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Genetic toxicity in vitro

Description of key information

The substance was negative in two Ames tests, negative in a chromosome aberration test and negative in an HPRT test.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Oct 2014 - 23 Oct 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and β-naphthoflavone induced rat S9 mix
Test concentrations with justification for top dose:
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
yes
Remarks:
Sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9: 2-aminoanthracene (60 µg/plate); without S9: 4-nitroquinoline-N-oxide (5 µg/plate)
Remarks:
with and without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes (PIT)
- Exposure duration: 37°C for 48 – 72 hours in the dark

NUMBER OF REPLICATIONS:
Three test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn
Evaluation criteria:
The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling of the spontaneous mutation rate in the tester strain either without S9 mix or after adding a metabolizing system.
A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for the tester strain were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found from about 2 500 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for the tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No bacteriotoxic effect (reduced trp- background growth, decrease in the number of trp+ revertants) was observed in the standard plate test and in the preincubation assay under all test conditions.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experimental Result

Revertants/plate (mean from three plates)
SPT PIT
Dose (µg/plate) with S9 without S9 with S9 without S9
DMSO 72.0 56.3 62.3 71.3
33 62.3 55.0 60.0 57.0
100 54.3 52.0 62.3 68.3
333 55.3 57.0 63.0 58.7
1000 64.7 54.3 69.0 59.0
2500 59.3 58.7 62.0 67.7
5000 51.0 41.7 63.0 64.7
Positive Control 257.0 860.7 172.7 660.7
Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of Escherichia coli strain WP2 uvrA in a reverse mutation assay at a dose range of 33 μg - 5000 μg/plate (SPT) and 33 μg - 5000 μg/plate (PIT)

Both Standard plate test (SPT) and preincubation test (PIT) were performed with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from about 2500 μg/plate onward with and without S9 mix. No bacteriotoxic effect was observed under all test conditions. A relevant increase in the number of trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000
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)
GLP compliance:
yes (incl. QA statement)
Remarks:
RCC Cytotest Cell Research GmbH, 64380 Roßdorf
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
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) supplemented with 10 % fetal calf serum (FCS).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 liver microsomal fraction from rats induced with phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
without S9 mix
Experiment I, 4h: 10, 20, 40, 60, (80), (120) µg/ml
Experiment III, 18h: (2.5), (5), 10, 15, (20), 30 µg/ml
Experiment III, 28h: (2.5), (5), 10, 15, (20), 30 µg/ml
with S9 mix
Experiment I, 4h: 10, 20, 40, 60, (80), (120) µg/ml
Experiment II, 4h: (15), (20), (25), 30, 35, 40 µg/ml
Experiment III, 4h: (5), 10, 20, 40, 60, (80) µg/ml
Concentrations in brackets were not evaluated.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation Migrated to IUCLID6: 600 µg/ml = 4.8 mM (continuous exposure); 1000 µg/mI = 8.0 mM (4 h exposure)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation Migrated to IUCLID6: 0.7 µg/mI = 2.5 µM (4 h exposure; 18 h preparation interval); 1.0 µg/ml = 3.5 µM (4 h exposure; 28 h preparation interval)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18 hours (without metabolic activation) and 4 h (with and without metabolic activation)
- Expression time (cells in growth medium): 14 and 24 hours (with metabolic activation); 18 and 28 hours (without metabolic activation).
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 hours

SPINDLE INHIBITOR (cytogenetic assays): 16 h and 26 h, respectively after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2 h later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCI) for 20 min at 37°C followed by fixation.

STAIN (for cytogenetic assays): Giemsa (E. Merck, D-64293 Darmstadt).

