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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria in vitro (OECD 471): negative in S. typhimurium TA 1535, TA 100, TA 1537, TA 98, and TA 102 with and without metabolic activation

Chromosome aberration in mammalian cells in vitro (OECD 743): negative in Chinese hamster lung fibroblasts (V79) with and without metabolic activation

Gene mutation in mammalian cells in vitro (OECD 476): negative in Chinese hamster lung fibroblasts (V79) with and without metabolic activation

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:
5 Feb - 11 Dec 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
mutant histidine gene
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 100, TA 1537, TA 98, and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 mix was made from the livers of male Sprague Dawley rats, which received a single intraperitoneal injection of 500 mg/kg bw Aroclor 1254, dissolved in corn oil, 5 days prior to sacrifice. The S9 mix comprised 10% S9 fraction supplemented with cofactors.
Test concentrations with justification for top dose:
plate incorporation assay:
0, 50, 158, 500, 1581, 5000 µg/plate with and without S9 mix
preincubation assay:
0, 50, 158, 500, 1581, 5000 µg/tube with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water (test item and positiv control Mytomycin C); DMSO (other positive controls)
- Justification for choice of solvent/vehicle: test item formed a clear colorless solution
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Na-azide (only TA 1535), nitrofurantoin (only TA 100), 4-nitro-1,2-phenylene diamine (TA 1537 and TA 98), mitomycin C (only TA 102 in plate incorporation assay), cumene hydroperoxide (only TA 102 in preincubation assay), 2-aminoanthracene
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
Statistics:
no statistics perfomed; evaluation based on criteria mentioned above.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 100, TA 1537, TA 98, and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
Total bacteria counts remained unchanged and no inhibition of growth was observed.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the plate incorporation test, none of the five strains showed a dose-related and/or biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials.

Additional data supporting the information provided are attached as pdf documents below under 'Attached background material'.

Conclusions:
The test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification tested in Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and TA 102.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 Aug - 3 Dec 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen, Düsseldorf, Germany
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9: isolated from Arochlor 1254 male Sprague Dawley rat liver microsomal fraction
Test concentrations with justification for top dose:
4 h treatment time, 18 h harvest time (+/- S-9 mix):
600, 1200 and 2400 µg/mL

18 h treatment time, 18 h harvest time (- S-9 mix):
600, 1200, 1600, 2000, 2400 µg/mL
Vehicle / solvent:
deionised water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
Positive controls:
without S9 mix: Mitomycin C, 0.1 µg/mL (for 4 h treatment) and 0.03 µg/mL (for 18 h treatment)
with S9 mix: Cyclophosphamide, 2 µg/mL
Chromosomes were prepared 18 and 30 h (for 4 h treatment) or 18 h (for 18 h treatment) after start of treatment with test substance. The treatment interval was 4 h with and without metabolic activation and 18 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations. Polyploid metaphases were recorded.
Evaluation criteria:
An increased incidence of gaps of both types without concomitant increase of other chromosomal aberration types was not considered as indication of a clastogenic effect.
A test was considered positive, if there was a relevant and statistically significant increase in the aberration rate.
A test was considered negative, if there was no such increase at any time interval.
A test was also considered negative, if there were statistical significant values, which were, however, within the range of historical negative controls.
In addition, a test was considered equivocal if there was an increase of aberrant metaphases above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. A test was also considered equivocal, if its result was implausible.
Statistics:
Statistical significance at the 5% level (p<0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
+S-9 mix: >= 1200 µg/mL (4 h treatment); 2400 µg/mL (18 h treatment); +S-9 mix: no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
- Effects on pH: none
- Effects of osmolality: no effects
- Water solubility: soluble up to 300 mg/mL
- Precipitation: none

Additional data supporting the information provided are attached as pdf documents below under 'Attached background material'.

Conclusions:
The test substance is considered not to be clastogenic in mammalian cells based on an in vitro chromosome aberration test with Chinese hamster V79 cells in the presence and absence of S9 mix.
Endpoint:
in vitro gene mutation 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
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)
Remarks:
Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Type and identity of media: MEM (minimal essential medium; SEROMED, Berlin, Germany) supplemented with 10 % fetal calf serum
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced male Wistar rat liver S9 mix
Test concentrations with justification for top dose:
Experiment I:
without S9 mix: 150, 300, 600, 1200, 2400 µg/mL
with S9 mix: 150, 300, 600, 1200, 2400 µg/mL

Experiment II
without S9 mix: 300, 600, 1200, 1800, 2400 µg/mL
with S9 mix: 300, 600, 1200, 1800, 2400 µg/mL
In both main experiments the cultures at the lowest concentrations with and without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guideline.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: deionised water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulfonate (EMS, 0.15 mg/mL, -S9); dimethylbenzanthracene (DMBA, 1.1 µg/mL, +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item is considered 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 considered 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 considered 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 in the range normally found (0.6 – 31.7 mutants per 10E6 cells) a concentration-related increase of the mutation 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 using SYSTAT®11 (SYSTAT Software, Inc., 501, Ganal Boulevard, Suite G, Richmond, GA 94804, USA) statistics software. The number of mutant colonies obtained for the cultures treated with the test item were compared to the solvent controls. 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.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not examined
Positive controls validity:
valid

Additional data supporting the information provided are attached as pdf documents below under 'Attached background material'.

