Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Bacterial gene mutation: negative; OECD test guideline 471, GLP (BASF SE, 2009);
- Mammalian gene mutation: negative; OECD test guideline 476 (CHO/HPRT assay), GLP (BASF SE, 2011);
- In vitro mammalian chromosome aberration test: negative; OECD test guideline 473, GLP (BASF SE, 2011).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-05-23 - 2011-09-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study (GLP)
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Adopted on July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
Adopted on Aug, 1998
Deviations:
no
GLP compliance:
yes
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):
- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL), 1% (v/v) amphotericin B (250 μg/mL).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
The cell cycle of the untreated V79 cells lasted for about 12 - 14 hours (measurement based on the BrdU method) under the selected culture conditions.
Metabolic activation:
with and without
Metabolic activation system:
1 part S9 (liver of 5 male Wistar rats pre-treated with 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally) fraction was mixed with 9 parts S9 supplement (cofactors)
Test concentrations with justification for top dose:
- First experiment: 31.3, 62.5, 125.0, 250.0, 500.0 and 750.0 µg/mL (without S9 mix; 4 h exposure and 18 h sampling time); 250.0, 500.0, 750.0, 1000.0 and 1300.0 µg/mL (with S9 mix; 4 h exposure and 18 h sampling time);
- Second experiment: 40.6, 81.3, 162.5, 325.0, 650.0 and 1300.0 µg/mL (without S9 mix; 4 h exposure and 18 h sampling time); 81.3, 162.5, 325.0, 650.0 and 1300.0 µg/mL (with S9 mix; 4 h exposure and 18 h sampling time);
- Third experiment: 40.6, 81.3, 162.5, 325.0, 650.0 and 1300.0 µg/mL (without S9 mix; 18 h exposure and 18 h sampling time); 162.5, 325.0, 650.0 and 1300.0 µg/mL (without S9 mix; 18 h exposure and 28 h sampling time); 81.3, 162.5, 325.0, 650.0 and 1300.0 µg/mL (with S9 mix; 4 h exposure and 28 h sampling time).

- The slides were not scored for mitotic indices or cytogenetic damage because the experiment did not fulfil the recommendations of the current guideline due to technical errors.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in water, and in comparison to other commonly used vehicles (e.g. DMSO, ethanol etc.) acetone was selected as the most suitable vehicle, which has been demonstrated to be suitable in the V79 in vitro cytogenetic assay and for which historical control data are available. The final concentration of the vehicle acetone in culture medium was 1% (v/v).
Untreated negative controls:
yes
Remarks:
MEM medium with or without FCS
Negative solvent / vehicle controls:
yes
Remarks:
medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9 mix: 500 μg/mL ethyl methanesulfonate (EMS); with S9 mix: 0.5 μg/mL cyclophosphamide (CPP); both dissolved in MEM without FCS (2.5 mg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: cells (run through max. 15 routine passages) visually checked for attachement (20-30 h incubation at 37°C, 5% (v/v) CO2 and ≥ 90% humidity in MEM incl. 10% (v/v) FCS, were used for treatment
- Exposure duration: see above (first to fourth experiment)
- Expression time (cells in growth medium): see above; samples were taken at 18 hours (about 1.5-fold of the normal cell cycle time) and 28 hours (more than 1.5-fold of the normal cell cycle time).
- Fixation time (start of exposure up to fixation or harvest of cells): cells were then fixed with methanol : glacial acetic acid (ratio 3 : 1; +4°C) for a total of 30 min.

SPINDLE INHIBITOR (cytogenetic assays): 2 - 3 hours prior to cell harvest, 100 μL colcemide (stock: 10 μg/mL colcemide dissolved in PBS [Phosphate Buffered Saline]) was added to each chamber in order to arrest mitosis in the metaphase.
STAIN (for cytogenetic assays): after drying, the slides were stained with 7.5% (v/v) Giemsa/Titrisol solution pH 7.2 for 10 minutes. After being rinsed twice in purified water and clarified in xylene, the slides were mounted in Corbit-Balsam.

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 100 metaphases per culture were evaluated for structural chromosome aberrations, whereby aneuploid and polyploid cells were recorded separately. Due to clearly positive findings (> 10% aberrant cells exclusive gaps) in all positive control cultures, the analysis of these test groups was restricted to 50 metaphases per culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (in general, 1 000 cells, incl. mitotic cells, were counted per culture); cloning efficiency (cells were seeded in cell culture flasks (2.5x10E5 cells per 25 cm2 flask) about 24 - 30 hours prior to exposure. The cells were treated similar to the slides using the same media and test substance concentrations. At the end of the recovery period or at the end of continuous treatment single cell suspensions were prepared from each flask and the cells were counted using a cell counter); at the end of the treatment period, the test cultures of all test groups were checked microscopically for cell morphology, which is an indication of attachment of the cells to the slides.
Evaluation criteria:
The test substance is considered as “positive” if the following criteria are met: (1) a statistically significant, dose-related and reproducible increase in the number of cells with structural chromosome aberrations (excl. gaps); (2) the number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle control value and the historical negative control data range. A test substance generally is considered as “negative” if the following criteria are met: the number of cells with structural aberrations (excl. gaps) in the dose groups is not statistically significant increased above the concurrent negative/vehicle control value and is within the historical negative control data range.
Statistics:
The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are statistically significant compared with the respective vehicle control, labels (* p ≤0.05, ** p ≤0.01) are printed in the tables.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
under the experimental conditions chosen here, the test substance is not a chromosome-damaging (clastogenic) substance nor did it unduce changes in the number of chromosomes under in vitro conditions using V79 cells.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
no clear mitotic activity suppression indicated by mitotic rates below 50% of control was observed; a dose-dependent growth inhibition was observed in all experiments under any of the experimental conditions.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was not influenced by test substance treatment.
- Effects of osmolality: osmolarity was not influenced by test substance treatment.
- Precipitation: no test substance precipitation in medium was observed.
- Other confounding effects: a dose-related increase in the aberration rates (1.5%, 2.0% and 3.5% aberrant metaphases, excl. gaps) was observed at the concentrations scored for cytogenetic damage (162.5 to 650 μg/mL) in the 2nd Experiment in the absence of S9 mix after 4 hours exposure at 18 hours sampling time. However, all values were equal or below the respective negative control value (3.5% aberrant metaphases, excl. gaps) and clearly within the range of our historical negative control data (0.0% - 5.5% aberrant metaphases, excl. gaps). Thus, this observation has to be regarded as biologically irrelevant.