NUMBER OF REPLICATIONS: duplicate cultures were used in each experiment

NUMBER OF CELLS EVALUATED: 100 cells per culture (total 200)

DETERMINATION OF CYTOTOXICITY
- Mitotic Index
- Additionally, two cultures per test substance and solvent control treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the test substance is given as reduction of % cells as compared to the solvent control.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
The chromosome aberration assay performed in our laboratory 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 our 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:
without S9: EMS 600 µg/mI, range of 9.0 - 39.0%
with S9: CPA 0.47 - 0.93 µg/ml, range of 7.5 - 49.5%


A test substance is classified as non-clastogenic if:
- the number of induced structurai chromosome aberrations in all evaluated dose groups are in the range of our 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 substance is classified as clastogenic if:
- the number of induced structural chromosome aberrations are 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.
Statistics:
Statistical significance was confirmed by means of the Fischer's exact test (10) (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test substance are not clearly met, the classification with regard to the historical data and the bioiogical relevance is discussed and/or a confirmatory experiment is performed.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxic from 75 µg/ml (4 h treatment, in the absence and presence of S9) and from 18.8 µg/ml (18 h treatment, in the absence of S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No influence of the test substance on the pH value or osmolarity was observed.
- Precipitation: In the pre-test on toxicity, precipitation of the test substance after 4 h treatment was observed at 150 µg/ml and above in the absence of S9 mix and at 300 µg/mI and above in the presence of S9 mix.

RANGE-FINDING/SCREENING STUDIES:
Clear toxic effects were observed after 4 h treatment with 75 µg/ml in the absence and presence of S9 mix. In addition, 24 h continuous treatment with 18.8 µg/ml and above in the absence of S9 mix induced strong toxic effects. Considering the toxicity data of the pre-test, 120 µg/ml (with and without S9 mix) were chosen as top concentrations in the main experiment I.

DOSE SELECTION RATIONAL
In the first experiment with S9 mix an aberration frequency slightly beyond our historical control data range (0 - 4 % aberrant cells exclusive gaps) was observed after 4 h treatment with 20 µg/ml (4.5 % aberrant cells excl. gaps). To verify this result a second experiment in the presence of S9 mix was performed, using 40 µg/ml as top treatment concentration for 4 h exposure.
Dose selection of experiment III was also influenced by test substance toxicity. In the range finding pre-test clearly reduced ceIl numbers were observed in the absence of S9 mix after 24 h exposure with 18.8 µg/ml and above. Therefore, 30 µg/ml were chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix 80 µg/mI were chosen as top treatment concentration with respect to the results obtained in experiments I and II.

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment I in the presence of S9 mix, a marginal increase of aberrant cells exclusive gaps slightly beyond the historical control data range (0.0 % - 4.0 % aberrant celI exclusive gaps) was observed at 20 µg/ml. This observation could not be reproduced in experiment II. Therefore, in accordance to OECD 473, a third experiment was performed, covering the continuous treatment in the absence of S9 mix and the late preparation interval, both in
the absence and presence of S9 mix. In the third experiment, no biologically relevant increases were observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I and III in the absence and presence of S9 mix toxic effects indicated by clearly reduced mitotic indices and/or reduced cell numbers were found at least at the highest evaluated concentrations.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Structural chromosome aberrations

                 Aberrant cells (%)
Experiment Preparation Interval Concentration (µg/ml) Polyploid cells (%) Cell numbers (%) Mitotic indices (% of control) incl. Gaps excluding Gaps exchanges

Exposure period 4 h without S9 mix

I 18 h neg. control 4.4 n.t. 100 1.5 1.5 0.0
solvent ctrl. 4.8 100 100 2.0 1.5 0.0
pos. control1 4.9 n.t. 96 12.0 11.5S 10.0
10 4.4 71 92 0.5 0.5 0.0
20 4.4 60 127 3.5 1.5 0.5
40 6.4 41 103 3.5 2.0 0.5
60P 4.5 53 108 1.5 1.5 0.5

Exposure period 18 h without S9 mix

III

18 h neg. control 4.2 n.t. 100 1.0 0.0 0.0
solvent ctrl. 3.8 100 100 2.5 0.5 0.0
pos. control2 3.9 n.t. 86 15.0 13.5S 8.0
10 3.1 71 94 4.0 2.5 0.0
15 5.3 54 81 1.5 0.5 0.5
30 4.9 31 60 4.0 2.0 0.0
III 28 h

neg. control

2.7 n.t. 100 1.0 0.0 0.0

solvent ctrl.

