2,2-dimethylpropane-1,3-diyl dibenzoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is a point mutation test in bacteria (Ames test), a point mutation test in mammalian cells (HPRT test) and a test for clastogenicity (MNT in vitro) available, which are all performed according to the respective guidelines under GLP conditions. All 3 tests show that under the experimental conditions used 2,2-dimethylpropane-1,3-diyl dibenzoate has no mutagenic activity.

Endpoint Conclusion: No adverse effect observed (negative).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study and GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

prepared from the livers of Aroclor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

Plate incorporation and preincubation methodoloy with and without S9-mix: 0, 50, 160, 500, 1600, 5000µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

other: 2-aminoanthracene; Mitomycin C for Salmonella typhimuriumTA102 in plate incorporation trials; Cumene for Salmonella typhimurium TA102 in preincubation trials

Details on test system and experimental conditions:

Preincubation method and plate incorporation method

Evaluation criteria:

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100, TA1537 and TA98 this increase should be about twice that of solvent controls. For TA102 an increase of about 100 mutants should be reached. Otherwise the result is evaluated as negative.

Statistics:

no data

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

of 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative

Executive summary:

2,2-Dimethylpropane-1,3-diyl dibenzoate was investigated for point mutagenic effects in the Salmonella/microsome test (plate incorporation test and preincubation method). The test item was dissolved in DMSO and was administered in doses of up to and including 5000 µg/plate without and with S9 mix on the five Salmonella typhimurium LT2 mutant strains TA 1535, TA100, TA1537, TA98 and TA102 according OECD TG 471 under GLP conditions.

Doses up to and including 5000 µg/plate did not cause any bacteriotoxic effects. Substance precipitation occurred at the concentration of 5000 µg/plate. Evidence of mutagenic activity of the test item was not seen. No biologically relevant increase in the mutant count in comparison to the solvent controls was observed in any of the strains tested without and with S9 mix, in the plate incorporation as well as in the preincubation modification under the experimental conditions applied.

The employed positive controls had a marked mutagenic effect as was seen by a biologically relevant increase in mutant colonies compared to the corresponding solvent controls.

Therefore, 2,2-dimethylpropane-1,3-diyl dibenzoate was considered to be negative (non-mutagenic) without and with S9 mix in the plate incorporation as well as in the preincucation modification of the Salmonella/microsome test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP conditions

Qualifier:

according to guideline

Guideline:

other: OECD TG 487

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

In vitro MNT with Chinese Hamster V79 cells.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

other: Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix was prepared from livers of Aroclor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

4 hour-treatment:
- without S9-mix: 5 - 100 µg/ml
- with S9-mix: 5 - 500 µg/ml

24 hour-treatment
- without S9-mix: 2.5 - 80 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: vinblastine sulfate salt

Details on test system and experimental conditions:

The purpose of this study was to investigate whether the test item can induce chromosome breakage (structural chromosome aberrations) or misdistribution of chromosomes leading to aneuploidy, both measured by an increase in the frequency of micronuclei containing mammalian cells in the absence and in the presence of a metabolizing system.
For the evaluation of the frequency of micronucleus containing cells, 2000 cells (1000 cells per slide) per concentration were scored. Only cells which divided at least once and, therefore, formed colonies of ≥ 2 cells were evaluated.

Evaluation criteria:

The test item is classified as mutagenic if one of the test substance concentrations induced a micronucleus frequency that is three times higher than the micronucleus frequency of the concurrent solvent control.
The test item is classified as mutagenic if there is a reproducible concentration-related increase in the micronucleus frequency. Such an evaluation may be considered independently of the enhancement factor for induced micronucleus frequencies.
In the evaluation of the test results historical control data obtained in the laboratory and scientific plausibility is taken into consideration.
Any positive test result should be evaluated for its biological relevance.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Without S9-mix, 4 hour treatment: cytotoxic effects at 60µg/ml and above; without S9-mix, 24 hour treatment: cytotoxic effects at 20 µg/ml and above; with S9-mix, 4 hour treatment: cytotoxic effects and precipitation at 350 µg/ml and above.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the presence of S9-mix (4 hour treatment), no biologically relevant increase of the frequency of micronucleus containing V79 cells treated with the test item was observed. The increase observed at 400 µg/ml and above exceeded the lowest insoluble concentration of 350 µg/ml. Consequently, micronucleus frequencies slightly above background at 400 µg/ml were not considered biologically relevant, because no increase was observed at non-cytotoxic concentrations and the effect was not concentration related. In the repeat experiment no increase in micronucleus frequency was observed at 350 µg/ml, the minimal insoluble concentration determined in this study with minimal signs of cytotoxicity. Therefore, according to OECD TG 487 observations at concentrations above 350µg/ml are not biologically significant and were not included into the assessment.

Conclusions:

Interpretation of results: negative

Executive summary:

2,2-Dimethylpropane-1,3-diyl dibenzoate dissolved in DMSO was tested in the in vitro mammalian cell micronucleus test according to OECD TG 487 under GLP conditions. The 4 hour treatment was conducted with concentrations ranging from 5 to 100 µg/ml without S9-mix showing cytotoxicity at 60 µg/ml and above. The 4 hour treatment in the presence of S9-mix with concentrations ranging from 5 to 500 µg/ml cytotoxicity and precipitation were observed at 350 µg/ml and above. In the 24 hours treatment without S9-mix concentrations ranging from 2.5 to 80 µg/ml were applied and showed cytotoxic effects at 20 µg/ml and above.

