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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:
experimental study
Adequacy of study:
key study
Study period:
2011-06-24 to 2011-08-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Reaction mass of 2,2,4-trimethylhexa-1,6-diyl diisocyanate and 2,4,4-trimethylhexa-1,6-diyl diisocyanate
EC Number:
915-277-1
Cas Number:
32052-51-0
Molecular formula:
C11H18N2O2
IUPAC Name:
Reaction mass of 2,2,4-trimethylhexa-1,6-diyl diisocyanate and 2,4,4-trimethylhexa-1,6-diyl diisocyanate
Test material form:
other: liquid
Details on test material:
99.73%: Trimethylhexamethylene-1,6-diisocyanate

Method

Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT)
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
V79 cells (MRC Radiobiology Unit, Cell and Molecular Biology Division, Chilton, Didcot, England)
Cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10% fetal calf serum, penicillin 3 (100 U/mL) and
streptomycin (100 µg/mL) called DMEM-FCS

- Periodically checked for Mycoplasma contamination: yes (cells were periodically checked by using the HOECHST stain 33258. The spontaneous
mutation rate was continuously monitored.)
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
post-mitochondrial supernatant fraction derived from livers of Aroclor 1254-treated rats and an NADPH-generating system (S9 mix)
Test concentrations with justification for top dose:
- S9 mix: 3.13, 6.25, 12.5, 25 or 50 µg VESTANAT TMDI/mL
+ S9 mix: 6.25, 12.5, 25, 50 or 100 µg VESTANAT TMDI/mL
Vehicle / solvent:
dimethyl sulfoxide (DMSO)
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethyl methanesulphonate (EMS) in direct mutagenicity experiment; 9,10-dimethyl-1,2-benzanthracene (DMBA) in S9 mix mediated assay; both EMS and DMBA were dissolved in DMSO. The applied concentrations were 600 or 700 µg EMS/mL medium or 20 or 30 µg DMBA/mL
Details on test system and experimental conditions:
CELLS AND TISSUE CULTURE MEDIA
- V79 cells were maintained in Dulbecco's modified Eagle-Mediumsupplemented with 10% fetal calf serum, penicillin 3 (100 U/mL) and streptomycin (100 µg/mL) called DMEM-FCS
- Incubation of cultures: at 37°C in a humidified atmosphere (90%) containing 10% CO2
- For subculturing, a trypsin (0.05%)-EDTA (ethylenediaminetetraacetic acid, 0.02%) solution in modified Puck's salt solution A was used.


METHOD OF APPLICATION:
- Exposure to the test item in the presence of S9 mix was performed in Dulbecco's phosphate buffered saline (PBS) which additionally contained 20 mM HEPES (N'-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid) pH 7.4 (PBS-HEPES).


DURATION
- Preincubation period: 1 day (in 30 mL DMEM-FCS)
- Exposure duration:
* 4 hours (1st experiment) and 24 hours (2nd experiment) without S9 mix, respectively;
* in the experiments with S9 mix, the medium was replaced by 18 mL S9 mix and the exposure limited to 4 hours.
* After removal of the test item and washing of the plates with PBS cells were trypsinised and a relative plating efficiency was determined for each
dose to obtain an accurate measure of the toxic effect of the chemical
- Expression time (cells in growth medium):
* Three replicate plates (60 mm diameter) were used with a known number of cells.
* Remaining cells were replated and the culture incubation continued until day 8 with 30 mL normal DMEM-FCS with one subcultivation on day 5.
* Afterwards cells were harvested by trypsinisation and replated at a density of 1 000 000 per 150mm diameter dish in DMEM-FCS containing
6-thioguanine (10 µg/mL) for selection of mutants (5 replicate plates), or at approx. 100 to 150 cells (exact number known) per 60 mm diameter dish in medium without 6-thioguanine for the estimation of plating efficiencies (PE 2), (3 replicate plates).
* Plates were fixed and stained after about 8 days (plating efficiency plates) or 12 days (6-thioguanine plates).


NUMBER OF REPLICATIONS: three
NUMBER OF CELLS EVALUATED: 1 500 000

DETERMINATION OF CYTOTOXICITY (same procedure was used as employed for the mutagenicity experiments, except that no mutant selection was carried out)
- Method: survival
- A concentration of the test item which produces a low level of survival (10 to 20%) would be used as highest concentration and the survival in the
lowest concentration being approximately the same as that in the negative control.
- Five adequately spaced concentrations are employed
- In this preliminary experiment without and with metabolic activation cytotoxicity was noted at concentrations of 25 or 100 µg VESTANAT TMDI/mL and higher, respectively. Hence, 50 or 100 µg test item/mL were employed as the top concentrations for the mutagenicity tests without and with metabolic activation, respectively.
Evaluation criteria:
The following pre-determined descriptive criteria are used for interpretation of the results:
- If in both independent experiments solvent and positive controls show results within the norm and if the test item does not increase the mutation
frequency 2-fold above the mean of the solvent controls under any condition, or if the mutation frequency is always lower than 40 x 10^-6 and if at least 1 000 000 cells per condition have been evaluated, the item is considered as negative in the test.
- In case of a dose-dependent increase of the mutation frequency in both independent experiments (at similar concentrations) to at least 2-fold
solvent control and at least 40 x 10^-6 both in the presence and/or absence of S9 mix, the item is considered as positive in the test.
Statistics:
No satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments.

