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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic potential of the substance was investigated in a battery of different in vitro tests:

The substance was weakly mutagenic in bacterial mutagenicity assays in bacteria (Ames tests) at high concentrations in two particular S. typhimurium strains (TA100 and TA102). One of these Ames tests includes an investigation on the effect of different kinds of proteins or different concentrations of these proteins on the mutagenic activity of the substance. The results indicate that the direct acting test material may be inactivated by mammalian metabolizing enzymes. This means that the mutation effects would not occur in an in vivo setting.

An in vitro mammalian cell gene mutation test in V79 cells (hprt locus) shows the test material to be non-mutagenic up to concentrations reaching the solubility limits of the compound with and without metabolic activation.

The test material shows to be non-clastogenic in an in vitro mammalian chromosome aberration assay.

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:
1983-07-06 to 1983-08-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 1983
Deviations:
yes
Remarks:
The highest dose tested was 10,000 µg/plate and, thus, exceeding the maximum test concentration of 5,000 µg/plate as recommended by the Guideline.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
The highest dose tested was 10,000 µg/plate and, thus, exceeding the maximum test concentration of 5,000 µg/plate as recommended by the Guideline.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
His Operon
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
his D 3052, uvrB, rfa + R-factor (pKM101)
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa + R-factor (pKM101)
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
his G428, rfa + R-factor (pKM101)
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
his C 3076, uvrB, rfa
Metabolic activation:
with and without
Metabolic activation system:
10% rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
Test concentrations with justification for top dose:
1st and 2nd test: 50, 250, 1250, 2500, 5000, and 10000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
methylmethanesulfonate
mitomycin C
other: N-Methyl-N-nitro-N-nitrososguanidine
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 4 plates per test concentrations, 8 (2x 4) plates per solvent control
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)
Evaluation criteria:
No evaluation criteria are provided in the original report. For the reevaluation of the results of this study the following criteria are taken into account:
A test material was to be defined as positive or mutagenic in this assay if
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, TA 102) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed.
- an increase exceeding the threshold at only one concentration is considered as biologically meaning ful if reproduced in a second independent experiment
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration
Statistics:
no
Key result
Species / strain:
S. typhimurium TA 98
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
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
at concentrations larger than 2500 µg/plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium 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
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
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
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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
Positive controls validity:
valid

The test material was examined for mutagenic activity in two series of in vitro microbial assays employing Salmonella typhimurium indicator organisms. The substance was tested with and without the presence of a metabolic activation system, i.e. liver microsomal enzyme preparations from Aroclor-induced rats.

The test material was tested over a series of concentrations. The choice of the concentration range was mainly influenced by the endeavor to achieve a toxic effect in the highest concentration. Such a toxic effect is generally considered important in this test system. This effect was not obtained in the present trials: No toxicity and no precipitation were observed up to the highest dose tested at both experiments.

During the routinely conducted microscopic examination of the plates no toxic effect was detected. The dose range employed for the evaluation of this compound was from 50 to 10000µg per plate, i.e. the highest dose exceeded the maximum test material concentration as recommended by the OECD guideline.

The results of the tests conducted on the test material in the absence and presence of a metabolic system were negative for Salmonella typh. TA 98, TA 102, TA 1535, and TA 1537. The repeat tests were also negative.

With the tester strains Salmonella typhimurium TA 100 the observed increase in the number of revertant colonies was interpreted as a positive result.

The validity of the mutation assay can be assessed by the results obtained for the positive and negative controls. The negative control mutant frequencies were all in the normal range, and the positive control compounds yielded normal mutant frequencies that were greatly in excess of the background.

Conclusions:
Based on the results of this study, the test material is considered to be weakly positive in this bacterial reverse mutation assay.
Executive summary:

The investigations were performed using Salmonella typhimurium TA 100, TA 102, TA 98, TA 1535 and TA 1537 as tester strains. The mutagenic potential was examined in the plate incorporation test with and without addition of a liver postmitochondrial fraction as the in vitro metabolizing system (S-9; male rats, induced with Aroclor 1254).

Concentrations: 50, 250, 1250, 2500, 5000, and 10000 µg/plate.

2-Aminoanthracene, 9-aminoacridine, benzo(a)pyrene, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanidine, methyl methanesulfonate, N-methyl-N-nitro-N-nitro­soguanidine, mitomycin c, and 2-nitrofluorene served as positive controls for testing the bacteria and the induced S-9 preparation.

