronucleated cells.

OTHER: Evaluation of the slides was performed using NIKON microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area.

Evaluation criteria:

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory’s historical control data and
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.
A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.

Statistics:

Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together.

Species / strain:

lymphocytes: from humans

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

In Experiment I no cytotoxicity was observed up to the highest applied concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: from humans

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

No relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

In Experiment II in the presence of S9 mix no cytotoxicity was observed up to the highest applied concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: from humans

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No relevant increase in the number of micronucleated cells was observed after treatment with the test item.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed at the two highest evaluated concentrations. However, in Experiment II in the presence of S9 mix, the highest applied concentration was not evaluable due to a reduced cell number.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the pre-test on toxicity, precipitation of the test item was observed at the end of treatment at 512.7 µg/mL and above in the presence of S9 mix.

RANGE-FINDING/SCREENING STUDIES: Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA: In Experiment I, in the presence of S9 mix and in Experiment II in the absence of S9 mix, statistically significant increases in cells carrying micronuclei were observed. These values were in the range of the laboratory’s historical solvent control data and therefore considered as being biologically irrelevant.

ADDITIONAL INFORMATION ON CYTOTOXICITY: no further data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Experiment I

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. The micronucleus rates of the cells after treatment with the test item (0.20 -1.10 % micronucleated cells) exceeded the range of the solvent control values (0.20 - 0.40 % micronucleated cells) but were within the range of laboratory’s historical control data. However, two single statistically significant increases were observed in Experiment I in the presence of S9 mix after treatment with 512.7 µg/mL (1.10 % micronucleated cells) and in Experiment II in the absence of S9 mix at 292.9 µg/mL (0.65 % micronucleated cells). The statistical significances have to be regarded as being biologically irrelevant, since the values were in the range of the laboratory’s historical solvent control data, furthermore the increased MNBN frequency was only observed in one of the parallel cultures. Additionally, the solvent control values were at the very low end of the historical range.

Summary of Results

Summary of results of the in vitro micronucleus study in human lymphocytes with Allylcapronate

Exp.	Preparation	Test item	Proliferation	Cytostasis	Micronucleated
	interval	concentration	index	in %*	cells
		in µg/mL	CBPI		in %**
Exposure period 4 hrs without S9 mix
I	40 hrs	Negative control	2.08		0.20 / 0.80***
		Solvent control^1	2.07		0.30
		Positive control^2	1.17	84.72	6.10***^S
		Positive control^4	1.93	13.89	7.00^S
		512.7	2.04	2.25	0.40
		897.1	2.05	1.87	0.25
		1570.0	2.06	0.75	0.30
Exposure period 20 hrs without S9 mix
II	40 hrs	Negative control	2.10		0.30
		Solvent control^1	2.07		0.20
		Positive control^3	1.79	28.48	3.85^S
		Positive control^5	1.58	47.50	14.15^S
		95.7	1.88	17.70	0.25
		167.4	1.95	11.14	0.20
		292.9	1.55	48.88	0.65^S
		512.7	1.48	54.96	0.20

*   For the positive control groups, the relative values are related to the negative controls; for the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleate cells

***The number of micronucleated cells was determined in a sample of 2000 mononucleate cells

^S   Number of micronucleated cells statistically significant higher than corresponding control values

¹       DMSO 0.5 % (v/v)

²       Demecolcin        4.0 µg/mL

³       Demecolcin       0.05 µg/mL

⁴       MMC             1.0 µg/mL

⁵       MMC             0.3 µg/mL

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, Allyl hexanoate is considered to be non-aneugenic/clastogenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations. Therefore, the test item should not be classified and labeled according to regulation (EC) No. 1272/2008.

Executive summary:

The GLP studay was performed according to OECD TG 487. Allyl hexanoate, dissolved in DMSO, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in two independent experiments:

	Without S9-Mix		With S9-Mix
	Exp. I	Exp. II	Exp. I and II
Exposure period	4 h	20 h	4 h
Recovery	16 h	-	16 h
Cytochalasin B exposure	20 h	20 h	20 h
Preparation interval	40 h	40 h	40 h
Total culture period	84 – 88 h	84 – 88 h	84 – 88 h

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenic damage and coded slides. The highest applied concentration in this study (1570.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and according to the guideline. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurence of test item precipitation.

In Experiment I, in the absence and presence of S9 mix and in Experimental II, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentrations. In Experiment II, in the absence of S9 mix, cytotoxicity was observed at the two highest evaluated concentrations. However, in Experiment II in the presence of S9 mix, the highest applied concentration was not evaluable due to a reduced cell number.

In all experimental parts, in the absence and the presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. However, in Experiment I, in the presence of S9 mix and in Experiment II in the absence of S9 mix, statistically significant increases in cells carrying micronuclei were observed. These values were in the range of the laboratory’s historical solvent control data and therefore considered as being biologically irrelevant.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (α < 0.05) in cells with micronuclei.

Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, Allyl hexanoate is nonsidered to be non-aneugenic/clestogenic in this in vitro micronucleus test, when tested up to cytotoxic or precipitating concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

- genetic toxicity in vitro:

No classification, negative for bacterial mutation (two studies; key study: Wild 1983), negative for in vitro micronucleus test (one key study: Bohnenberger 2010), negative for mammalian cell gene mutation assay (one key study: Wollny 2013).

- genetic toxicity in vivo:

No classification, negative for in vivo micronucleus test (one supporting study: Wild 1983a) and negative for gene mutation Drosophila SLRL test (one supporting study: Wild 1983b).

Justification for selection of genetic toxicity endpoint
The selected study was performed under GLP and in accordance with OECD TG 487.

Justification for classification or non-classification

Based on the above stated assessment of the genotoxic potential of allyl hexanoate is deemed non-genotoxic and do not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (Regulation (EC) No 1272/2008 of the European Parliament and of the Council) as implementation of UN-GHS in the EU.