4.0 100 100 1.0 0.5 0.0
pos. control1 4.9 n.t. 66 17.5 17.5S 11.0
10 3.4 88 96 0.0 0.0 0.0
15 3.8 56 90 2.0 1.0 0.0
30 4.2 23 48 0.5 0.5 0.0

Exposure period 4 h with S9 mix

I 18 h neg. control 5.4 n.t. 100 4.0 4.0 1.5
solvent ctrl. 5.1 100 100 4.0 3.0 0.5
pos. control3 5.8 n.t. 108 10.0 10.0S 4.5
10 5.3 75 90 3.5 2.0 0.5
20 5.4 81 88 4.5 4.5 2.0
40 6.9 70 72 5.0 4.0 2.0
60P 3.4 26 25 4.0 1.5 0.5
II 18 h neg. control 2.1 n.t. 100 3.0 1.5 0.5
solvent ctrl. 3.1 100 100 1.0 0.0 0.0
pos. control3 3 n.t. 86 18.0 16.0S 7.5
30 2.7 90 89 0.5 0.0 0.0
35 3.0 92 90 2.5 2.5S 0.5
40 2.9 93 72 2.0 1.0 0.5
III 28 h neg. control 4.3 n.t. 100 1.5 1.5 0.5
solvent ctrl. 4.2 100 100 0.0 0.0 0.0
pos. control4 3.1 n.t. 104 12.5 12.5S 1.5
10 4.6 98 105 0.5 0.5 0.0
20 3.7 101 98 2.0 1.0 0.0
40 3.9 76 101 1.0 1.0 0.0
60 3.7 37 82 4.0 3.0S 1.5

n.t. = not tested

p = precipitation occured

s = aberration frequency statistically significant

solvent control = 0.5% DMSO

positive controls: 1 EMS (100 µg/ml); 2 EMS (600 µg/ml); 3 CPA (0.7 µg/ml); 4 CPA (1.0 µg/ml)

Two significant increases (p < 0.05) were observed in experiment II and III in the presence of S9 mix. These statistical significant increases were caused by the low response in the corresponding solvent control (0.0 % aberrant cells exclusive gaps). Since this response at low concentrations is clearly within the historical control data range, the statistical significance has to be regarded as being biologically irrelevant.

No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test substance as compared to the rates of the solvent controls.

In both experiments, EMS (600 and 1000 µg/ml, respectively) and CPA (0.7 and 1 µg/ml, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:
In the study described and under the experimental conditions reported, the test substance did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).
Executive summary:

In a GLP-compliant study following OECD guideline 473, the test substance dissolved in DMSO was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro. Three independent experiments were performed. Experiment II was performed to verify the results of experiment I in the presence of S9 mix. The exposure period was 4 h with metabolic activation (exp. I, II and, III), 4 h without metabolic activation (exp. I) and 18 h without S9 mix (exp. III). The chromosomes were prepared 18 h (exp. I, II, and, III) and 28 h (exp. III) after start of treatment with the test substance. In each experimental group two parallel cultures were set up. Per culture 100 metaphase cells were scored for structural chromosome aberrations. In a range finding pre-test on toxicity cell numbers 24 h after start of treatment were scored as indicator for cytotoxicity. Concentrations between 9.4 and 1200 µg/ml were applied. The top concentration was limited by the solubility of the test substance in DMSO. Clear toxic effects were observed after 4 h treatment with 75 µg/ml in the absence and presence of S9 mix. In addition, 24 h continuous treatment with 18.8 µg/ml and above in the absence of S9 mix induced strong toxic effects. In the range finder, precipitation of the test substance in culture medium was observed after treatment with 150 µg/ml and above in the absence and 300 µg/ml and above in the presence of S9 mix. No influence of the test substance on the pH value or osmolarity was observed. In experiment I and III in the absence and presence of S9 mix toxic effects indicated by clearly reduced mitotic indices and/or reduced cell numbers were found at least at the highest evaluated concentrations. In the confirmatory experiment II, no clear toxicity was observed since only the relevant concentrations for a verification of the results obtained in experiment I were evaluated for cytogenetic damage.