With and without addition of S-9 as the metabolizing system, the test material did show mutagenic activity with Salmonella typhimurium TA 100 in the concentration range used. With Salmonella typhimurium TA 98, TA 102, TA 1535 and TA 1537 no mutagnic activity was found. With Salmonella typhimurium TA 100 the number of revertant colonies was increased 2.4 - 3.8 -fold. The maximum concentration level in this study exceeded the highest test concentration as recommended by the current OECD-Guideline for this system. Toxicity and precipitation was not observed up to the highest dose tested.

The substances used as positive controls showed normal reversion properties of all strains and good metabolic activity of the S-9 mix used.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1983-06-06 - 1983-08-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 193
Deviations:
yes
Remarks:
The highest dose tested was 10,000 µg/plate and, thus, exceeding the maximum test concentration of 5,000 µg/plate as recommended by the Guideline.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
The highest dose tested was 10,000 µg/plate and, thus, exceeding the maximum test concentration of 5,000 µg/plate as recommended by the Guideline.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
His Operon
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
his D 3052, uvrB, rfa + R-factor (pKM101)
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa + R-factor (pKM101)
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
his G428, rfa + R-factor (pKM101)
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
his G 46, uvrB, rfa
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
his C 3076, uvrB, rfa
Metabolic activation:
with and without
Metabolic activation system:
10% rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)
Test concentrations with justification for top dose:
50, 250, 1250, 2500, 5000, and 10000 µg/plate
Vehicle / solvent:
DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-aminoanthracene
Remarks:
with S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
mitomycin C
other: Daunomycin, 2-aminoacridine, N-methyl-N-nitro_N-nitrosoguanidine
Remarks:
without S9
Details on test system and experimental conditions:
in agar (plate incorporation)
Evaluation criteria:
No evaluation criteria are provided in the original report. For the reevaluation of the results of this study the following criteria are taken into account:
A test material was to be defined as positive or mutagenic in this assay if
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, TA 102) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed.
- an increase exceeding the threshold at only one concentration is considered as biologically meaning ful if reproduced in a second independent experiment
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration
Statistics:
no
Key result
Species / strain:
S. typhimurium TA 98
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
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
only at concentrations >=5,000 µg/plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium 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
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
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
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
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
Positive controls validity:
valid
Additional information on results:
In S. typhimurium TA 102, the test item was positive according to the above mentioned evaluation criteria only at a concentration of 10,000 µg/plate, exceeding the maximum concentration of 5,000 µg/plate as recommended by the OECD Guideline. Therefore, this strain was evaluated as negative.

The test material was examined for mutagenic activity in two series of in vitro microbial assays employing Salmonella typhimurium indicator organisms. The substance was tested with and without the presence of a metabolic activation system, i.e. liver microsomal enzyme preparations from Aroclor-induced rats.

The test material was tested over a series of concentrations. The choice of the concentration range was mainly influenced by the endeavor to achieve a toxic effect in the highest concentration. Such a toxic effect is generally considered important in this test system. This effect was not obtained in the present trials: No toxicity and no precipitation were observed up to the highest dose tested at both experiments.

During the routinely conducted microscopic examination of the plates no toxic effect was detected. The dose range employed for the evaluation of this compound was from 50 to 10000µg per plate, i.e. the highest dose exceeded the maximum test material concentration as recommended by the OECD guideline.

The results of the tests conducted on the test material in the absence and presence of a metabolic system were negative for Salmonella typh. TA 98, TA 102, TA 1535, and TA 1537. The repeat tests were also negative.

With the tester strains Salmonella typhimurium TA 100 the observed increase in the number of revertant colonies was interpreted as a positive result.

The validity of the mutation assay can be assessed by the results obtained for the positive and negative controls. The negative control mutant frequencies were all in the normal range, and the positive control compounds yielded normal mutant frequencies that were greatly in excess of the background.

Conclusions:
Based on the results of this study, the test material is considered to be positive in this bacterial reverse mutation assay.
Executive summary:

The investigations were performed using Salmonella typhimurium TA 100, TA 102, TA 98, TA 1535 and TA 1537 as tester strains. The mutagenic potential was examined in the plate incorporation test with and without addition of a liver postmitochondrial fraction as the in vitro metabolizing system (S-9; male rats, induced with Aroclor 1254).

