Propanoic acid, 2-hydroxy-, C12-13-branched alkyl esters

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

migrated 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: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

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

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexyl lactate

EC Number:

228-503-2

EC Name:

2-ethylhexyl lactate

Cas Number:

6283-86-9

IUPAC Name:

2-ethylhexyl 2-hydroxypropanoate

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Analytical purity: 99%
- Lot/batch No.: 0805000655

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media: culture medium: Culture medium consisted of RPMI 1640 (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/mL and 50 U/mL, respectively) (Invitrogen Corporation) and 30 U/mL heparin (Sigma, Zwijdrecht, The Netherlands).
Lymphocytes cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin (Remel, Europe Ltd., United Kingdom) was added.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and β-naphtoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

Dose range finding test: 10, 33, 100, 333 and 1000 µg/mL (3 h exposure time) and 10, 33, 100, 333, 1000 and 2023 µg/ml (24 and 48 h exposure time)

Main assay:
First cytogenicity assay: with and without S9-mix: 100, 125, 150, 175, 200, 235, 270 and 300 µg/mL culture medium (3 h exposure time, 24 h fixation time)
Cytogenetic assay 1A: with S9-mix: 150, 200, 250, 270, 275, 280, 285, 290, 295 and 300 µg/mL culture medium (3 h exposure time, 24 h fixation time).
Second cytogenetic assay: Without S9-mix: 30, 100, 125, 150, 175, 200, 225, 250, 275 and 300 µg/mL culture medium (24 and 48 h exposure time, 24 and 48 h fixation time) and with S9-mix: 150, 200, 250, 270, 280, 290, 300, 310, 320 and 330 µg/mL culture medium (3 h exposure time, 48 h fixation time).
Cytogenicity assay 2A: Without S9-mix: 150, 200, 250, 300, 310, 320, 330, 340 and 350 µg/mL culture medium (48 h exposure time, 48 h fixation time)

The following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 100, 225 and 250 µg/mL culture medium (24 h exposure time, 24 h fixation time)
Without S9-mix: 150, 250 and 300 µg/mL culture medium (48 h exposure time, 48 h fixation time)
With S9-mix: 150, 300 and 330 µg/mL culture medium (3 h exposure time, 24 h fixation time)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO. PURASOLV®EHL dilutions were used within 2.5 h after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

-S9: mitomycin C; +S9: cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION:
- Exposure duration: dose range finding test: 3 h, 24 h and 48 h in the absence of S9-mix or 3 h in the presence of S9-mix. First cytogenetic assay: 3h in the presence and absence of S9-mix. Second cytogenetic assay: 24 h and 48 h in the absence of S9-mix and 3 h in the presence of S9-mix.
- Fixation time (start of exposure up to fixation or harvest of cells): Dose range finding test: 24 h (3 h and 24 h exposure) or 48 h (48 h exposure). First cytogenetic assay: 24 h. Second cytogenetic assay: 24 h (24h exposure) or 48 h (3 and 48 h exposure).

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/mL medium) (Acros Organics, Belgium)
STAIN (for cytogenetic assays): slides were stained for 10-30 min with 5% (v/v) Giemsa (Merck) solution in tap water

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 100 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) it induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) a statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria were not absolute and other modifying factors might have entered into the final evaluation decision.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results

Species / strain:

lymphocytes: cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: the highest tested concentration was determined by the solubility of the test substance in the culture medium at the 3 h exposure time. At the 24 and 48 h exposure time, the test substance was tested beyond the limit of solubility to obtain adequate toxicity data. Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
First cytogenicity assay: in the presence of S9-mix, no appropriate dose level could be selected for scoring of chromosome aberrations since at a concentration of 270 µg/mL no cytotoxicity was observed (9%), whereas the next higher concentration of 300 µg/mL was too toxic for scoring (79%). This part of the experiment was repeated (Cytogenetic assay 1A).

The following dose levels were selected for scoring to chromosome aberration:
Without S9-mix: 0, 200, 270 and 300 µg/ml culture medium (3 h exposure time, 24 h fixation time)
With S9-mix: 0, 150, 270 and 280 µg/ml culture medium (3 h exposure time, 24 h fixation time)

Second cytogenetic assay:
In the absence of S9-mix, at the 48 h continuous exposure time, no appropriate dose levels could be selected for scoring of chromosome aberrations since no concentration was available with the appropriate cytotoxicity greater than 50%. This part of the experiment was repeated (Cytogenetic assay 2A).

The following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 0, 100, 225 and 250 µg/ml culture medium (24 h exposure time, 24 h fixation time)
Without S9-mix: 0, 150, 250 and 300 µg/ml culture medium (48 h exposure time, 48 h fixation time)
With S9-mix: 0, 150, 300 and 330 µg/ml culture medium (3 h exposure time, 24 h fixation time)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

All results are presented in Tables 1 -10 in the attached background material (NOTOX Project 494584).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

In a mammalian cell cytogenetic assay (chromosome aberration), primary peripheral human lymphocytes cultures were exposed to the PURASOLV®EHL (2-ethylhexyl-S-lactate, purity 99%) at concentrations of 0, 100, 150, 200, 225, 250, 270 and 300 µg/mL without metabolic activation and 0, 150, 270, 280, 300 and 330 µg/mL with metabolic activation (phenobarbital and β-naphtoflavone induced rat liver S9 -mix). The test substance was tested up to cytotoxic concentrations. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background. Based on the results of the study, PURASOLV®EHL is not considered clastogenic in human lymphocytes.