In this study, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In experiment I in the presence of S9 mix, a marginal increase of aberrant cells exclusive gaps slightly beyond our historical control data range (0.0 % - 4.0 % aberrant cell exclusive gaps) was observed at 20 µg/ml. This observation could not be reproduced in experiment II. Therefore, in accordance to OECD 473, a third experiment was performed, covering the continuous treatment in the absence of S9 mix, and the late preparation interval with and without S9 mix. In the third experiment, no biologically relevant increases were observed. The apparent dose related increase within our historical control data range in the presence of S9 mix must be regarded as statistically occurring artifact. In addition, two significant increases (p < 0.05; see pages 34 and 35) were observed in experiment II and III in the presence of S9 mix. These statistical significant increases were caused by the low response in the corresponding solvent control (0.0 % aberrant ceils exclusive gaps). Since this response at low concentrations is clearly within the historical control data range, the statistical significance has to be regarded as being biologically irrelevant. No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test substance as compared to the rates of the solvent controls. In both experiments, EMS (600 and 1000 µg/ml, respectively) and CPA (0.7 and 1 µg/ml, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test substance did not induce structural chromosome 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:
June 5, 1980 - June 20, 1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to guideline
Guideline:
other: Ames, B. N., J. McCann, and E. Yamasaki. Mutation Research, 1975, 31: 347-364.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no strain with AT base pair at the primary reversion site was used (TA 102 or E.coli WP2). No confirmatory experiment performed.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from direct light.
Target gene:
histidine
Species / strain / cell type:
other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced microsomal enzyme preparations (S9) from rat liver
Test concentrations with justification for top dose:
1000 µg, 500 µg, 100 µg, 10 µg, and 1 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: see "details on test system"
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours at 37 °C.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- By "filter paper disc-agar overlay zone of inhibition method"

POSITIVE CONTROLS
- Without S9:
N-methyl-N-nitro-N-nitrosoguanidine (MNNG): 5 µg/plate (TA 1535, TA 100)
9-Aminoaoridine (9-AA): 100 µg/plate (TA 1537)
2-Nitrofluorene (2-NF): 0.5 µg/plate (TA 1538, TA 98)
- With S9:
2-Aminoanthracene (2-AA): 5 µg/plate (all strains)
Evaluation criteria:
The following criteria were used in the evaluation and reporting of the mutagenic potential of the test material:

a. the spontaneous revertant levels for each strain when used in either the direct plate assay or the activated plate assay must be within the acceptable limits as defined by the historical data.
b. all sterility controls must be negative
c. all positive controls must demonstrate that the indicator strains are functional with known mutagens as evidenced by an increase of at least three times the number of revertant colonies per plate as the spontaneous revertant controls.
d. to be considered positive for mutagenic activity, the test material should exhibit a dose reponse effect (increasing numbers of revertant colonies with increased amounts of the test sample).
e. for strains TA 1535, TA 1537, TA 1538, and TA 98, the test sample should produce a positive dose response over the conoentrations with the lowest increase in revertants/plate greater than or equal to 3x the solvent control value or the S-9 fraction control value, as applicable, to be considered mutagenic.
f. to be considered mutagenic for strain TA 100, the test sample should produce a positive dose response over three conoentrations with at least one dose producing an increase in revertants/plate greater than or equal to 3.5x the solvent control value or the S-9 fraction control value, as applicable.

