Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

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:

20 Feb 2017 - 01 jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

EC Number:

268-582-0

EC Name:

Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

Cas Number:

68130-25-6

Molecular formula:

C10 H20 O2 . x C8 H16 O2 . x C8 H16 O2 . x C5 H12 O4

IUPAC Name:

Decanoic acid, ester with 2,2-bis(hydroxymethyl)-1,3-propanediol 2-ethylhexanoate octanoate

Test material form:

liquid

Details on test material:

Identification: H-32
CAS No.: 68130-25-6
Batch: 64296
Purity: > 99%
Appearance: Pale yellow liquid
Expiry Date: 30 September 2018
Storage Conditions: At room temperature
Stability in Solvent: Not relevant
Purpose of Use: Industrial chemical

Specific details on test material used for the study:

- Identification: H-32
- CAS Number: 68130-25-6
- Sponsors Description: Pale yellow liquid
- Batch: 64296
- Purity: > 99%
- Molecular Weight: Theoretical value 704.6
Measured value 706.0
- Expiry Date: 30 September 2018
- Storage Conditions: Room temperature in the dark

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:human lymphocytes
- Sex, age and number of blood donors if applicable:
Preliminary Toxicity Test: female, aged 33 years
Main Experiment: male, aged 29 years



MEDIA USED
- CO2 concentration if applicable: 5%

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate metabolizing system (S9)

Test concentrations with justification for top dose:

i) 4-hour exposure without S9-mix - the dose range of test item used was 0, 2, 4, 8, 16, 32, 64, 128μg/mL.
ii) 4-hour exposure with S9-mix (2%) - the dose range of test item used was 0, 4, 8, 16, 32, 64, 128 and 256 μg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix - the dose range of test item used was 0, 2, 4, 8, 16, 32, 64 and 128 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Mitomycin C used in abcence of S9-mix. Cyclophosphamide (CP) used in the presance of S9-mix

Details on test system and experimental conditions:

DURATION
- Exposure duration: 4 and 24 hours at approx 37 ºC, 5% CO2 in humidified air
NUMBER OF REPLICATIONS: duplicates
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: dry slides were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium
NUMBER OF CELLS EVALUATED: 2000

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level
A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Results and discussion

Test results

Key result

Species / strain:

other: Human lymphocytes

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

Any other information on results incl. tables

Dose level (µg/mL)	24 Hour without S9
	Mean	% Control Group
0	5.65	100
2	-	-
4	-	-
8	7.50	133
16	8.33	147
32	8.65	153
64	-	-
128	-	-
MMC 0.1	2.9	51

 

Applicant's summary and conclusion

Conclusions:

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

The test item,was considered to be non-clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1991).

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at three dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected for the Main Test were as follows:

Group	Final concentration of test item H-32 (μg/mL)
4(20)-hour without S9	0, 2, 4, 8, 16, 32, 64, 128
4(20)-hour with S9 (2%)	0, 4, 8, 16, 32, 64, 128, 256
24-hour without S9	0, 2, 4, 8, 16, 32, 64, 128

Results

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

Conclusion

The test item, H-32 was considered to be non-clastogenic to human lymphocytes in vitro.