Under the experimental conditions 2,2-dimethylpropane-1,3-diyl dibenzoate does not induce biologically relevant increase in the frequencies of micronucleus containing V79 cells in vitro when tested up to the maximum recommended concentrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study and GLP

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

other: Chinese hamster lung fibroblasts (V79). The cells have a stable karyotype with a modal chromosome number of 22.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-naphthoflavone indused rat liver S9-mix

Test concentrations with justification for top dose:

PRETEST ON TOXICITY
4 h in the presence and 4 and 24 h in the absence of a metabolic activation system:
25.0 - 3200 µg/ml (approximately equal to 10 mM)

GENE MUTATION ASSAY
- EXPERIMENT I
4 h without S9 mix: 0.2, 0.4, 0.8, 1.5, 3.0, 6.0, 12.0 µg/ml
4 h with S9 mix: 12.5, 25.0, 50.0, 100.0, 200.0(P), 400.0(P) µg/ml

- EXPERIMENT II
24 h without S9 mix: 1.6, 3.1, 6.3, 12.5, 25.0, 50.0, 75.0, 100.0 µg/ml
4 h with S9 mix: 12.5, 25.0, 50.0, 100.0, 200.0(P), 400.0(P) µg/ml

P = Precipitation visible to the unaided eye

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

The assay was performed in 2 independent experiments, using 2 parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

Evaluation criteria:

A test item is classified as positive, if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points, that is three times above the spontaneous mutation frequency.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant, whenever the p-value (probability value) is below 0.05.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

EXPERIMENT I: without S9 mix, 4 h at 3.0 µg/ml and above and EXPERIMENT II: without S9 mix, 24h 25 µg/ml and above; with S9-mix, 4h: precipitation at 200.0 µg/ml and above.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the pre-experiment relevant cytotoxic effects, indicated by a relative cloning efficiency I (survival) of 50 % and below, were observed at the two lowest concentration of 25.0 and 50.0 µg/ml following 4 hours treatment without metabolic activation. At higher concentration the cell growth was completely inhibited. No relevant cytotoxic effect occurred up to the maximum concentration of 3200 µg/ml with metabolic activation (4 hour treatment). Following 24 hour treatment performed without metabolic activation cytotoxic effects occurred at the two lowest concentrations; at all of the higher concentrations the cell growth was completely stopped.

Conclusions:

Interpretation of results: negative

Executive summary:

The study was performed to investigate the potential of 2,2-dimethylpropane-1,3-diyl dibenzoate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD TG 476 under GLP conditions.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The dose-range of the main experiments was limited by cytotoxic effects and precipitation of the test item at the end of treatment.

No relevant and reproducible dose dependent increase in mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, 2,2-dimethylpropane-1,3-diyl dibenzoate is considered to be negative (non-mutagenic) in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No study available.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

2,2-Dimethylpropane-1,3-diyl dibenzoate was investigated for point mutagenic effects in the Salmonella/microsome test (plate incorporation test and preincubation method). The test item was dissolved in DMSO and was administered in doses of up to and including 5000 µg/plate without and with S9 mix on the five Salmonella typhimurium LT2 mutant strains TA 1535, TA100, TA1537, TA98 and TA102 according OECD TG 471 under GLP conditions.

Doses up to and including 5000 µg/plate did not cause any bacteriotoxic effects. Substance precipitation occurred at the concentration of 5000 µg/plate. Evidence of mutagenic activity of the test item was not seen. No biologically relevant increase in the mutant count in comparison to the solvent controls was observed in any of the strains tested without and with S9 mix, in the plate incorporation as well as in the preincubation modification under the experimental conditions applied.

The employed positive controls had a marked mutagenic effect as was seen by a biologically relevant increase in mutant colonies compared to the corresponding solvent controls.

Therefore, 2,2-dimethylpropane-1,3-diyl dibenzoate was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincucation modification of the Salmonella/microsome test.

 

HPRT test

The study was performed to investigate the potential of 2,2-dimethylpropane-1,3-diyl dibenzoate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD TG 476 under GLP conditions.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The dose-range of the main experiments was limited by cytotoxic effects and precipitation of the test item at the end of treatment.

No relevant and reproducible dose dependent increase in mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, 2,2-dimethylpropane-1,3-diyl dibenzoate is considered to be non-mutagenic in this HPRT assay.

 

MNT in vitro

2,2-Dimethylpropane-1,3-diyl dibenzoate dissolved in DMSO was tested in the in vitro mammalian cell micronucleus test according to OECD TG 487 under GLP conditions. The 4 hour treatment was conducted with concentrations ranging from 5 to 100 µg/ml without S9-mix showing cytotoxicity at 60 µg/ml and above. The 4 hour treatment in the presence of S9-mix with concentrations ranging from 5 to 500 µg/ml cytotoxicity and precipitation were observed at 350 µg/ml and above. In the 24 hours treatment without S9-mix concentrations ranging from 2.5 to 80 µg/ml were applied and showed cytotoxic effects at 20 µg/ml and above.

Under the experimental conditions 2,2-dimethylpropane-1,3-diyl dibenzoate does not induce biologically relevant increase in the frequencies of micronucleus containing V79 cells in vitro when tested up to the maximum recommended concentrations.

 

Overall evaluation

Based on the available test results it can be concluded that 2,2-dimethylpropane-1,3-diyl dibenzoate does not induce mutagenic activity.

 

Overall conclusion

2,2-Dimethylpropane-1,3-diyl dibenzoate does not induce chromosomal aberrations in the in vitro MNT test and does not induce gene mutations in bacteria or in mammalian cell systems. The available in vitro studies are of high quality (Klimisch Score 1). Therefore, there is no reason to believe that these results would not be applicable in humans.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the negative test results of the available in vitro tests (Ames test, HPRT test, MNT) it can be concluded that 2,2-dimethylpropane-1,3-diyl dibenzoate does not induce mutagenic activity.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.