All of the criteria noted above must be met before the results of testing conducted with any test sample can be considered valid.
Statistics:
Least squares linear regression analysis was used to compute the "best fit" regression line of dose response on dose level for the test sample. These regression lines represent the strength of dose response obtained against each bacterial indicator strain. For each response line.
y = mx +b,
the sample regression coefficient, m, was tested for a statistically significant deviation from the value zero (0) which would indicate mutagenic, activity. The null hypothesis and the alternative hypothesis are stated below:

H0: m = 0, the data do not suggest mutagenic activity
HA: m > 0, the data suggest increasing mutagenic aotivity dose

Significance level = 0.05
Species / strain:
other: TA 1535, TA 1537, TA 1538, TA 98, TA 100
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:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No cytotoxicity was observed in the pre-test.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Average colony counts

  TA 1535 TA 1537 TA 1539 TA 98 TA 100
Conc. (µg/plate) -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
DMSO 10 10 4 4 5 5 14 14 100 100
Positive Control * 1272 120 329 102 792 976 620 1049 1243 992
1000 7 10 2 7 8 15 21 19 85 85
500 4 17 3 6 12 13 16 19 79 108
100 7 8 3 7 9 19 17 22 82 99
10 15 12 6 4 7 18 21 26 82 103
1 11 9 4 8 10 15 21 31 91 113

* For control substance and concentrations details see: "details on test system"

Conclusions:
The test substance was found to be non mutagenic to strains TA 1535, TA 1537, TA 1538, TA 98, TA 100 with and without S9 mix.
Executive summary:

The potential of the test substance to cause reverse point mutations in bacteria was investigated in a pre-GLP Ames test. The test article was applied to Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 by plate incorporation method at 1000, 500, 100, 10 and 1 µg/plate with and without the addition of an induced rat liver fraction (S9). After incubation for 48 hours at 37°C, the plates were observed for revertant colonies and the colonies were counted and recorded. The test article tested in this assay was found to be nonmutagenic to all five of the Salmonella strains with and without the metabolic activation system (S-9).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
JuIy 24, 2000 - November 6, 2000
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)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Remarks:
BASF, Experimental Toxicology and Ecology
Type of assay:
mammalian cell gene mutation assay
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media:
1. Ham's F12 medium with glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS). During exposure to the test substance, Ham's F12 medium was used without FCS supplementation. In case of continuous treatment Ham's F12 medium with FCS supplementation was used.
2. Pretreatment medium ("HAT medium"): FCS-supplemented Ham's F12 medium with glutamine and hypoxanthine containing Hypoxanthine (13.6 µg/ml), Aminopterin (0.18 µg/ml) and Thymidine (3.88 µg/ml).
3. Selection medium ("TG medium"): Glutamine- and FCS-supplemented, hypoxanthine-free Ham's F12 medium with 6-thioguanine at a final concentration of 10 µg/mI.
All media were supplemented with 1% (v/v) penicillin/streptomycin (10,000 IU /10,000 µg/ml) and 1 % (v/v) amphotericine B (250 µg/mI)

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced rat liver fraction (S9)
Test concentrations with justification for top dose:
1st experiment
without S-9 mix: 0; 0.313; 0.625; 1.25; 2.5; 5.0; 10.0 µg/ml
with S-9 mix (S-9 fraction : cofactors = 3 : 7): 0; 15.625; 31.25; 62.5; 125; 250; 500 µg/ml

2nd experiment
without S-9 mix: 0; 0.625; 1.25; 2.5; 5; 10; 20 µg/mI
with S-9 mix (S-9 fraction : cofactors = 1 : 9): 0; 12.5; 25; 50; 100; 200; 400 µg/mI
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation Migrated to IUCLID6: 300 µg/mI culture medium added in a volume of 1.0 ml.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
With metabolic activation Migrated to IUCLID6: 10 µg/mI culture medium added in a volume of 1.0 ml.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

