Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three GLP and OECD guideline conform in vitro assays were performed to examine the genotoxic potential of the test item. The test item was negative for mutagenicity in an Ames test (OECD 471) and in a HPRT assay in V79 cells (OECD 476). The test material did also not induce clastogenicity in mammalian cells (OECD 473).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
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)
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/p-Naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Concentration range in Experiment I (without metabolic activation, 4 h exposure period, 14 h recovery time, 18 h preperation interval): 3.1, 6.3 and 12.5 µg/mLConcentration range in Experiment I (with metabolic activation, 4 h exposure period, 14 h recovery time, 18 h preperation interval): 12.5, 25 and 50 µg/mLConcentration range in Experiment II (without metabolic activation, 18 h exposure period, 0 h recovery time, 18 h preperation interval): 3.1, 6.3 and 12.5 µg/mLConcentration range in Experiment II (without metabolic activation, 28 h exposure period, o h recovery time, 28 h preperation interval): 6.3 and 12.5 µg/mLConcentration range in Experiment II (with metabolic activation, 4 h exposure period, 24 h recovery time, 28 h preperation interval): 12.5, 25 and 50 µg/mL
Vehicle / solvent:
Tetrahydrofuran (THF)
Untreated negative controls:
yes
Remarks:
culture medium
Negative solvent / vehicle controls:
yes
Remarks:
THF
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumSTAIN (for cytogenetic assays): with GiemsaNUMBER OF REPLICATIONS: two independent experimentsNUMBER OF CELLS EVALUATED: per culture 100 metaphase plates
Evaluation criteria:
Acceptability of the Test:The chromosome aberration test performed is considered acceptable if it meets the following criteria:a) The number of structural aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.0 - 4.0 %.b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratories historical control data.Evaluation of Results:A test item is classified as non-clastogenic if:- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of historical control data (0.0 - 4.0 % aberrant cells exclusive gaps) and/or- no significant increase of the number of structural chromosome aberrations is observed.A test item is classified as clastogenic if:- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps) and- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:A test item can be classified as aneugenic if:- the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance are considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- CYTOTOXICITY: in the pre-test the toxicity of the test item was examined using the determination of the cell number. Cell numbers of two cultures (10 coordinate defined fields per culture) were determined for each experimental group.- PRECIPITATION: In the pre-test on toxicity, precipitation of the test item after treatment was observed at 16 µg/ml and above in the absence of S9 mix and at 63 µg/ml and above in the presence of S9 mix. Since no clear toxicity was observed in the pre-test on toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation as recommended in the OECD Guideline 473. Therefore, 50 µg/ml (without S9 mix) and 200 µg/ml (with S9 mix) were chosen as top treatment concentrations in the main experiment I.In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test item did not induce structural Schromosome aberrations in V79 cells (Chinese hamster cell line).
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
His-: Salmonella typhimuriumTrp-: E. coli
Species / strain / cell type:
other: TA 1537, TA 98, TA1535, TA100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/p-Naphthoflavone induced rat liver S9 mix
Test concentrations with justification for top dose:
Experiment I (plate incorporation test): 33; 100; 333; 1000; 2500; and 5000 µg/plateExperiment II (preincubation test): 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: see: "Details on test system" in IUCLID
Details on test system and experimental conditions:
Positive Control Substances:Without metabolic activation:Strains: TA 1535, TA 100Name: sodium azide, NaN3Supplier: SERVA, D-69042 HeidelbergCatalogue No.: 30175Purity: at least 99 %Dissolved in: water deionisedConcentration: 10 µg/plateStrains: TA1537,TA98Name: 4-nitro-o-phenylene-diamine, 4-NOPDSupplier: SIGMA. D-82041 DeisenhofenCatalogue No.: N9504Purity: > 99.9 %Dissolved in: DMSO (MERCK. D-64293 Darmstadt; purity > 99 %)Concentration: 10 µg/plate in TA 98. 50 µg/plate in TA 1537Strains: WP2 uvrAName: methyl methane sulfonate. MMSSupplier: Merck-Schuchardt, D-85662 HohenbrunnCatalogue No.: 820775Purity: > 99.0 %Dissolved in: water deionisedConcentration: 4 µl/plateWith metabolic activation:Strains: TA 1535, TA 1537, TA 98. TA 100, WP2 uvrAName: 2-aminoanthracene, 2-AASupplier: SIGMA, D-82041 DeisenhofenCatalogue No.: A 1381Purity: 97.5 %Dissolved in: DMSO (MERCK, D-64293 Darmstadt; purity > 99 %)Concentration: 2.5 µg/plate, 10 µg/plate in strain WP2 uvrA
Evaluation criteria:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:- regular background growth in the negative and solvent control- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data- the positive control substances should produce a significant increase in mutant colony frequenciesA test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count ofthe corresponding solvent control is observed.A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experimentA dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
No statistical evaluation of the data is required.
Key result
Species / strain:
other: Salmonella typhimurium strains TA 1535, TA 1537, TA98, and TA 100, and Escherichia coli strain WP2 uvrA.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In experiment I: toxic effects (below the factor of 0.5) were observed at higher concentrations in strain TA 1535 without S9 mix and in strains TA 1537 and TA 98 with and without S9 mix. In experiment II: toxic effects were observed at the two highest concentrations in strain TA 1535 without S9 mix.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
hprt
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
3.9, 7.8, 15.6, 31.3, 62.5, 125 and 250 mg/L (experiment 1; 4h)7.8, 15.6, 31.3, 62.5, 125 and 250 mg/L (experiment 2; 24h, without S9)12, 25, 50, 75, 100, 125 mg/L (experiment 2; 4h, with S9)
Vehicle / solvent:
Tetrahydrofuran
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in mediumDURATION- Preincubation period: Two days (experiment I and experiment II with S9 mix) or three days (experiment II without S9 mix) after sub-cultivation stock cultures- Exposure duration: 4h or 24h- Expression time (cells in growth medium): 7 days- Selection time (if incubation with a selection agent): 8 days- Fixation time (start of exposure up to fixation or harvest of cells): 19 daysSELECTION AGENT (mutation assays): 6-thioguanineNUMBER OF REPLICATIONS: Population doubling time 12 - 14hNUMBER OF CELLS EVALUATED: not applicableDETERMINATION OF CYTOTOXICITY- Method: cloning efficiencyOTHER: The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
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 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.
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The maximum concentration of the test item was limited by the solubility of the test item in a suitable solvent or aqueous medium. No cytotoxicity was observed in the pre-experiment when tested up to concentrations far above the limit of solubility of the test item in culture medium (2666.6 mg/L).Precipitation of the test item was observed in the first experiment at 62.5 mg/L and above in the presence and absence of metabolic activation. In the second experiment precipitation was noted at 31.3 mg/L and above without and at 100.0 mg/L and above with metabolic activation.No relevant cytotoxic effects indicated by a relative cloning efficiency I or relative cell densitiy below 50% occurred. However, in the absence of S9 mix in both experiments the cell density was reduced to approximately 63 % in one culture at the highest applied concentration of 250.0 mg/L. This considerable variability of the cell density between parallel cultures is most likely based on precipitation of the test item leading to mechanical cell damage by shear forces.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There are reliable in vitro studies available to assess the potential of the test substance for gene mutations in bacteria and mammalian cells and cytogenicity in mammalian cells.