Conclusions:
The test substance did not induce mutagenic effects in the in vitro gene mutation assay (HPRT test) with Chinese hamster V79 cells in the presence and absence of a metabolic activation system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in bacteria in vitro

In an in vitro assessment of the mutagenic potential of ethane-1,2-diol, propoxylated (CAS No.31923-84-9, EC No. 500-078-0) in bacteria according to OECD guideline 471 under GLP conditions (rel 1-key, Ames, OECD 471, Bayer Schering Pharma, 2010b, T7080063), histidine-dependent auxotrophic mutants of Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98, and TA 102 were exposed to the test substance diluted in deionised water. Deionised water was also used as a negative control. Two independent experiments were performed in the presence and absence of liver preparations (S9 mix) from rats treated with aroclor 1254. The first test was a standard plate incorporation assay; the second included a pre-incubation stage. Concentrations of 0, 50, 158, 500, 1581, 5000 µg/plate with and without S9 mix were tested in both experiments. No signs of toxicity were observed towards the tester strains in either mutation test. No evidence of mutagenic activity was seen at any concentration of the test substance in either mutation test, in the absence or presence of metabolic activation. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that ethane-1,2-diol, propoxylated showed no evidence of mutagenic activity in this bacterial system under the test conditions used.

Chromosome aberration in mammalian cells in vitro

The clastogenic potential of ethane-1,2-diol, propoxylated (CAS No.31923-84-9, EC No. 500-078-0) was evaluated in a chromosome aberration test in vitro according to OECD guideline 473 under GLP conditions (rel 1-key, CA, OECD 473, Bayer Schering Pharma, 2010c, T6080062). Chinese hamster V79 cells were exposed in the absence and in the presence of S9 mix for 4 h to concentrations of 600, 1200 and 2400 µg/mL of the test substance. Cultures of all concentrations were harvested 18 h after the beginning of the treatment. In addition, cells treated with 2400 µg/mL were harvested 30 h after the beginning of the treatment. Without S9 mix an additional experiment was performed using continuous treatment for 18 h, harvest at the same time, and test substance concentrations of 600, 1200, 1600, 2000 and 2400 µg/mL. Based on cytotoxicity test substance concentrations were selected for reading of metaphases. Without S9 mix cytotoxic effects were observed at 1200 µg/mL and above (4 h treatment) and at 2400 µg/mL (18 h treatment). With S9 mix no cytotoxic effects were observed. Precipitation in the medium did not occur. Therefore, concentrations of 600, 1200 and 2400 µg/mL (18 h treatment) were chosen for reading in the absence of S9 mix. All of the cultures harvested 18 h after the beginning of the treatment were included. None of the cultures treated with the test substance in the absence and in the presence of S9 mix showed biologically relevant or statistically significant increased numbers of aberrant metaphases. The positive controls induced clastogenic effects and demonstrated the sensitivity of the test system and the activity of the used S9 mix. Based on this test, ethane-1,2-diol, propoxylated is considered not to be clastogenic in mammalian cells in vitro.

Gene mutation in mammalian cells in vitro

A study according to OECD guideline 476 under GLP conditions was performed to investigate the potential of ethane-1,2-diol, propoxylated (CAS No.31923-84-9, EC No. 500-078-0) to induce gene mutation at the HPRT locus in V79 cells of the Chinese hamster (rel 1-key, HPRT, OECD 476, Harlan CCR, 2009, 1280201). The assay was performed in two independent experiments, using two parallel cultures each. Both experiments were performed with and without liver microsomal activation and a treatment period of 4 h. The highest applied concentration (2400 µg/mL) was equal to a molar concentration of approximately 10 mM. The tested concentrations were as follows: Experiment I: 150, 300, 600, 1200, 2400 µg/mL (experiment I, with and without S9 mix) and 300, 600, 1200, 1800, 2400 µg/mL (experiment II, with and without S9 mix). No precipitation was observed up to the maximal concentration. No substantial and reproducible dose-dependent increase of the mutation frequency was observed in both 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 conclusion it can be stated that under the experimental conditions reported ethane-1,2-diol, propoxylated did not induce gene mutations at the HPRT locus in V79 cells.

Justification for classification or non-classification

The available in vitro data on genetic toxicity with ethane-1,2-diol, propoxylated (CAS No.31923-84-9, EC No. 500-078-0) do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 (CLP) and are therefore conclusive but not sufficient for classification.