Hexanedioic acid, mixed esters with decanoic acid, heptanoic acid, octanoic acid and pentaerythritol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Aug 2017 - 07 Nov 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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

Hexanedioic acid, mixed esters with decanoic acid, heptanoic acid, octanoic acid and pentaerythritol

EC Number:

268-597-2

EC Name:

Hexanedioic acid, mixed esters with decanoic acid, heptanoic acid, octanoic acid and pentaerythritol

Cas Number:

68130-55-2

Molecular formula:

C36H64O10

IUPAC Name:

6-[3-(decanoyloxy)-2-[(heptanoyloxy)methyl]-2-[(octanoyloxy)methyl]propoxy]-6-oxohexanoic acid

Test material form:

liquid

Specific details on test material used for the study:

Based on the information provided by sponsor, the test item is considered as an Unknown
or Variable Composition, Complex Reaction products and Biological materials (UVCBs),
test item is able to be well dissolved in acetone.
EC#268-597-2, CAS #68130-55-2
Purity: >99% I UVCB, Homogenous liquid
Pale yellow liquid, no odour

Method

Target gene:

thymidine kinase gene

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The test system used in the study was L5178Y TK+1
- clone (3.7.2C) from Mus
musculus (mouse) lymphoma (ATCC®CRL-9518™), passage 7.

Metabolic activation:

with and without

Metabolic activation system:

The cofactor supplemented post-mitochondrial fraction (S9) prepared from the liver of Aroclor 1254 induced Sprague-Dawley rats was used as metabolic activation system.

Test concentrations with justification for top dose:

To prepare test substances, the \est item was dissolved in acetone at concentration of 60%,
20%, 6. 7% and 2.3% (v/v) respectively, four concentrations in total. The concentration
interval is approximately 3.

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

The test system used in the study was L5178Y TK+/- clone (3.7.2C) from Mus
musculus (mouse) lymphoma (ATCC®CRL-9518™), passage 7.

L5178Y TK+1- cells were maintained in the complete growth medium R1 OP [ROP
(RPMI 1640 Medium containing 2g/L Sodium Bicarbonate, 0.1% Poloxamer 188, 1 mM Sodium Pyruvate and 1 % Penicillin-Streptomycin) with 10% Fetal Bovine Serum (FBS)] in exponentially proliferating status at 37 °C, 5% C02.

L5178Y TK+/- cells were sub-cultured in the complete growth medium R10P
1- day before being used for treatment. 6 x 10E6 cells were used for each
treatment. The complete growth medium R1 OP was replaced by treatment
medium R3P (ROP with 3% FBS) with test substances, negative control
substances, and positive control substances so as to expose the cells to these
substances.

The exposure was performed with and without metabolic activation. The cofactor
supplemented post-mitochondrial fraction (S9) prepared from the liver
of Aroclor 1254 induced Sprague-Dawley rats was used as metabolic
activation system. The concentration of S9 fraction in the S9 mixture was 10%
(v/v) while the concentration of S9 fraction in final test medium was 1 %.

The exposure was conducted for 3 hrs. After the exposure, the above
solutions were removed, the cells were washed, added with fresh complete
growth medium R10P at seeding density of 30 x 10E4 cells/ml and then
incubated for another 48 hrs at 37 °C in 5% C02 incubator. During 2-day
expression, the cells was counted on each day and cell density was adjusted
to 30 x 10E4 cells/ml after Day 1 counting.

Results and discussion

Test results

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:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Under the condition of this study, the above results show that, using acetone as solvent, the test item -
Mixed esters with CAS No. 68130-55-2, Batch or Lot No: 026X6 is non-mutagenic to the L5178Y TK +/-
clone (3.7.2C) cells at 3-hour treatment both in the absence and presence of S9 metabolic activation
system.

Executive summary:

At 3-hour treatment with and withous S9, MF of negative control substance is within the range of

35 to 140 x 10E-6 , cloning efficiency of negative control substance is within the range of 65-120%,

suspension growth of negative control substance is within the range of 8-32 fold, which indicates

acceptability of data.

Induced MF (IMF) of positive control is more than 300 x 1 over the concurrent negative control,

IMF of small colony of positive control is 265 x 10E-6 (> 150 x 10E-6), which indicates positive

response.

At 3-hour treatment with and without S9, there is no mutant frequency (MF) of test substances

more than 90 x 10 E-6 (Global Evaluation Factor) over the concurrent negative control, which

indicates no positive response in all test substances.