RATIONAL FOR DOSE SELECTION:
The experimental doses were determined from an appropriate pretest with cultures exposed for the duration of 4 hours to a wide dose range of the test article, i.e. 1 µg/ml - 3,000 µg/ml culture medium both without S-9 mix and after adding a metabolizing system. The cloning efficiency was strongly affected without S-9 mix from about 10 µg/ml onward. With metabolic activation the cloning efficiency was only slightly influenced from about 500 µg/ml onward but the colony size was very small demonstrating a test substance influence an colony growth. Test substance precipitation was observed from about 50 µg/ml onward. On the basis of the findings from the pretest, the following doses were selected as top doses:
- Without and with S-9 mix, 4 hours exposure time: 10 µg/ml
- With S-9 mix, 4 hours exposure time: 500 µg/ml

DURATION
- Attachment period: 24 hours
- Exposure duration: 4 hours (with and without S9 mix)
- Expression time (cells in growth medium): 7-9 days
- Selection time (if incubation with a selection agent): 1 week (in TG Medium)
- Fixation time (start of exposure up to fixation or harvest of cells): about 2 weeks

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN (for cytogenetic assays): Colonies were fixed with methanol and stained with Giemsa.

NUMBER OF REPLICATIONS: 2 per dose

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (survival of treatment and cell viability without mutant selection); mutant frequency; cell morphology after test substance exposure.

OTHER
pH values and osmolality were measured. The solubility of the test substance in the vehicle used and in the aqueous culture medium about 3 hours after treatment was checked to ensure proper culturing and to avoid extreme treatment conditions.
Evaluation criteria:
EVALUATION CRITERIA:
The criteria for a positive response are:
- Increases of the corrected mutation frequencies above the concurrent negative control values and above 15 mutants per 10^6 clonable cells and/or the evidence of a dose-response relationship
- Reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.
Isolated increases of mutant frequencies above 15 mutants per 10^6 clonable cells or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

The test chemical is considered non-mutagenic according to the following criteria.
-The corrected mutation frequency in all dose groups is within the historical control range and is not significantly above the concurrent negative control.
Statistics:
Due to the negative findings, a statistical evaluation was not carried out.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The number of colonies was decreased from about 250 µg/mI onward in the 1st experiment and from about 25 µg/mI onward in the 2nd experiment. Cell density was reduced from about 250 µg/mI onward in the 1st experiment and from 50 µg/ml in the 2nd experiment
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 20 µg/ml number of colonies was decreased and ceII density was reduced.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH remained unaffected during the experiments
- Effects of osmolality: Osmolality remained unaffected during the experiments.
- Precipitation: Test substance precipitation was observed from about 50 µg/ml onward.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutant frequencies at any concentration were within the range of the historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Cell morphology: Slightly reduced attachment observed with S9 mix at 500 µg/ml

EXPERIMENTAL RESULT

Experiment I, 4 h exposure:

concentration (µg/ml) S9 Mix absolute cloning efficiency I (%) relative cloning efficiency I (%) absolute cloning efficiency II (%) relative cloning efficiency II (%) mutant frequency / 106cells
DMSO - 79.3 100 98.3 100 7.56
Positive control EMS 300 - 67.9 85.6 89.5 91 377.75
Test item 0.313 - 81.8 103.2 91.5 93.1 1.81
Test item 0.625 - 75 94.6 98.8 100.5 3.85
Test item 1.25 - 79.2 99.9 92.9 94.5 7.83
Test item 2.5 - 84 105.9 91.2 92.8 15.41
Test item 5 - 80.6 101.6 103.7 105.5 7.06
Test item 10 - 80.6 101.6 98.2 99.9 9.52
DMSO + 91.2 100 100.4 100 1.11
Positive control MCA 10 + 95.5 95.5 91.4 91 81.11
Test item 15.625 + 89.5 98.1 95.9 95.5 6.38
Test item 31.25 + 88.7 97.3 100.6 100.2 3.76
Test item 62.5 + 83.9 92 98.9 98.5 1.73
Test item 125 + 89.4 98 96.2 95.8 11.61
Test item 250 + 32.7 35.9 96.3 95.9 8.18
Test item 500 + 0 0 98.8 98.4 10.5

