Diisopropyl adipate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains and E.coli strain in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

In vitro mammalican chromosome aberration study:

The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

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.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98

Remarks:

1

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Remarks:

2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Remarks:

2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

The liver microsome fraction (S-9) was prepared from the liver of Fischer rats

Test concentrations with justification for top dose:

1. 6 different concentrations were used; 10 mg/plate was the maximum concentration
2. 0, 312.5, 625, 1250, 2500 or 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: AF-2 (2- (2-furyl) -3- (5-nitro-2-furyl) acrylamide), 2-aminoanthracene

Remarks:

2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

2. METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

1. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated).

2. When the increase increased more than 2 times and the increase was found to be reproducible or dose dependent, it was decided that the test substance has mutagenicity (positive) in this test system.

Statistics:

No data

Species / strain:

S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94 and TA98

Remarks:

1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

S. typhimurium, other: TA98, TA1535, TA1537, TA100

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1. ADDITIONAL INFORMATION ON CYTOTOXICITY: The maximum dose for negative results represents the highest non-cytotoxic dose used in the experiment

2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The ratio was set to about 3 in the range of 50 to 5000 μg / plate, and the test was carried out. As a result, no
antibacterial activity was observed in all the test bacteria at all doses. Therefore, the maximum dose in this test was determined to be 5000 μg / plate for both direct method and metabolic activation method in all test bacteria.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains and E.coli strain in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 10 mg/plate being the maximum concentration. The chemical was dissolved in DMSO. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

In another study,bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 312.5, 625, 1250, 2500 or 5000µg/plate with and without S9. Concurrent solvent and positive controls were also included in the study.The test chemical did not induce gene mutation inSalmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains and E.coli strain in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on data from various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE for the target CAS is summarized based on data from various test chemicals

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration test

Target gene:

No data

Species / strain / cell type:

mammalian cell line, other: Chinese hamster fibroblast cell line CHL

Remarks:

1/2

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum
Essential Medium (MEM; GIBCO) supplemented by 10% calf serum
- Properly maintained: yes by 4 day passages
- Periodically checked for Mycoplasma contamination: No data available
- Periodically checked for karyotype stability: No data available
- Periodically "cleansed" against high spontaneous background: No data available

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

1. At three different doses with 0.25 mg/mL being the maximum dose concentration
2. At three different doses with 2.0 mg/mL being the maximum dose concentration

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The chemical was soluble in ethanol

2. At three different doses with 2.0 mg/mL being the maximum dose concentration

Untreated negative controls:

yes

Remarks:

Untreated cells served as negative control

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

1

Untreated negative controls:

yes

Remarks:

Untreated cells served ar negative control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

2

Details on test system and experimental conditions:

1./2. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): Giemsa solution (1.5%, pH 6.8)
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: 100 well spread metaphases

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

1/2. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%.

Statistics:

No data

Species / strain:

mammalian cell line, other: Chinese hamster fibroblast cell line CHL

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature. The studies are as mentioned below:

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical 1 and test chemical 2. The cells were exposed to the test material at three different doses with 0.25 mg/mL being the maximum concentration for test chemical 1 and 2.0 mg/mL beinf the maximum concentration for test hemical 2 for 48 hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The test chemical 1 and 2 did not induce chromosomal aberration in chinese hamster fibroblast cell line CHL and hence it is not likely to classify as a gene mutant in vitro.

Based on the observations made, the test chemical did not induce chromosomal aberration in Chinese hamster fibroblast cell line CHL and hence it is not likely to classify as a gene mutant in vitro.

Reference 3

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

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

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 / 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

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

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

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

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

  

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Data available for the various test chemicals was reviewed to determine the mutagenic nature. The studies are as mentioned below:

Ames assay:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 with and without S9 metabolic activation system. The test was performed as per the preincubation assay at six different concentrations with 10 mg/plate being the maximum concentration. The chemical was dissolved in DMSO. Preincubation was performed for 20 mins and the exposure duration was for 48 hrs. The result was considered positive if the number of colonies found was twice the number in the control (exposed to the appropriate solvent or untreated). The test chemical did not induce a doubling of revertant colonies over the control using S. typhimurium strains TA92, TA1535, TA100, TA1537, TA94 and TA98 in the presence and absence of S9 metabolic activation system and hence the chemical is not likely to classify as a gene mutant in vitro.

In another study,bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0, 312.5, 625, 1250, 2500 or 5000µg/plate with and without S9. Concurrent solvent and positive controls were also included in the study.The test chemical did not induce gene mutation inSalmonella typhimurium strains TA98, TA1535, TA1537, TA100 and Escherichia Coli WP2uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian chromosmome aberration study:

Chromosomal aberration study was performed to determine the mutagenic nature of the test chemical 1 and test chemical 2. The cells were exposed to the test material at three different doses with 0.25 mg/mL being the maximum concentration for test chemical 1 and 2.0 mg/mL beinf the maximum concentration for test hemical 2 for 48 hr. Colcemid (final concn 0.2µg/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution for 12-15 min. A hundred well-spread metaphases were observed under the microscope. In the present studies, no metabolic activation systems were applied. The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Untreated cells and solvent-treated cells served as negative controls, in which the incidence of aberrations was usually less than 3.0%. The results were considered to be negative if the incidence was less than 4.9%, equivocal if it was between 5.0 and 9.9%, and positive if it was more than 10.0%. The test chemical 1 and 2 did not induce chromosomal aberration in chinese hamster fibroblast cell line CHL and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

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.

Based on the data available and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.