In a GLP conform study according to OECD guideline 471 the potential of the test substance (purity: >98 weight-%) to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA (RCC Ltd., 2002). The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration (10 - 5000 µg/plate), including the controls, was tested in triplicate. No visible reduction of the background growth was observed up to the highest concentration. Cytotoxicity was observed in experiment I and II at the two highest concentrations in presence and absence of S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

In a GLP conform study according to the OECD guideline 476 the potential of the test item (purity: >98.8 weight-%) to induce mutations V 79 cells was assessed. 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. Due to limited solubility of the test item in a suitable solvent the highest concentration of the test item in the pre-experiment was 2666.6 mg/L resulting in strong test item induced precipitation at the end of the treatment period. As no cytotoxicity was observed the highest concentration applied in the main experiments was far above the limit of solubility of the test item in culture medium. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system

In a GLP conform study according to OECD guideline 473, the test item, dissolved in THF, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments (RCC Ltd., 2002). Since no relevant toxicity was observed in the pre-test on toxicity, the test item was tested up to a concentration exhibiting clear test item precipitation (2000 µg/ml). No toxic effects indicated by reduced mitotic indices or reduced cell numbers were observed after treatment with the test item. In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the test substance is considered to be non-clastogenic in this chromosome aberration test neither with nor without S9 mix.


Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on this data the substance does not need to be classified for genotoxicity under Regulation (EC) No. 1272/2008.