Experiment II, 4 h exposure:

concentration (µg/ml) S9 Mix absolute cloning efficiency I (%) relative cloning efficiency I (%) absolute cloning efficiency II (%) relative cloning efficiency II (%) mutant frequency / 106cells
DMSO - 79.8 100 81.2 100 1.37
Positive control EMS 300 - 76.4 95.7 88.5 109 211.78
Test item 0.625 - 78.7 98.6 89.4 110.1 0.29
Test item 1.25 - 78.4 98.2 96.5 118.8 2.61
Test item 2.5 - 84.4 105.8 90.7 111.7 7.03
Test item 5 - 69.9 87.6 77 94.8 0.71
Test item 10 - 77.7 97.4 91.7 112.9 7.22
Test item 20 - 9.6 12 88.6 109.1 0.96
DMSO + 80 100 90.7 100 2.17
Positive control MCA 10 + 75.5 94.4 73.6 81.1 140.51
Test item 12.5 + 58.5 73.1 83.8 92.4 0.62
Test item 25 + 23.9 29.9 95.7 105.5 0.52
Test item 50 + 15.6 19.5 80.6 88.9 7.31
Test item 100 + 8.5 10.6 91.2 100.6 0.59
Test item 200 + 6 7.5 91.7 101.1 1.73
Test item 400 + 3.4 4.3 87.2 96.1 2.41

- Mutant frequency was corrected on the basis of the absolute cloning efficiency II at the end of the expression period

- Cloning efflciency I (survival): Determined for each test group after the exposure period in parallel cultures.

- Cloning efficiency II (viability): Determined for each test group at the end of the expression period in parallel cultures.

Conclusions:
Under the experimental conditions of this assay, the test substance has no mutagenic activity in vitro in the CHO/HPRT forward mutation assay.
Executive summary:

In a GLP compliant study following OECD guideline no. 476, the test article was tested for its ability to induce gene mutations at the

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of Aroclor-induced rat liver S-9 mix (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiment, the following doses were evaluated in the 1st experiment after an exposure period of 4 hours:

without S-9 mix: 0; 0.313; 0.625; 1.25; 2.5; 5.0; 10.0 µg/ml

with S-9 mix (S-9 fraction cofactors = 3 7): 0; 15.625; 31.25; 62.5; 125; 250; 500 µg/ml

In a 2nd experiment for confirmation of the results of the 1st experiment, the following doses were evaluated after an exposure period of 4 hours with S-9 mix and without metabolic activation:

without S-9 mix: 0; 0.625; 1.25; 2.5; 5; 10; 20 µg/mI

with S-9 mix (S-9 fraction cofactors = 1: 9): 0; 12.5; 25; 50; 100; 200; 400 µg/mI

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation, an expression phase of about 7 - 9 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The negative controls (vehicle controls) gave mutant frequencies within the range

expected for the CHO cell line. Both of the positive control chemicals, i.e. EMS and MCA, led to the expected increase in the frequencies of forward mutations. On the basis from the results of the present study, the test substance did not cause any increase in the mutant frequencies either without S-9 mix or after adding a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this assay, the test substance has no mutagenic activity in vitro in the CHO/HPRT forward mutation assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Point mutation in bacteria

The potential of the test substance to cause reverse point mutations in bacteria was investigated in a pre-GLP Ames test. The test article was applied to Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 by plate incorporation method at 1000, 500, 100, 10 and 1 µg/plate with and without the addition of an induced rat liver fraction (S9). After incubation for 48 hours at 37°C, the plates were observed for revertant colonies and the colonies were counted and recorded. No increases in revertant colonies were observed when compared to the control in any strain at any concentration tested. No cytotoic effects were observed in the pre-test. The positive control substances led to the expected increases in revertant colony counts. In conclusion, the test article tested in this assay is considered to be non-mutagenic to all five of the Salmonella strains either in presence or in absence of a metabolic activation system (S-9).

