2-ethylhexyl 3,5,5-trimethylhexanoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2012 - January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl 3,5,5-trimethylhexanoate

EC Number:

275-073-7

EC Name:

2-ethylhexyl 3,5,5-trimethylhexanoate

Cas Number:

70969-70-9

Molecular formula:

C17H34O2

IUPAC Name:

2-ethylhexyl 3,5,5-trimethylhexanoate

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy, non-smoking donors not receiving medication. For this study, blood was collected from a male donor (31 years old) for the first experiment and from a 29 year-old male donor for Experiment II. All donors had a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Exp. I (without S9): 17.5*, 30.7*, 53.7*, 94.0*, 164.5*, 287.9*, 503.8*, 881.6*, 1542.9*, 2700.0* µg/mL
Exp. II (without S9): 10.0, 17.5, 30.7, 53.7*, 94.0*, 164.5*, 287.9*, 503.8*, 881.6*, 1542.9* µg/mL
Exp. I (with S9): 17.5, 30.7, 53.7*, 94.0*, 164.5*, 287.9*, 503.8*, 881.6*, 1542.9*, 2700.0* µg/mL
Exp. II (with S9): 17.5, 30.7, 53.7, 94.0, 164.5, 287.9, 503.8, 881.6, 1542.9, 2700.0 µg/mL
* indicates that phase separation was observed at the end of the treatment

Vehicle / solvent:

On the day of the experiment (immediately before treatment), the test item was dissolved in ethanol. The final concentration of ethanol in the culture medium was 0.5 % (v/v). The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. The osmolarity and pH-value were determined in the solvent control and the maximum concentration without metabolic activation: Concentration 2700 µg/mL, osmolarity 390 and 404 mOsm in solvent control of experiment 1 and 2 and 354 and 355 mOsm in test solution. pH was 7.5 – 7.6 in all solutions.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

experiments without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

experiments with S9

Details on test system and experimental conditions:

