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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item 1,3,5-Triisopropylbenzene is considered as “not mutagenic under the conditions of the test” following an in vitro bacterial reverse mutation assay.

 

Under the test conditions to induce structural chromosomal aberrations in human lymphocytes in vitro, the test item is considered as “mutagenic”.

QSAR prediction reports no chemical structure activity relationship alerts know to be mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 Dec 2014 to 24 Mar 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No. 6F11027000
- Expiration date of the lot/batch: 17. Nov. 2016
- Purity: 96 %
Target gene:
Five genetically changed strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535)
Species / strain / cell type:
S. typhimurium TA 97
Remarks:
TA97a
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
S. typhimurium TA 1535
Metabolic activation:
with and without
Test concentrations with justification for top dose:
1st Experiment: 5000 / 1500 / 500 / 150 / 50 µg/plate.

2nd Experiment: 5000 / 2500 / 1250 / 625 / 313 / 156 µg/plate.

To verify the results of the first experiment, the second experiment was performed. The test item did not show mutagenic effects in the second experiment, either.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol and DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-Nitro-1,2-phenylene diamine CAS-No.: 99-56-9 2-Amino-Anthracene CAS-No.: 613-13-8
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for 1st experiment; preincubation for 2nd experiment

DURATION
- Preincubation period: 20 minutes
- Incubation time: 48 hrs

NUMBER OF REPLICATIONS: 3 replicates, with/without S9
Evaluation criteria:
The colonies were counted visually, the numbers were recorded. The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.
Statistics:
A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.
Key result
Species / strain:
S. typhimurium TA 97
Remarks:
97a
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:
S. typhimurium TA 102
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 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
Conclusions:
The test item 1,3,5-Triisopropylbenzene is considered as “not mutagenic under the conditions of the test” following an in vitro bacterial reverse mutation assay.
Executive summary:

The mutagenic potential of the test item with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14 with two independent repeats was investigated. Five concentrations of the test item (up to 5000 µg/plate) were used in the first test following the plate incorporation method and in the second test following the pre-incubation method. Five genetically changed strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 (genetically manipulated) and TA1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9-mix, rat liver S9-mix induced by Aroclor 1254) for 48 hours.

None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Under the conditions of the test, the test item did not show mutagenic effects towards Salmonella typhimurium, strains TA97a, TA98, TA100, TA102 and TA1535. Therefore, no concentration-effect relationship could be determined. The test item 1,3,5 -Triisopropylbenzene is considered as “not mutagenic under the conditions of the test”.

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:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2014
Deviations:
yes
Remarks:
2 deviations noted. Considered uncritical. Refer to results for additional details
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
2008
Deviations:
yes
Remarks:
2 deviations noted. Considered uncritical. Refer to results for additional details
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch No. 6F11027000
- Expiration date of the lot/batch: 17. Nov. 2016
- Purity: 96 %
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
Human lymphocytes
Human whole blood treated with anti-coagulant (heparin).
Blood samples were obtained from healthy donors who neither smoke nor receive medication.
Metabolic activation:
with and without
Test concentrations with justification for top dose:
Experiment 1:
With S9: 2036, 1018, 509, 16 µg/mL
Without S9: 16, 4, 2 µg/mL

Experiment 2:
With S9: 1994, 499, 8 µL/mL
Without S9: 31, 23, 16 µL/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: because the test item was sufficiently soluble, and this solvent does not affect the viability of cells in the test system
Untreated negative controls:
yes
Remarks:
solvent
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Exp 1: 4hrs (without S9) and 4hrs (with S9); Exp 2: 23hrs (without S9) and 4hrs (with S9)
- Expression time: Exp 1: 19hrs (without S9) and 20hrs (with S9); Exp 2: 19hrs (with S9)
- Culture harvest time: Exp 1: 23hrs (without S9) and 24hrs (with S9); Exp 2: 23hrs (without S9) amd 23 hrs (with S9)

