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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 IUCLID pick-list as this should be the maximum score for read-across.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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
Type:
Constituent
Details on test material:
- Stability under test conditions: The dilutions of the stock solutions were prepared on the day of the experiment and used immediately.

Method

Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM supplemented with 10 % FCS, neomycin (5 µg/mL) and amphotericin B (1%); for the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of Phenobarbital/beta-naphthoflavone induced male Wistar rats.
Test concentrations with justification for top dose:
Experiment I: without S9-mix 0.3, 0.5, 1.0, 2.0, 4.0, 6.0 and 8.0 µg/mL ; with S9-mix 1.0, 2.0, 4.0, 8.0, 16.0, 24.0 and 32.0 µg/mL
Experiment II: without and with S9-mix 1.0, 2.0, 4.0, 8.0, 16.0, 24.0 and 32.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethylene glycol dimethyl ether (EGDE)
- Justification for choice of solvent/vehicle: Ethylene glycol dimethylether (EGDE, dried with a molecular sieve, 0.3 nm) was selected as solvent, based on test item characteristics and as indicated by the sponsor.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
EGDE
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
ethylmethanesulphonate (1.2 mM) used without metabolic activation, 7,12-dimethylbenzanthracene (4.3 µM) used with metabolic activation
Details on test system and experimental conditions:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE). The highest applied concentration in the pre-test on toxicity (500 µg/mL) was chosen based on the limited solubility of the test item. The pre-test was performed in a concentration range from 3.9 and 500 µg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.

The main test was performed in two independent experiments. 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.

METHOD OF APPLICATION: in medium; All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Treatment Protocol without Metabolic Activation: Approximately 1.5 x 10exp6 (single culture) and 5 x 10exp2 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment. After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps. In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation. The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below. Three or four days after treatment 1.5 x 10exp6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5 x 10exp5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DURATION
- Exposure duration: 4 hours.
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): about 8 days, then colonies were stained.

DETERMINATION OF CYTOTOXICITY: cloning efficiency
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.
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.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
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. However, both, biological and statistical significance were considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The cell cultures were evaluated at the following concentrations: Exp. I without S9-mix 1.0, 2.0, 4.0, 6.0, 8.0 µg/mL and with S9-mix 2.0, 4.0, 8.0, 16.0, 24.0 µg/mL; Exp. II without and with S9-mix 2.0, 4.0, 8.0, 16.0, 24.0 µg/mL.

No relevant and reproducible increase in mutant colony numbers/1exp6 cells was observed in the main experiments up to the maximum concentration with and without metabolic activation. The induction factor exceeded the threshold of three times the mutation frequency of the corresponding solvent control at 8.0 µg/mL (culture I) and at 16.0 µg/mL (culture II) in the second experiment with metabolic activation. However, the mutation frequency did not exceed the historical range of solvent controls. The effects were judged to be based upon the rather low solvent controls of 7.9 and 7.4 mutant colonies/1exp6 cells, respectively. In culture I of the first experiment with metabolic activation the absolute values of the mutation frequency (41.6 mutant colonies per 1exp6 cells) slightly exceeded the historical range of solvent controls (3.4 - 36.6 mutant colonies/1exp6 cells). However, the effect was judged as biologically irrelevant artefact as it was not reproduced in the parallel culture performed under identical experimental conditions and the threshold of three times the mutation frequency of the corresponding solvent control was not exceeded. A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 7.4 up to 40.4 mutant colonies per 1exp6 cells; the range of the groups treated with the test item was from 11.1 up to 41.6 mutant colonies per 1exp6 cells. So, the solvent controls with EGDE remained within the historical range. The positive controls used showed a distinct increase in induced mutant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50 % in both parallel cultures occurred in the second experiment at 24.0 µg/mL without metabolic activation and at 16.0 µg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10-20 % relative cloning efficiency I was covered with metabolic activation. The cytotoxic gradient was very steep without metabolic activation leading to a relative cloning efficiency I of 37.1 to 40.2 % at the maximum analysable concentration of 24.0 µg/mL and just 0.5 to 0.7% at the next higher concentration of 32.0 µg/mL. In the first experiment without metabolic activation only moderate cytotoxic effects occurred at the maximum concentration of 8.0 µg/mL (relative cell density down to 59.8 % of the solvent control in culture II). However, in the pre-experiment the relative cloning efficiency at 7.8 µg/mL was just 5.8 % so, the concentration range of the first experiment without metabolic activation went right up to the steep onset of cytotoxicity.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) of the pre-test prior to removal to the test item. Phase separation occurred at 250 μg/mL and above in the presence and absence of metabolic activation following 4 and 24 hours treatment.
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

For assessing skin irritation in vivo a read across to a substance with a very similar chemical composition (EC No. 900 -066 -9; comparable allophanate-type HDI oligomerisation product) is applied.The read across is based on physicochemical and toxicological similarity of the two substances. Especially a comparative pulmonary irritant potency study according to TRGS 430 (Technical Rule for Hazardous Substances 430; published by the German Federal Ministry of Labour and Social Affairs, 2009), as the key study for justification of read-across, confirmed for both substances the same toxicological mode of action and nearly identical potency. For further justification of the grouping and read-across according to regulation (EC) No 1907/2006, Annex XI, 1.5 see document attached to chapter "Assessment Reports".

 

Applicant's summary and conclusion

Executive summary:

The substance was tested in an in vitro gene mutation assay (HPRT) in V79 cells according to OECD TG 476. The assay was performed in two independent experiments: 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 test item was dissolved in ethylene glycol dimethyl ether (EGDE, dry). The concentration range of the main experiment was limited by cytotoxic effects. 

No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. 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.