Blood samples were obtained from healthy, non-smoking donors not receiving medication. For this study, blood was collected from a male donor (31 years old) for the first experiment and from a 29 year-old male donor for Experiment II. All donors had a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes. Blood samples were drawn by venous puncture and collected in heparinized tubes. The tubes were sent to Harlan CCR to initiate cell cultures within 24 hrs after blood collection. If necessary, the blood was stored before use at 4 °C.
S9 (Preparation by Harlan CCR): Phenobarbital/ß-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar rats (Hsd Cpb: WU, Harlan Laboratories B. V., 5960 AD Horst, The Netherlands), weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. phenobarbital i.p. (Desitin; 22335 Hamburg, Germany) and ß-naphthoflavone orally (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1 part plus 3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test. The protein concentration of the S9 preparation was 45.8 mg/mL (Lot no. 150612) for Experiment I and 29.8 mg/mL (Lot no. 280912) for Experiment II.
S9 Mix: An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL (0.71 mg/mL in Experiment I) in the cultures. Cofactors were added to the S9 mix to reach the following concentrations: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, and 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4. During the experiment, the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. (Ames, B.N., McCann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test. Mutation Res., 31, 347-363).
Range-finder: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the mutagenicity assay. The pre-test phase was performed with 10 concentrations of the test item and a solvent and positive control. All cell cultures were set up in duplicate. Exposure time was 4 hrs (with and without S9 mix). The preparation interval was 22 hrs after start of the exposure.
Dose Selection: The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced mitotic indices of approx. 50 % of control, and/or the occurrence of precipitation. In the case of non-toxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, provided that the substance can be formulated in an appropriate solvent.
With regard to the molecular weight of the test item, 2700.0 µg/mL of 2-ethylhexyl 3,5,5-trimethylhexanoate (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 17.5 and 2700.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item wasobserved. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using reduced mitotic indices as an indicator for toxicity in Experiment I, no cytotoxic effects were observed after 4 hours treatment in the absence and presence of S9 mix. Therefore, 2700.0 µg/mL was chosen as top treatment concentration for Experiment II.
Experimental Performance Cytogenetic Experiment
Culture Initiation: Blood cultures were set up in bulk within 24 hrs after collection in 75 cm² cell culture flasks (Nunc GmbH & Co. KG, 65203 Wiesbaden, Germany). The culture medium was DMEM:F12 (Dulbecco's modified eagle medium / Ham's F12 medium; mixture 1:1; Life Technologies GmbH (Invitrogen division), 64293 Darmstadt, Germany) already supplemented with 200 mM Glutamax. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 Pg/mL) (SEROMED, 12247 Berlin, Germany), the mitogen phytohemagglutinin (PHA, final concentration 3 Pg/mL, SEROMED), 10 % FBS (fetal bovine serum) provided by PAA Laboratories GmbH (35091 Cölbe, Germany), 10 mM HEPES, and the anticoagulant heparin (125 IE/mL, NATTERMANN, 50829 Köln, Germany).
The following volumes were added to the flasks (per 10 mL): 7.60 mL culture medium, 1.00 mL fetal bovine serum, 0.10 mL antibiotic solution, 0.05 mL phytohemagglutinin, 0.05 mL heparin, 0.10 mL HEPES, and 1.10 mL whole blood. All incubations were done at 37 °C in a humidified atmosphere with 5.5 % CO 2 (94.5 % air).
Treatment for exposure time of 4 hours: About 72 hrs after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks (Nunc GmbH & Co. KG, 65203 Wiesbaden, Germany). The culture medium was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 hours the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in "saline G". The washing procedure was repeated once as described. The "saline G" solution was composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, glucose•H2O 1100 mg, Na2HPO4 •2H2O 192 mg, and KH2PO 4 150 mg; pH was adjusted to 7.2.
After washing the cells were re-suspended in complete culture medium and cultured until preparation.
Treatment for exposure time 22 hours (without S9 mix): About 72 hours after seeding for each test group 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks (Nunc GmbH & Co. KG, 65203 Wiesbaden, Germany). The culture medium was replaced with complete medium (with 10 % FBS) containing the test item without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed. All cultures were incubated at 37 °C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).
Preparation of the Cultures: Three hours before harvesting, colcemid (Fluka, 89203 Neu-Ulm, Germany) was added to the cultures (final concentration 0.2 Pg/mL). The cultures were harvested by centrifugation 22 hours after beginning of treatment. The supernatant was discarded and the cells were re-suspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37 °C for 20 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part, respectively). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide. The cells for evaluation of cytogenetic damage were stained with Giemsa (MERCK, 64293 Darmstadt, Germany).
Analysis of Metaphase Cells: The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphases were scored. Only metaphases with 46 ± 1 centromer regions were included in the analysis. To describe a cytotoxic effect, the mitotic index (% cells in mitosis) was determined.
Data Recording: The generated data were recorded in the laboratory protocol. The results were presented in tabular form, including experimental groups with the test item, solvent controls and positive controls, respectively.

Evaluation criteria:

Acceptability of the Assay: The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) The number of aberrations found in the solvent controls falls within the range of the historical laboratory control data.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations.
A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the historical control data.
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of the historical control data and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (9) (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In Exp. II (without S9) cytotoxicity was observed at the highest evaluated concentration (37.8 % of control).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test item, dissolved in ethanol, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without S9 mix. In Experiment II the exposure period was 4 hours with S9 mix and 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. At least 100 metaphases per culture were scored for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were scored. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 2700.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests. No visible precipitation of the test item in the culture medium was observed. Phase separation was observed in Experiment I at 17.5 µg/mL and above in the absence of S9 mix and at 53.7 µg/mL and above in the presence of S9 mix. In Experiment II in the absence of S9 mix phase separation was observed at 53.7 µg/mL and above. In Experiment II in the presence of S9 mix no phase separation was observed. No relevant influence on pH value and osmolarity was observed.
In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix cytotoxicity was observed at the highest evaluated concentration (37.8 % of control).
In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.5 - 2.8 % aberrant cells, excluding gaps) were close to the range of the solvent control values (0.0 - 0.5 % aberrant cells, excluding gaps) and within the range of the laboratory historical solvent control data. However, in Experiment I in the presence of S9 mix, one statistically significant increase was observed after treatment with 30.7 µg/mL (2.0 % aberrant cells, excluding gaps). In Experiment II in the absence of S9 mix statistically significant increases were observed at all evaluated concentrations (2.5, 2.8, 2.0 % aberrant cells, excluding gaps). The values were clearly in the range of the laboratory historical solvent control data (0.0 - 3.0 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (770.0 µg/mL) or CPA (15.0 or 20.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item Dragoxat ® 89 did not induce structural chromosomal aberrations in human lymphocytes in vitro, when tested up to cytotoxic or the highest required concentration.

