Diisopropyl adipate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Data is from study report

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

The purpose of this study was to assess toxic and genotoxic effects of the test chemical on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Diisopropyl adipate

EC Number:

230-072-0

EC Name:

Diisopropyl adipate

Cas Number:

6938-94-9

Molecular formula:

C12H22O4

IUPAC Name:

1,6-bis(propan-2-yl) hexanedioate

Test material form:

other: Colorless liquid

Details on test material:

- Name of test material (as cited in study report): Diisopropyl adipate
- Molecular formula : C12H22O4
- Molecular weight : 230.30 g/mol
- Substance type: Organic
- Physical state: Liquid
- Analytical purity: Not available
- Activity (Clinical Indication): Industrial Chemical
- Safety Precautions: Safety precautions included use of protective clothing, gloves, masks and eye protection (glasses).
- Stability Data: The test chemical was dissolved in 99.5% ethanol and this freshly made stock solution was stored at -20°C until day of testing. On this day, the stock solution was thawed and the appropriate volume of the test chemical was taken for further dilutions in PBS or in cell culture medium (depending on assay used). The stock solution was then immediately returned to the freezer. No changes in chemical stability was observed since the chemical did not precipitate upon thawing
- Storage Condition: At room temperature (RT) in ventilated cabinets

Method

Target gene:

Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Not applicable
- Periodically checked for karyotype stability: Not applicable

Additional strain / cell type characteristics:

other: Hypodiploid, modal No. 20

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

0, 0.1, 0.25, 0.5 or 1.0 mM

Vehicle / solvent:

Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle: The test chemical was dissolved in ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Pre-incubation: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
- Expression time: 7 days
- Selection time: 14 days
- Fixation time: 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
SPINDLE INHIBITOR (cytogenetic assays): Not applicable
STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.

OTHER EXAMINATIONS: Not applicable
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

The cell line was observed for gene mutation at the specified locus

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

Positive at 1 mM concentration

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Table 1A.Effect of exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO₂. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO₂. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

	With S9		Without S9
	with TG	without TG	with TG	without TG
Neg. control	0/4	643/4	0/4	773/4
Pos. control	0/4	688/4	20/4	698/4
0.1 mM	0/4	596/4	0/4	733/4
0.25 mM	0/4	600/4	0/4	605/4
0.5 mM	0/4	551/4	0/4	666/4
1.0 mM	0/4	503/4	9/4	384/4

 

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to the test chemical in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

	With S9	Without S9
Neg. control	N/A	N/A
Pos. control	N/A	3.12x10^-4
0.1 mM	N/A	N/A
0.25 mM	N/A	N/A
0.5 mM	N/A	N/A
1.0 mM	N/A	1.78x10^-4

  

Applicant's summary and conclusion

Conclusions:

The test chemical in the concentration of 0, 0.1, 0.25, 0.5 or 1.0 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical in presence of metabolic activation and did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical upto 0.5 mM in abscence of metabolic activation.

Executive summary:

With S9 metabolic activation

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.1, 0.25, 0.5 or 1.0 mM and S9-induced metabolic activation for 3 hours. The results showed no evidence of cytotoxicity after treatment with the test chemical. Independently of tested concentrations of Diisopropyl adipate, the results showed no evidence of gene toxicity. Therefore, it is considered that the test chemical in the concentration of 0, 0.1, 0.25, 0.5 or 1.0 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence of metabolic activation.

Without S9 metabolic activation

In a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.1, 0.25, 0.5 or 1.0 mM and without S9-induced metabolic activation for 3 hours. The results showed no evidence of cytotoxicity after treatment with the test chemical. After the tested concentrations of Diisopropyl adipate, the results showed no evidence of gene toxicity upto 0.5 mM. Therefore, it is considered that the test chemical in the concentration of 0, 0.1, 0.25, 0.5 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the absence of metabolic activation.

Conclusion

At time of exposure, the test chemical was added in the absence or presence of S9 liver microsomal fraction. The test chemical was added to each applicable well to give a final concentration of 0, 0.1, 0.25, 0.5 or 1.0 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. pH and osmolality was not determined in the gene mutation test.

The positive control ENU showed indication of gene mutations as well as treatment with the test chemical at 1 mM in the absence of S9 liver microsomal fraction, while no other treatment gave rise to gene toxicity.

When the mutation frequency was determined, a frequency of 4.18 x 10^-4was shown after a 3 hour exposure of ENU and 1.78 x 10^-4for 1 mM, both in the absence of S9 liver microsomal fraction. Thus, it is concluded that the test chemical in the concentration of 0, 0.1, 0.25, 0.5 mM does not cause genetic mutation(s) when CHO cells are exposed to the test chemical in the presence or absence of metabolic activation.