Concentrations: 50, 250, 1250, 2500, 5000, and 10000 µg/plate.

2-Aminoanthracene, 9-aminoacridine, benzo(a)pyrene, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanidine, methyl methanesulfonate, N-methyl-N-nitro-N-nitro­soguanidine, mitomycin c, and 2-nitrofluorene served as positive controls for testing the bacteria and the induced S-9 preparation.

With and without addition of S-9 as the metabolizing system, the test material did show mutagenic activity with Salmonella typhimurium TA 100 in the concentration range used. With Salmonella typhimuriumTA 98, TA 102, TA 1535 and TA 1537 no mutagnic activity was found. With Salmonella typhimurium TA 100 the number of revertant colonies was increased 2.4 - 3.8 -fold. The maximum concentration level in this study exceeded the highest test concentration as recommended by the OECD-Guideline for this system.Toxicity and precipitation was not observed up to the highest dose tested.

The substances used as positive controls showed normal reversion properties of all strains and good metabolic activity of the S-9 mix used.

Based on the results obtained, the test item is considered positive in this bacterial mutagenicity assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-07-23 to 1994-05-26
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)
Version / remarks:
adopted 4 April 1984
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT-locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type of cells: V79 cell line
- Source of cells: Istituto di Mutagenesi e Differenziamento, Consiglio Nazionale delle Ricerche, Pisa, Italy
- Suitability of cells: suitable for this type of test
- Normal cell cycle time (negative control): approx. 21 hours

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable:
- Methods for maintenance in cell culture:
- Cell cycle length, doubling time or proliferation index:
- Periodically checked for karyotype stability: yes
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
The cells were cultured in supplemented Dulbecco's Minimal Essential Medium (DMEM). The DMEM was supplemented with L-glutamine (4 mM), sodium bicarbonate (0.375%), antibiotics (penicillin/streptomycin) and 5% fetal calf serum (FCS). All incubations were performed at +37°C in a 4-5% carbon dioxide atmosphere (100% humidity)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from livers of rats pretreated with Aroclor 1254Type and composition of metabolic activation system:
- source of S9 : prepared at the test facility
- method of preparation of S9 mix : prepared from Aroclor 1254-treated male Wistar rats according to Ames et al (1975)
- concentration or volume of S9 mix and S9 in the final culture medium : S9-concentration: 10% (v/v): In experiments +S9 mix, the medium is replaced by 2,6 mL PBS-HEPES and 0.4 mL S9-mix (= 0.3 mL S9)
- quality controls of S9: metabolic capability (positive control DMBA)
Test concentrations with justification for top dose:
31.6, 100, 316, and 1000 µg/mL for 24 hours in the absence of S9-mix
158, 500, 1580, and 5000 µg/mL for 2 hours in the presence of S9
Vehicle / solvent:
distilled water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
other: N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)
Details on test system and experimental conditions:
According to guideline

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: test medium: duplicate; control: triplicate
- Number of independent experiments : 2 Series

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 1.5 E6
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 24 hours (-S9-mix) and 2 hours (+S9 mix)
- Harvest time after the end of treatment: Day 8 after start of treatment

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 days
- Selection time (if incubation with a selective agent): 8 - 12 days
- Staining: Giemsa
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: 6-thioguanine (7 µg/mL)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 1 000 000 cells per 150 mm Petri dish

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: no
Evaluation criteria:
So far no satisfactory mathematical methods are available for the statistical analysis of mammalian cell mutagenicity experiments such as those performed here. The experience has shown that the following predetermined descriptive criteria are the most useful for the interpretation of experimental results.
The effects of the test material upon the mutation frequency is defined as
- "No increase" in the mutation frequency if the mean frequency of the two parallel incubations of a given test material concentration is less than 2.0-fold above the mean of the actual negative controls or the mutation frequency is < 20.0 x 10-6 .
- "Clear increase" in the mutation frequency if the test material induces at least a 5.0-fold increase above the mean of the actual negative controls and the mean mutation frequency for a given test material concentration is > 40.0 x 10-6.
- All other results are defined as a "weak increase" of the mutation frequency.