In a GLP-compliant guideline study investigating the mutagenic potential of the test article towards E. coli strain WP2 uvrA, the test article was applied to bacterial plates at a dose levels of 33 -5000 µg/plate. A standard plate test and a preincubation test were performed, both with and without metabolic activation. No bacteriotoxic effect was observed under all test conditions. Test substance precipitation was found from about 2 500 μg/plate onward with and without S9 mix. The test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other. The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study. The number of revertant colonies in the negative controls was within the range of the historical negative control data for the tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data. Thus, under the experimental conditions chosen here, it is concluded that the test article is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

In a publication from 1982 (Bonin et al., Mutation research), the test substance was reported to be negative in a bacterial reverse mutation assay following the method laid down by Ames et al (1975). The test substance was tested among other chemicals in Salmonella typhimurium TA 100, TA 98; TA1535, TA1537 and TA 1538. No details were given regarding control substances, dosing levels and colony counts. However, the reported data is in line with the negative Ames test performed conditions and is therefore used as supporting evidence.

Point mutation in mammalian cells

In a GLP compliant study following OECD guideline no. 476, the test article was assessed for its ability to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with and without the addition of Aroclor-induced rat liver S-9 mix. In the dose range finding pretest, the cloning efficiency was strongly effected without S-9 mix from about 10 µg/ml onward. With metabolic activation the cloning efficiency was only slightly influenced from about 500 µg/ml onward but the colony size was very small demonstrating a test substance influence an colony growth. Test substance precipitation was observed from about 50 µg/ml onward. On the basis of the findings, the following doses were selected as top doses: 10 µg/ml for exposure without S9 mix and 500 µg/ml with S9 mix. After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation, an expression phase of about 7 - 9 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The test substance did not cause any increase in the mutant frequencies either without S-9 mix or after adding a metabolizing system in two experiments performed independently of each other. The negative controls (vehicle controls) gave mutant frequencies within the range expected for the CHO cell line. Both of the positive control chemicals, i.e. EMS and MCA, led to the expected increase in the frequencies of forward mutations. Thus, under the experimental conditions of this assay, the test substance has no mutagenic activity in vitro in the CHO/HPRT forward mutation assay.

Chromosome damage in mammalian cells

In a GLP-compliant study following OECD guideline 473, the test substance dissolved in DMSO was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in three independent experiments. Dose selection was based on the toxicity of the test item observed in a range finding pre-test. Here, clear toxic effects were observed after 4 h treatment with 75 µg/ml in the absence and presence of S9 mix and after 24 h continuous treatment with 18.8 µg/ml and above in the absence of S9 mix. Based on the pre-test results, 120 µg/ml was chosen as top concentrations in the initial main experiment. The exposure periods assessed were 4 h with and without metabolic activation and 18 h without S9 mix. The chromosomes were prepared 18 h and 28 h after start of treatment with the test substance.

In the main study, clear toxic effects indicated by strongly reduced mitotic indices and/or reduced cell numbers below 50 % of control were observed in experiment I and III in the absence and presence of S9 mix at least at the highest evaluated concentrations. No biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test substance. In experiment I with S9 mix, a marginal increase of aberrant cells exclusive gaps slightly beyond the historical control data range (0.0 % - 4.0 % aberrant cells exclusive gaps) was observed at 20 µg/ml. This observation could not be reproduced in experiments II and III. Two significant increases (p < 0.05) were observed in experiment II and III in the presence of S9 mix. These statistically significant increases were caused by the low response in the corresponding solvent control. Since this response at low concentrations is clearly within the historical control data range, the statistical significance has to be regarded as being biologically irrelevant. No increase in the frequencies of polyploid metaphases was found after treatment with the test substance as compared to the frequencies of the controls. The positive controls induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test substance 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 article is considered to be non-clastogenic in this chromosome aberration test.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the data, classification for genetic toxicity is not warranted under Regulation (EC) No.1272/2008.