Any other information on results incl. tables

Table: Summary of results:

Experiment	preparation interval	conc. [µg/mL]	mitotic indices [%]	incl. gaps*	aberrant cells [%]	carrying exchanges
I (without S9)	22 hrs	solv. control^1	100	1.0	0.5	0.5
		pos. control^2	83.2	13.0	13.0^S	1.5
		881.6^PS	105.6	0.5	0.5	0.0
		1542.9^PS	92.9	0.5	0.5	0.0
		2700.0^PS	95.0	2.0	2.0	0.0
II (without S9)	22 hrs	solv. control^1	100.0	0.0	0.0	0.0
		pos. control^2#	42.1	42.0	42.0^S	5.0
		10.0	89.6	3.0	2.5^S	0.0
		17.5^##	76.2	2.8	2.8^S	0.0
		30.7	37.8	2.0	2.0^S	0.0
I (with S9)	22 hrs	solv. control^1	100.0	0.0	0.0	0.0
		pos. control^3	78.9	22.5	22.5^S	5.0
		881.6^PS	100.0	0.5	0.5^S	0.0
		1542.9^PS	83.3	0.5	0.5	0.0
		2700.0^PS	88.2	1.5	1.5	0.0
II (with S9)	22 hrs	solv. control^1	100.0	0.5	0.5	0.0
		pos. control^4	61.1	9.0	8.0^S	0.0
		881.6	96.9	2.5	2.5	0.0
		1542.9	103.8	1.5	1.5	0.0
		2700.0	90.1	2.5	2.5	0.0

* Including cells carrying exchanges

PS Phase separation occurred at the end of treatment

S Aberration frequency statistically significant higher than corresponding control values

# Evaluation of 50 metaphases per culture

## Evaluation of 200 metaphases per culture

1 Ethanol 0.5 % (v/v)

2 EMS 770.0 Pg/mL

3 CPA 15.0 Pg/mL

4 CPA 20.0 Pg/mL*

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

The test substance is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or the highest required concentration.

Executive summary:

The test item, dissolved in ethanol, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments. The following study design was performed:

Without S9 mix: Exposure period 4 hrs in Experiment 1 and 22 hrs in Experiment 2, Recovery period of 18 hrs in Experiment 1 and none in Experiment 2

With S9 mix: Exposure period 4 hrs and 18 hrs Recovery period in Experiment 1 and 2

Preparation interval was 22 hrs in all experiments.

In each experimental group two parallel cultures were analysed. Per culture at least 100 metaphases were evaluated for structural chromosomal aberrations, except for the positive control in Experiment II, in the absence of S9 mix, where only 50 metaphases were evaluated. The highest applied concentration in this study (2700.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 473. The chosen treatment concentrations and the rationale for the dose selection as well as evaluated experimental points and the results were reported.

In Experiment I in the absence and presence of S9 mix and in Experiment II in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix cytotoxicity was observed at the highest evaluated concentration. Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. However, in Experiment I in the presence of S9 mix, one statistically significant increase was observed after treatment with 30.7 µ g/mL (2.0 % aberrant cells, excluding gaps). In Experiment II in the absence of S9 mix statistically significant increases were observed at all evaluated concentrations (2.5, 2.8, 2.0 % aberrant cells, excluding gaps). The values were clearly in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and therefore considered as being biologically irrelevant.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, teh test substance is considered to be non-clastogenic in this chromosome aberration test, when tested up to cytotoxic or the highest required concentration.