Test materials are assessed as negative or non-mutagenic in this test system if
- no effect (no increase in the mutation frequency) occurs in the two independent experiments performed or
- a weak effect (weak increase) occurs in one series and no effect (no increase) in the other series of experiments.

Test materials are assessed as positive or mutagenic in this test system if
- a clear effect (clear increase in the mutation frequency) occurs at similar concentrations of the test material in the two independent experiments performed,
- a clear effect (clear increase) occurs in one series and a weak effect (weak increase) in the other series of experiments at identical concentrations, or
- weak effects (weak increases) occur dose-dependently (over at least two test material concentrations) and reproducibly at identical concentrations in the two experiments performed.
Statistics:
no statistics performed; see evaluation criteria
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for the highest test material concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with
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 examined
True negative controls validity:
not examined
Positive controls validity:
valid

Mutation frequencies in the solvent negative control remained within normal ranges, and treatment with the positive controls MNNG and DMBA yielded distinct increases in mutant frequencies. Accordingly, the study was considered to be valid.

When tested up to 1000 or 5000 µg/mL, the test material did not increase the mutant frequencies in the hprt locus of V79 cells either in the absence or the presence of S9 -mix, respectively (For results tables, please refer to the attached background material).

Conclusions:
The test material was not mutagenic in this mammalian cell gene mutation test in V79 cells (hprt locus) either in the absence or presence of metabolic activation under conditions where the positive controls exerted potent mutagenic effects.
Executive summary:

The test item was investigated for induction of gene mutations in V79 Chinese hamster cells in vitro (hprt locus).

The test item was tested in two main series of experiments in both the presence and absence of an exogenous metabolizing system (S9-Mix from livers of rats pretreated with Aroclor 1254). The cells were exposed to the following test material concentrations:

31.6, 100, 316, and 1000 µg/ml (without S9-Mix)

158, 500, 1580 and 5000 µg/ml (with S9-Mix)

In the absence of S9-Mix, the highest test material concentration exhibited a clear toxic response of the V79 cells: the cloning efficiency measured in the range finding experiment was reduced to 47 % and the growth rates of the cells measured on day 5 of the two experiments of the main study were reduced to values ranging from 60 to 83 % of the respective solvent controls. No clear signs of toxicity were seen in the experiments with S9-Mix.

N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG, 1 µg/ml) and 7,12-dimethylbenz[a]anthracene (DMBA, 20 µg/ml) served as positive control compounds in the absence and presence of S9-Mix, respectively.

The V79 cells were exposed to the test material, the respective positive control or the solvent for 24 hours in the absence and 2 hours in the presence of S9-Mix. Two parallel cultures per concentration were used for test concentrations and the positive controls. Three parallel cultures were established for the negative, solvent control. Two independent experiments were performed.

The positive control compounds induced the expected clear increase in the mutation frequency.

In the absence and presence of S9-Mix the test item did not increase the mutation frequency of the V79 cells as compared to the actual solvent controls. According to the predetermined criteria for the evaluation of results the test material was clearly negative in this test system.

In conclusion, the test material was not mutagenic in the V79 mammalian cell gene mutation test under conditions where the positive controls exerted potent mutagenic effects.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993-12-12 to 1994-07-29
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)
Version / remarks:
adopted April 1984
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Type of cells: V79
- Source of cells: Technische Hochschule Darmstadt (H. Miltenburger)
- Suitability of cells: Suitable for this type of assay
- Normal cell cycle time (negative control): 16-20 h

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: 1st passage
- Methods for maintenance in cell culture: maintained in DMEM
- doubling time: 16-20 h
- Modal number of chromosomes: 22
- Periodically checked for karyotype stability: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
The cells were cultured in supplemented Dulbecco's Minimal Essential Medium (DMEM). The DMEM was supplemented with L-glutamine (4 mM), sodium bicarbonate (0.375%), antibiotics (penicillin/streptomycin) and 5% fetal calf serum (FCS). All incubations were performed at +37°C in a 4-5% carbon dioxide atmosphere (100% humidity)
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from livers of rats pretreated with Aroclor 1254
- Type and composition of metabolic activation system:
- source of S9: prepared at the test facility
- method of preparation of S9 mix: prepared from Aroclor 1254-treated male Wistar rats according to Ames et al (1975)
- concentration or volume of S9 mix in the final culture medium: 10% (v/v) (1% S9 fraction)
- quality controls of S9: metabolic capability (positive control Cyclophosphamide)
Test concentrations with justification for top dose:
1st series: 140, 445, and 1400 µg/ml
2nd series: 140, 445, 1400, and 2500 µg/ml
Vehicle / solvent:
- Vehicle/solvent used: water (aqua bidest)
- Justification for choice of solvent/vehicle: good solubility and stability of test item
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
According to guideline
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : Two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in solvent water

