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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Klimisch reliability of study is 1 (GLP guideline study); according to ECHA Practical Guide 6 rel. 2 is selected from the pick-list as this should be the maximum score for read-across.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2007
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1997)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Stability under test conditions: The stability of the test substance in the vehicle over a period of 4 hours was analytically verified.

Method

Target gene:
HGPRT locus
Species / strain
Species / strain:
Chinese hamster Ovary (CHO)
Details on mammalian cell lines (if applicable):
- Type and identity of media: Ham's F12 medium with supplements; for the selection of mutant cells the medium was supplemented with 10 µg/mL 6-thioguanine.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain characteristics:
other: substrain K1
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of male Aroclor 1254 induced Sprague-Dawley rats.
Test concentrations with justification for top dose:
Experiment I: without S9 mix 1.5, 3.0, 6.0, 12.0, 18.0, and 24.0 μg/mL; with S9 mix 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 μg/mL
Experiment II: without S9 mix: 2.5, 5.0, 7.5, 10.0, 15.0, and 20.0 μg/mL; with S9 mix: 0.25, 0.50, 1.00, 2.00, 4.00, and 6.00 μg/mL
Vehicle:
Dimethylsulfoxide (DMSO; dried over molecular sieve)
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethyl methanesulfonate (EMS; 300 µg/mL), methylcholanthrene (MCA; 10 µg/mL)
Remarks:
EMS was used without and MCA with metabolic activation
Details on test system and conditions:
PRE-TEST: A pre-test with concentrations up to 1000 µg/mL was performed in order to determine the concentration range for the mutagenicity experiments.

METHOD OF APPLICATION: in medium
For each test group, about 1x10exp6 logarithmically growing cells per flask (175 cm²; after the 2nd passage in the 1st and 2nd Experiment, respectively) were seeded into about 20 mL medium supplemented with 10% (v/v) FCS and incubated for about 20 - 24 hours with 5% (v/v) CO2 at 37°C and > 90% humidity. Two flasks were used for each test group. After the attachment period, the medium was removed from the flasks and the treatment medium was added. The exposure period in both main experiments was 4 hours with and without S9 mix in an incubator (5% [v/v] CO2, 37°C and ≥ 90% humidity). After the exposure period, the cultures were rinsed several times with Hanks' balanced salt solution (HBSS). Then the flasks were topped up with at least 20 mL medium with 10% (v/v) FCS. After an incubation period of 65 – 72 hours (5% [v/v] CO2, 37°C and ≥ 90% humidity) the 1st passage was carried out. After an entire expression period of 6 - 8 days the cells were transferred into selection medium (2nd passage). For the selection of the mutants, six 75 cm² flasks with 3x10exp5 cells each from every treatment group, if possible, were seeded in 10 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 - 8 days (5% [v/v] CO2, 37°C and ≥ 90% humidity). At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

time schedule:
Day 1 - Seeding of the cells pretreated with "HAT" medium: in 175 cm² flasks (1x10exp6 cells in 20 mL) and in 25 cm² flasks (200 cells in 5 mL)
Day 2 - Test substance incubation (20-24 hours after seeding), exposure period 4 hours, then washing of the cultures and 1st cytotoxicity determination (cloning efficiency 1 = survival)
Day 5 - 1st passage of the treated cells
Day 9 - 2nd passage of the treated cells with seeding in the selection medium, 2nd cytotoxicity determination (cloning efficiency 2 = viability)
From Day 16 - drying, fixation, stainig and counting of the selected colonies.

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 6-8 days
- Selection time: 6-8 days

DETERMINATION OF CYTOTOXICITY
- Method: absolute and relative cloning efficiencies (%); This was initially assessed in the pre-test, secondly the survival was determined after the exposure period and thirdly the viability was assessed after the expression period.
Evaluation criteria:
A finding is assessed as positive if the following criteria are met:
- Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative/vehicle control values and the historical negative control data range
- Evidence of reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose response relationship.
The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency in the dose groups is not statistically significant increased above the concurrent vehicle control and is within the historical negative control data range.
Statistics:
The number of mutant colonies of the dose groups and the positive controls were compared with that of the solvent control groups using the Fisher-Pitman Test for the hypothesis of equal means. The test was performed one-sided.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: Chinese hamster Ovary/HPRT cells, substrain K1
Remarks:
Migrated from field 'Test system'.
Additional information on results:
In this study, no increase in the number of mutant colonies was observed either without or with a metabolizing system. In both experiments after 4 hours treatment with the test substance the values for the corrected mutation frequencies were within the range of the respective vehicle control values and of the historical negative control data. No statistical significance was observed. The positive control substances EMS and MCA induced clearly increased mutant frequencies as expected.

TEST-SPECIFIC CONFOUNDING FACTORS
- Osmolarity and pH values were not influenced by test substance treatment.
- Precipitation: In the absence and the presence of S9 mix no precipitation in culture medium was observed up to the highest applied test substance concentration.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control were observed in both experiments in the absence and in the presence of S9 mix at least in the highest applied concentrations.

Any other information on results incl. tables

For genetic toxicity/mammalian cell mutagenicity a read across to HDI oligomers, isocyanurate type (EC 931 -274 -8) is applied. This substance is a close structural analogue to HDI oligomers, iminooxadiazindione type, also derived from catalytic oligomerisation of 1,6 -hexamethylene diisocyanate (HDI; CAS 822 -06 -0) and also belonging to the CAS number 28182-81-2 (Hexane, 1,6 - diisocyanato-, homopolymer).The read across is based on physicochemical and toxicological similarity. In fact, comparison of the toxicological endpoints, that are available for both of the two substances (Acute oral toxicity, Acute inhalation toxicity, Skin and Eye Irritation/Corrosion, Skin Sensitisation, Bacterial mutagenicity (Ames)) reveal good correlation. With respect to Inhalation Toxicity an expert statement is available justifying the read across (Pauluhn, Comparison of pulmonary irritation potency..., Bayer HealthCare AG, 2008).

Therefore, test results obtained for HDI oligomers, isocyanurate type can be transferred to HDI oligomers, iminooxadiazindione type and the results on mammalian cell mutagenicity of HDI oligomers, isocyanurate type are also valid for HDI oligomers, iminooxadiazindione type. This approach is in accordance with Annex XI, section 1.5 of the REACH Regulation (Regulation (EC) No 1907/2006).

Applicant's summary and conclusion

Executive summary:

An in vitro Mammalian Cell Gene Mutation Test (HPRT assay) according to OECD TG 476 was conducted in order to assess the potential of the substance to induce gene mutations in mammalian cells. Based on a pre-test doses up to 24.0 μg/mL without S9 mix and up to 6.4 μg/mL with S9 mix were selected for the initial experiment. An independent repeat was conducted with doses up to 20 µg/mL without S9 mix and up to 6.0 µg/mL with S9 mix.

In this study, no increase in the number of mutant colonies was observed either without or with a metabolizing system. In both experiments after 4 hours treatment with the test substance the values for the corrected mutation frequencies were within the range of the respective vehicle control values and of the historical negative control data. No statistical significance was observed. The positive control substances EMS and MCA induced clearly increased mutant frequencies as expected. Thus, under the experimental conditions of the assay the substance was considered not to be mutagenic.