STAIN: 10% solution of Giemsa

NUMBER OF REPLICATIONS: In each experiment, all cell cultures were set up in duplicates.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The slides were prepared by dropping the cell suspension onto clean microscope slides. The cells were then stained with a 10% solution of Giemsa. All slides, including those of all controls, were independently coded before microscopic analysis.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: In Exp 1, 100 well spread metaphases per culture were analysed. In Exp 2, 150 well spread metaphases per culture were analysed.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Although this test is not designed to measure aneuploidy, an evaluation of possible polyploidy was performed. About 1000 metaphases (500 from each parallel culture) per concentration of the test item were evaluated to determine the proportion of polyploid cells.
Evaluation criteria:
A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is below 5.0 % aberrant cells (excluding gaps) resp. lies in the range of the historical laboratory control data for solvent controls.
- no significant or concentration-related increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is above 5.0 % aberrant cells (excluding gaps) resp. lies above the historical laboratory control data for solvent controls.
- either a concentration-related or a significant increase in the number of cells with structural chromosome aberrations is observed.
Statistics:
The number of metaphases with structural aberrations in each treatment group was compared with the solvent control value. Statistical significance was tested using Fisher’s exact test at the 5% level (p < 0.05). The resulting probability of the respective distribution was halved and the probabilities of the more extreme distributions (down to a value of 0 in the controls) were added to give the cumulated p-value of the tail of the distribution.
Key result
Species / strain:
lymphocytes: Human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The following deviation of the study plan was observed:
- The test item was stored at 16 ± 6°C instead of 20 ± 5°C. This is considered uncritical. The test item is considered to be stable at this temperature.

The following deviations from the Guideline were observed:
- It was not possible to achieve the highest evaluable concentration of 55 ± 5 % cytotoxicity. This is considered uncritical, because the test item showed mutagenicity even in less cytotoxic concentrations.
- It was not possible to achieve the required 200 (experiment I) resp. 300 (experiment II, according to updated guideline) metaphases for structural evaluation in all analysed concentrations. This is uncritical, because there is no influence on the out-come of the study.

Cytotoxicity

The test item showed cytotoxicity, especially in the experimental parts without metabolic activation. By contrast, the test item showed no or only low cytotoxicity in the experimental parts with metabolic activation in the higher and the lower concentrations, but stronger cytotoxicity in the intermediate concentrations. A possible attempt to explain this observation may be that a certain relation between amount of test item and solvent may have an influence on the test system.

Genotoxicity

The test item is considered to have mutagenic properties. Though the ratio of aberrant cells did only in one treatment (experiment II without metabolic activation, 16 μg/mL) exceed 5%, in a total of 3 treatments a statistically significant increase of structural chromosomal aberrations could be observed: in experiment II without metabolic activation (extended exposure) in 2 concentrations (31 μg/mL and 16 μg/mL) and in experiment I with metabolic activation in 1 concentration (1018 μg/mL). In all cultures treated with test item the ratio of aberrant cells lay above the solvent controls. Furthermore, in 2 evaluated treatments (experiment I with metabolic activation, 509 μg/mL, and experiment II without metabolic activation, 16 μg/mL) chromatid exchanges could be found. This is considered to be a further indication of the mutagenic properties of the test item, because this type of aberration is practically never observed in untreated cultures. Moreover it should be taken into account, that the evaluated concentrations in the experimental parts without metabolic activation were very low, due to the cytotoxicity of the test item. Solvent controls and positive controls showed numbers within the range of the historical data and met the acceptability criteria.

Although in experiment I without metabolic activation 2 concentrations (16 and 4 μg/mL) showed significantly increased ratios of polyploid cells (compared with the concurrent solvent control) and additionally, single cells with endoreduplication could be observed, the obtained values are not sufficient, to prove that the test item can influence mitotic processes and/or cell cycle progress.

Conclusions:
Under the experimental conditions reported, the test item 1,3,5-Triisopropylbenzene has the ability to induce structural chromosome aberrations in human lymphocytes in vitro in the chosen test solutions.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
(Q)SAR
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Guideline:
other: QSAR prediction
Principles of method if other than guideline:
- Software tool(s) used including version: Toxtree (Estimation of Toxic Hazard - A Decision Tree Approach) Version 2.6.6
- Model(s) used: Toxtree: Benigni-Bossa rule base for genotoxic and non-genotoxic carcinogenicity
- Model description: see 'Attached justification'
- Justification of QSAR prediction: Attached justification
Specific details on test material used for the study:
SMILES = CC(C)c1cc(cc(c1)C(C)C)C(C)C
Key result
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
QSAR prediction reports no chemical structure activity relationship alerts know to be mutagenic.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Results are available from a bacterial reverse mutation assay and an in vitro mammalian chromosome aberration test with human lymphocytes. The bacterial reverse mutation assay was negative for mutagenicity and the in vitro mammalian chromosome aberration test was determined to be clastogenic. According ECHA’s Guidance on the Application of the CLP Criteria, in vitro results can only lead to a Category 2 mutagen classification in a case where there is support by chemical structure activity relationship to known germ cell mutagens. The test item does not possess chemical structure activity relationship alerts according to the Toxtree: Benigni-Bossa rule base. Therefore, the test substance does not meet the EC 1278/2008 as amended criteria for classification for germ cell mutagenicity.