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 hours
- Harvest time after the end of treatment (sampling/recovery times): 0, 11 and 21 hours

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): Colchicin at 0.1 µg/mL for 2 hours
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): Medium was replaced by KCl (0.4%) and cells were fixed by metahnol:acetic acid (3:1). Cells were stained with aceto-orcein
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 2 slides per test item concentration and control. 100 cells were analysed per slide
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Only well spread metaphases were used for analysis. The metaphases were analysed for gaps, breaks, exchanges, multiple aberrations, and specific aberrations.
- Determination of polyploidy: yes (1000 mitoses per slide)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: Mitotic index
Evaluation criteria:
A test material is defined as being unambiguously negative or non-clastogenic in this test system if no statistically significant increase in the number of aberrant metaphases per 100 cells, as compared to the actual negative control, occurs at any test material concentration.
A test material is positive or clastogenic in this test system if
- a statistically significant, dose-related increase in the number of aberrant metaphases per 100 cells occurs or
- a statistically significant increase in the number of aberrant metaphases per 100 cells is reproduced at the same test material concentration in independent experiments.
In both cases, however, the number of aberrant metaphases should be above the range defined as the historical negative controls of the laboratory and the biological relevance of the results has to be discussed.
Statistics:
Pairwise comparisons within each experiment
Each treatment group was compared with the negative control. For the negative control the solvent groups and the medium groups were pooled. For the comparisons the Exact Fisher Test (Sachs, 1984) was performed against one-sided alternatives.

Combination of first and second experiment
Identical treatment groups (i.e. groups with identical concentration and sampling time) of first and second experiment were combined. Therefore the nonparametric combination test from Fisher (Sonnemann, 1983) was carried out. The p-values of the combination test together with the corresponding chi-squared values, the degrees of freedom and the p-values from the single experiments are presented.

Multiple test procedure
As there is more than one treatment group, the p-values obtained are considered jointly to maintain an error rate (multiple level of significance)
of a = 5%. For this purpose Holm's multiple test procedure was used, which is an improvement of the Bonferroni procedure (Holm, 1979).
Key result
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 examined
True negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

For results tables, please refer to attached background material

Conclusions:
The test item was not clastogenic in this test system under conditions where the positive controls exerted potent clastogenic effects
Executive summary:

The test item was investigated for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro.

V79 cells cultured on glass slides were exposed to the test material for 5 hours in both the presence and absence of an exogenous metabolizing system (S9-Mix from livers of rats pretreated with Aroclor 1254).

The test item was tested in two main series of experiments. With and without metabolic activation metaphases of cells exposed to the following test material concentrations were evaluated in both experimental series performed: 140, 445, and 1400 µg/ml. The concentration of 2500 µg/ml was additionally tested in the second series.

Ethylmethane sulfonate (EMS, 500 µg/ml) and cyclophosphamide (CPA, 2.0 µg/ml) served as positive control compounds.

Four slides (= cultures) per concentration (2 with and 2 without S9-Mix) per time were investigated by scoring 100 well spread metaphases per slide. Preparation of the cells was performed 18 hours (medium control, low and medium concentration of test material) or 7, 18, and 28 hours (solvent control and high test material concentration) after addition of the test material or the solvent, respectively.

The positive control compounds induced the expected clear increase in the proportion of cells with chromosome aberrations.

The test item decreased the mitotic index at the highest test material concentration tested (2500 µg/ml) to relative values ranging from 34 to 67 % (with and without S9-Mix) as compared to the respective solvent control values.

In the absence and presence of the in vitro metabolizing system the test item did not increase the proportion of cells with aberrant chromosomes as compared to the actual controls. Furthermore, the test item did not lead to an increase in the number of polyploid cells.

