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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

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.

The test item was considered to be non-mutagenic under the conditions of this test in the Ames study.

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

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

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

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

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.

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

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

 

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.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2017 - 01 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

- Identification: H-32
- Physical state/Appearance: Pale yellow slightly viscous liquid
- Batch: 64296
- Purity: >99%
- Expiry Date: 30 September 2018
- Storage Conditions: Room temperature, in the dark

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: 5%
- Properly maintained: yes

Metabolic activation:

with and without

Test concentrations with justification for top dose:

1.95; 3.91; 7.81; 15.63; 31.25 and 62.5 μg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation.

Details on test system and experimental conditions:

- Cell density at seeding: 1 x 10^6 cells/mL for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation.

- Exposure duration: 4h with and without metabolic activation (S9), and 24 h without metabolic activation with continuous shaking using an orbital shaker within an incubated hood.

- NUMBER OF REPLICATIONS: duplicates, both with and without metabolic
activation at eight dose levels of the test item (0.49 to 62.5 μg/mL for all three of the exposure groups), vehicle and positive controls.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

 Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In VitroMammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (acetone), and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

 

Group	Concentration of H-32 (µg/mL) plated for viability and mutant frequency
4-hour without S9	1.95, 3.91, 7.81, 15.63, 31.25, 62.5
4-hour with S9 (2%)	1.95, 3.91, 7.81, 15.63, 31.25, 62.5
24-hour without S9	1.95, 3.91, 7.81, 15.63, 31.25, 62.5

Results……..

The maximum dose level used in the Mutagenicity Test was limited by the onset of test item precipitate. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 Jan 2017 - 23 Feb 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
Identification: H-32
Chemical Name:
Physical state/Appearance: Pale yellow slightly viscous liquid
Batch Number: 64296
Purity: > 99%
Expiry: 30 September 2018
Storage Conditions: Room temperature in the dark

Direct acting compounds in the absence of S9-mix:
Identity: N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)
Batch number: 67F-3700
Expiry date: 18 September 2017
Solvent: DMSO
Concentration: 2 μg/plate for WP2uvrA 3 μg/plate for TA100 5 μg/plate for TA1535

Identity: 9-Aminoacridine (9AA)
Batch number: S32398-438
Purity: 99.9%
Expiry date: 01 October 2017
Solvent: DMSO
Concentration: 80 μg/plate for TA1537

Identity: 4-Nitroquinoline-1-oxide (4NQO)
Batch number: 030M1206
Purity: 100%
Expiry date: 08 October 2017
Solvent: DMSO
Concentration: 0.2 μg/plate for TA98

Indirect-acting compounds in the presence of S9-mix:
Identity: 2-Aminoanthracene (2AA)
Batch number: STBB1901M9
Purity: 97.5%
Expiry date: 08 October 2017
Solvent: DMSO
Concentration: 1 μg/plate for TA100 2 μg/plate for TA1535 and TA1537 10 μg/plate for WP2uvrA

Identity: Benzo(a)pyrene (BP)
Batch number: 090M1400V
Purity: 96%
Expiry date: 12 October 2017
Solvent: DMSO
Concentration: 5 μg/plate for TA98



TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

FORM AS APPLIED IN THE TEST (if different from that of starting material)

OTHER SPECIFICS:

Target gene:

histidine synthesis

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation);
- Cell density at seeding (if applicable):
DURATION
- Exposure duration: 48h
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: revertant colonies

Evaluation criteria:

The test material may be considered positive in this test system if it induces a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

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

Key result

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

Key result

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

Key result

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

Key result

Species / strain:

E. coli WP2 uvr A

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

Conclusions:

The test item was considered to be non-mutagenic under the conditions of this test

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate.

Six test item dose levels per bacterial strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Results

The vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). A test item precipitate (globular in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in Experiment 1 (plate incorporation method). Similarly, no toxicological significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix), in Experiment 2 (pre-incubation method). Small, statistically significant increases in TA98 revertant colony frequency were observed in the second mutation test (presence of S9-mix only) at and above 150 μg/plate. These increases were considered to be of no biological relevance because there was no clear evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.7 times the concurrent vehicle control.

Conclusion

H-32 was considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

All three in-vitro studies were concluded as non-mutagenic therefore we can confirm that H-32 does not cause genetic toxicity. Therefore according to the CLP regulation the test substance is classified as non-hazardous for genetic toxicity.