Results and discussion

Test results
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without and with metabolic activation cytotoxicity was noted at concentrations of 25 or 100 µg test item/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES (Preliminary cytotoxicity test):
A concentration of the test item which produces a low level of survival (10 to 20%) would be used as highest concentration and the survival in the
lowest concentration being approximately the same as that in the negative control. In the case of no cytotoxicity, the highest concentration would be
determined by the solubility of the test item. Five adequately spaced concentrations are employed.
In this preliminary experiment without and with metabolic activation cytotoxicity was noted at concentrations of 25 or 100 µg VESTANAT TMDI/mL
and higher, respectively. Hence, 50 or 100 µg test item/mL were employed as the top concentrations for the mutagenicity tests without and with
metabolic activation, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA:
All the mutation frequencies obtained for the test item are within the negative control ranges. The mean mutation frequency of the control background data is 14.11 ± 7.42 x 10^-6 clonable cells with a range of 1.30 - 34.80 x 10^-6 clonable cells for the experiments without metabolic activation and 14.88 ± 8.20 x 10^-6 clonable cells with a range of 2.18 - 38.36 x 10^-6 clonable cells for the experiments with metabolic activation.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

 Mutagenicity experiments

Experiment 1:

Hours

Absence of S9

4-h exposure / 4-h sampling

Hours

Presence of S9

4-h exposure / 4-h sampling

0

Commence treatment

      0

Commence treatment

    + 4

Remove treatment medium, wash, trypsinisation and replating

    + 4

Remove treatment medium, wash, trypsinisation and replating

 

Experiment 2:

 

Hours

Absence of S9

24-h exposure / 24-h sampling

Hours

Presence of S9

4-h exposure / 4-h sampling

0

Commence treatment

       0

Commence treatment

 

24-hour continuous treatment

    + 4

Remove treatment medium, wash, trypsinisation and replating

+ 24

Remove treatment medium, wash, trypsinisation and replating

-

-

 

Applicant's summary and conclusion

Conclusions:
negative

Under the present test conditions, 2,2,4(or 2,4,4)-trimethylhexane-1,6-diisocyanate tested up to cytotoxic concentrations of 50 or 100 µg/mL in the experiments without and with metabolic activation, respectively, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.
 
Executive summary:

Test item was tested for mutagenic potential in a gene mutation assay in cultured mammalian cells (V79, genetic marker HPRT) both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 -induced animals. The duration of the exposure with the test item was 4 hours or 24 hours in the experiments without S9 mix and 4 hours in the experiments with S9 mix.

The test item was completely dissolved in dimethyl sulfoxide (DMSO).

The concentrations employed were chosen based on the results of a cytotoxicity study.In this preliminary experiment without and with metabolic activation cytotoxicity was noted at concentrations of 25 or 100 µg test item/mL and higher, respectively. Hence, 50 or 100 µg test item/mL were employed as the top concentrations for the mutagenicity tests without and with metabolic activation, respectively.

Five concentrations ranging from 3.13 to 50 or 6.25 to 100 µg test item/mL were selected for the experiments without and with metabolic activation, respectively.

 

Cytotoxicity

In the main study, cytotoxicityin form of decreased plating efficiency (PE1) and (PE2) was noted in the first and second experiments at the top concentrations of 50 or 100 µg/mL in the absence and presence of metabolic activation, respectively.

 

Experiments without metabolic activation

The mutation frequency of the negative controlDMSO was 7.52 and 5.26 x 10 -6clonable cells. Hence, the negative controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 3.13, 6.25, 12.5, 25 or 50 µg test item/mL culture medium ranged from 3.70 to 11.08 x 10‑6clonable cells. These results are within the normal range of the negative controls.

 

Experiments with metabolic activation

The mutation frequency of the negative control DMSO was 4.91 and 5.89 x 10 -6clonable cells. Hence, the negative controls were well within the expected range (see below).

The mutation frequency of the cultures treated with concentrations of 6.25, 12.5, 25, 50 or 100 µg test item/mL culture medium ranged from 1.12 to 9.60 x 10 -6clonable cells. These results are within the normal range of the negative controls.

The positive controls EMS (ethyl methanesulfonate) in the direct test and DMBA (9,10 -dimethyl-1,2 -benzanthracene), a compound which requires metabolic activation, caused a pronounced increase in the mutation frequencies ranging from 1082.00 to 2799.13x 10 -6clonable cells in the case of EMS and ranging from 253.23 to 533.91x 10 -6clonable cells in the case of DMBA, indicating the validity of this test system.

The background mutation frequency at LPT ranges from 1.30 to 38.36 x 10 -6 clonable cells for the negative controls. The mutation frequency of the positive controls at LPT ranges from 112.1 to 1708.4 x 10 -6 clonable cells for EMS and 130.0 to 2693.3 x 10 -6 clonable cells for DMBA.

 

Under the present test conditions, test item tested up to cytotoxic concentrations of 50 or 100 µg/mL in the experiments without and with metabolic activation, respectively, was negative in the HPRT-V79 mammalian cell mutagenicity test under conditions where positive controls exerted potent mutagenic effects.

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