In conclusion, the test material was not clastogenic in this test system under conditions where the positive controls exerted potent clastogenic effects.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus test in the mouse reveals no statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei in any of the test item-treated groups compared to the negative control.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986-02-4 to 1986-03-19
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to
Guideline:
other: JAPAN: Guidelines for Toxicity Studies of Drugs
Version / remarks:
1984-02-15, Yakushin 118
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: ddY
Sex:
male
Details on test animals and environmental conditions:
Seven to eight-week-old ddY male and female mice were purchased from SLC Inc. and used at 8 to 10 weeks of age. The mice were given commercial food pellets and tap water ad libitum throughout acclimation and experimental periods. For the main study, only male mice were used. Male and female mice were used for the acute toxicity test and the pilot study.
Route of administration:
intraperitoneal
Vehicle:
- Vehicle/solvent used: physiol. saline (0.9% NaCl)
- Justification for choice of solvent/vehicle: good solubility and stability of test item
- Concentration of test material in vehicle: 125, 250 and 500 mg/mL (main study)
1, 10, 50, 100, and 500 mg/mL (pilot study)
100, 200, 300, 400, and 500 mg/mL (acute toxicity test)
- Volume of vehicle administered: 10 mL/kg bw
Details on exposure:
The test chemical solutions and physiological saline (negative control) were given as single doses intraperitoneally to mice in a volume of 10 mL/kg.
Duration of treatment / exposure:
Sampling time was 24 hours after administration of the test material based on the results of a small acute test and the pilot micronucleus test.
Frequency of treatment:
The animals were treated once.
Post exposure period:
24 h after administration the animals were sacrificed. The post exposure time was shorter than recommended by the guideline. However, the substance is very hydrophilic; DHA was administered intraperitoneally at doses far above the limit dose and the results showed a clear negative response. Based on these considerations, the shorter post exposure time does not have influenced the result of the study.
Dose / conc.:
1 250 mg/kg bw/day (nominal)
Dose / conc.:
2 500 mg/kg bw/day (nominal)
Dose / conc.:
5 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 male mice per dose in the main test
Control animals:
yes, concurrent vehicle
Positive control(s):
Main Study: Mitomycin C
- Justification for choice of positive control(s): Mitomycin C is recommended by Guideline
- Route of administration: i.p.
- Doses / concentrations: 2 mg/kg

Pilot Study:
Mitomycin C (2 mg/kg, i.p., sampling at 24h after treatment)
6-Mercaptopurine (50 mg/kg, i.p., sampling at 48h after treatment)
7,12-Dimethylbenz[a]anthracene (40 mg/kg, i.p., 72h after treatment)
Tissues and cell types examined:
For microscopic investigation, at least two specimens from each animal were prepared and coded. The number of micronucleated polychromatic erythrocytes (MNPCEs) per 1000 polychromatic erythrocytes (PCEs) per animal was determined. The proportion of PCEs to total erythrocytes b(PCEs and normochromatic erythrocytes) based on 1000 erythrocytes per animal was also determined to evaluate the bone marrow toxicity of DHA. The number of evaluated cells is below the figures in the OECD guideline, however, based on the very high test dose exceeding the limit dose of the guideline (5000 mg/kg bw) and the clear negative result, this deviation is not considered to have an impact on the reliability of the study.
Details of tissue and slide preparation:
Mice were euthanized by cervical dislocation at each sampling time and the femoral marrow cells were flushed out with fetal bovine serum. The cell suspension was centrifuged at 1000 rpm for 5 minutes, and the supernatant discarded to get appropriate cell suspension concentrations. The suspensions were smeared onto clean glass slides and allowed to dry. The specimens were fixed by addition of methanol for 5 minutes, and then stained with 3% Giemsa solution containing 0.004% citric acid for 30 minutes. The slides were then rinsed and allowed to dry before micronucleus examination. At least two specimens were prepared per animal.
Evaluation criteria:
The result would be judged to be positive when the frequency of MNPCEs in treated animals was statistically significantly different from controls at the probability level of 5%.
Statistics:
Statistical analysis was only performed in the main study micronucleus test. The statistical significance of the difference between the incidence of MNPCEs in DHA treatment groups or the positive control group and the negative control group was evaluated by the methods of Kastenbaum and Bawman.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei was observed in any of the test item-treated groups compared to the negative control (0.17 % in control vs 0.17, 0.25, 0.2 % for the low mid and high dose group, respectively). The PCE ratio was not changed in the treatment groups. The number of micronucleated PCE in the positive control was 2.12 % and statistically significantly different to control.

For details please refer to attached table.

Conclusions:
DHA was not mutagenic in this in vivo micronucleus assay in male mice under conditions where the positive controls exerted potent mutagenic effects.
Executive summary:

An in vivo mouse bone marrow micronucleus test was conducted with the test material to evaluate its potential for clastogenicity in male ddY mice. The maximal dose level of the test material was 5000 mg/kg on the basis of the maximal tolerated dose estimated by an acute toxicity test, with intermediate and low dose levels set at 2500 and 1250 mg/kg, respectively. The sampling time was 24 hr after administration, based upon the result of a pilot micronucleus test. The main study micronucleus test was performed under the optimal experimental conditions, which were based on the results from the small-scale acute toxicity test and the pilot micronucleus test. Dihydroxyacetone was intraperitoneally singly administered to mice, and the induction of micronuclei was evaluated in femoral bone marrow. The number of micronucleated PCE in animals treated with Dihydroxyacetone was in the control range and did not show a statistically significant increase compared to the negative control.

The results suggest that dihydroxyacetone is not clastogenic in vivo under these experimental conditions

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The mutagenic/genotoxic potential of the test material was investigated in an extensive battery of studies including in vitro tests (bacterial reverse mutation assays, mammalian cell mutation test in Chinese Hamster V79 cells (hprt locus) and chromosomal aberration test in Chinese Hamster V79 cells) and an in vivo assay (bone marrow micronucleus test in mice). The robustness of this test battery was evaluated as discussed below.

(1) Bacterial reverse mutation tests

The test material was evaluated for bacterial mutagenicity in several different bacterial reverse mutation tests. Initially, two tests were carried out on two different batches of the test item. Each test comprised of two independent experiments in the presence and absence of Aroclor-induced rat S9-mix. For both tests, negative results were obtained with the strains TA98, TA1535 and TA1537 up to a maximum concentration of 10000 µg/plate, equivalent to 111 mMol/plate. In both studies, reproducible and dose-related increases in revertant counts were observed at high test concentrations ( 2500 µg/plate, i.e. 28 mMol/plate) with the test strains TA100 and TA102 in the presence and absence of S9-mix. The presence of S9-mix had no modifying effects in these tests. These results were confirmed in a third bacterial reverse mutation test performed with test strain TA100 and different modifications to the in vitro metabolizing system, i.e. different concentrations of Aroclor-induced rat S9-mix, inactivated Aroclor-induced rat S9-mix, BSA or combinations of these proteins. It was shown that the use of different amounts of S9-mix and the other proteins specified had no significant effect on the quantitative response of TA100. A fourth bacterial reverse mutation test was carried out in order to investigate a possible role for photo-activation on the observed mutagenic activity. In this test, DHA was exposed to UV-light before testing. Essentially the same results were obtained as in the previous tests: negative results were obtained with strains TA98, TA1535, and TA1537, whilst increases in revertant counts were obtained with test strains TA100 and TA102 in the presence and absence of S9-mix at high concentrations. A possible photomutagenic potential of dihydroxyacetone was investigated in a Photoames assay using the four bacterial strains S. typhimurium TA102, TA1537, TA100 and E. coli WP2 in the absence of a mammalian metabolizing system. No mutagenicity was observed in strains TA1537 and WP2 either with or without UV irradiation. As expected, DHA increased the number of revertant counts of TA100 and TA102 in the absence of UV irradiation. The magnitude of the mutagenic effects was not influenced by exposure to UV-light. The results from these studies are consistent with the lack of UV absorption shown by DHA in the UVA/UVB range of wavelength (290-400 nm). Based on all these studies it is concluded that DHA is a weak direct acting base-substitution mutagen in S. typhimurium strains TA100 and TA102. The mutagenic effects are not modified by the presence of Aroclor-induced rat S9-mix and there is no detectable photo-activation after exposure to UV-light.

(2) Other in vitro genetic toxicitity tests

The genotoxic potential of the test item to mammalian cells was evaluated in an hprt gene mutation assay using Chinese Hamster V79 cell. Cells were exposed in two independent experiments at test concentrations ranging from 31.6 to 1000 µg/mL (i.e. 0.3 to 11 mM) for 24 hrs in the absence of Aroclor-induced S9-mix, and concentrations ranging from 158 to 5000 µg/mL (i.e. 1.8 to 56 mM) for 2 hrs in the presence of S9. No significant increases in mutation frequencies were observed in cells exposed to various concentrations of DHA in the absence or presence of a metabolic activation system. Dihydroxyacetone was not mutagenic at the hprt locus in Chinese Hamster V79 cells. It should be noted that in the main study, no clear evidence of cytotoxicity was seen at the limit dose of 5000 µg/mL (56 mM). However this test concentration was in excess of that recommended in modern test guidelines, which state a maximal test concentration of 5000 µg/mL or 10 mM, whichever is the lowest. The clastogenic potential of DHA was evaluated in Chinese hamster V79-cells in the presence and absence of a mammalian metabolic activation system (S9). The cells were treated in two independent experiments with test concentrations up to 1400 and 2500 µg/mL (i.e. 16 to 28 mM), respectively. Cells were sampled for metaphase analysis at various time points after start of treatment. No increases in chromosomal aberrations or in the frequency of polyploidy were obtained in this study at any sampling time. Therefore, DHA was evaluated as not clastogenic in this in vitro chromosomal aberration assay. At 2500 µg/mL (28 mM) the level of cytotoxicity was slightly lower than recommended for this study. However, as with the previous studies the test concentrations were well in excess of the 5000 µg/mL or 10 mM limit stipulated by current test guidelines.

(3) In vivo genetic toxictity

DHA has been evaluated for its ability to induce clastogenic events and/or disruption of the mitotic spindle apparatus in vivo using the mouse bone marrow micronucleus assay. DHA was administered intraperitoneally as a single dose to groups of 6 male ddY mice at 1250, 2500, and 5000 mg/kg bw. Males were selected on the basis of a preliminary toxicity study in which no sex-related difference in toxicity was observed. The highest dose level used exceeded the current limit dose of 2000 mg/kg bw for non-toxic chemicals in this assay (OECD, 1997). Animals were killed 24 hrs after dosing and bone marrow was sampled for further analysis. There was no significant reduction in the PCE:NCE ratio at any dose level and, therefore, no indication of bone marrow cytotoxicity. No significant increases in micronucleus frequency were observed at any dose level. Plasma analyses were not carried out and, hence, direct evidence of bone marrow exposure is lacking, however, considering the route of administration (intraperitoneal), the high doses used (above the limit dose level recommended for this assay) and the physico-chemical properties of DHA (high water solubility, low molecular weight), sufficient systemic exposure thus bone marrow exposure to DHA can be assumed. Based on the results of this study, it is concluded that DHA did not induce micronuclei in the PCE of mouse bone marrow up to a maximum intraperitoneal dose of 5000 mg/kg bw. It should be noted that this study does not comply with current regulatory requirements, because the cell sample size of 1000 PCE was lower than currently recommended, and evaluation at 48-hours after treatment was not performed. However, considering the hydrophilic test compound and the very high test dose (far above the limit dose), these deviations are considered not to influence the reliability of the study.

(4) Summary/Conclusion

In conclusion, there exists an extensive battery of studies addressing the genotoxic potential of dihydroxyacetone. The overall package is considered to be robust, and shows that DHA causes base-pair substitutions at very high concentrations in bacterial cells, but is not mutagenic in mammalian in vitro test systems or in vivo.


Short description of key information:
The genetic toxicity of dihydroxyacetone was studied using an extensive battery of tests addressing the genotoxic potential. The overall package is considered to be robust, and shows that dihydroxyacetone causes base-pair substitutions at very high concentrations in bacterial cells, but is not
mutagenic in mammalian in vitro test systems or in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The negative results obtained with the test material in the in vitro genotoxicity assays using Chinese hamster V79 cells, and in vivo in the mouse bone marrow micronucleus test, indicate that the weak mutagenic effect of this chemical for bacterial cells is not likely to be relevant to any potential mutagenic or carcinogenic risk in mammalian species including humans. The absence of a mutagenic or carcinogenic potential of the substance in vivo was confirmed by the negative results observed in a lifetime skin painting study in mice. (See IUCLID5 section 7.7 - Carcinogenicity). Therefore, the substance must not be classified as genotoxic/mutagenic.