RANGE-FINDING/SCREENING STUDIES:
In the initial range-finding cytotoxicity test for the determination of the experimental doses, the test substance did not exhibit any pronounced toxicity up to the highest required concentration, 5100 μg/mL (approx. 5000 μg/mL of the active ingredient), at which distinct test substance precipitation was observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The structural chromosome aberration rates of the vehicle control groups were within the historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of structural chromosome aberrations induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed. The values were also within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Strongly reduced cell numbers were observed from 1250 μg/mL onward in the absence of S9 mix and from 5000 μg/mL onward in the presence of S9 mix in the 1st experiment after 4 hours treatment. The cell numbers were also strongly reduced at 650 μg/mL (37.4% of control) and above in the absence of S9 mix and at 1 300 μg/mL (28.4% of control) in the presence of S9 mix In the 2nd experiment after 4 hours treatment at 18 hours preparation interval.
Strong growth inhibition was observed in the 3rd experiment at 650 μg/mL (14.4% of control) and above after 18 hours continuous exposure in the absence of S9 mix. In addition, after 18 hours exposure at 28 hours sampling time in the absence of S9 mix and after 4 hours exposure at 28 hours sampling time in the presence of metabolic activation, cytotoxicity was observed at 1300 μg/mL (52.5% or 12.7% of control, respectively). However, in the 3rd experiment concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.
Distinct changes in cell attachment and/or cell morphology were observed in the 2nd experiment in the absence of S9 mix at 1 300 μg/mL only. Changes in cell attachment and/or cell morphology were found at 325 μg/mL and above at 18 and 28 hours preparation interval.
In the 3rd experiment in the absence of S9 mix. In the presence of S9 mix at 28 hours preparation interval no changes in cell attachment and/or cell morphology were found up to the highest applied concentration.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Summary of the results without S9 mix

 

Exp.

Schedule

Test groups

P

Genotoxicity [%]

Cytotoxicity

Exposure/ preparation period

Aberrant cells

Polyploid cells

Cell number [%]

Mitotic index [%]

incl. gaps

excl. gaps

with exchanges

 

 

 

2

 

 

 

4/18 h

Medium

-

6.5

3.5

0.5

0.0

100.0

100.0

40.6 µg/mL

-

n.d.

n.d.

n.d.

n.d.

104.3

n.d.

81.3 µg/mL

-

n.d.

n.d.

n.d.

n.d.

98.0

n.d.

162.5 µg/mL

-

6.0

1.5

1.0

0.0

111.8

122.1

325.0 µg/mL

-

5.5

2.0

0.5

0.0

89.1

106.3

650.0 µg/mL

+

4.5

3.5

1.0

0.0

37.4

85.8

1300.0 µg/mL

+

n.s.

n.s.

n.s.

n.s.

11.2

n.s.

Positive control

-

22.0

18.0

9.0

0.0

n.t.

93.2

 

 

 

 

 

3

 

 

 

 

18/18 h

Medium

-

6.0

4.0

0.5

0.5

100.0

100.0

40.6 µg/mL

-

4.5

2.0

0.5

0.0

106.4

139.6

81.3 µg/mL

-

4.0

0.5

0.5

0.0

99.7

111.1

162.5 µg/mL

-

4.0

3.0

0.5

0.0

100.2

84.5

325.0 µg/mL

-

n.s.

n.s

n.s

n.s

72.6

n.s

650.0 µg/mL

-

n.d.

n.s.

n.s.

n.s.

14.4

n.s.

1300.0 µg/mL

+

n.d.

n.s.

n.s.

n.s.

1.3

n.s.

Positive control

-

31.0

28.0

18.0

0.0

n.t.

78.7

 

 

 

 

 

3

 

 

 

 

18/28 h

Medium

-

5.5

3.0

0.0

0.0

100.0

100.0

162.5 µg/mL

-

n.d.

n.d.

n.d.

n.d.

89.9

n.d.

325.0 µg/mL

-

3.5

1.5

0.5

1.0

68.2

109.7

650.0 µg/mL

-

n.s.

n.s.

n.s.

n.s.

67.7

n.s.

1300.0 µg/mL

+

n.s.

n.s.

n.s.

n.s.

52.5

n.s.

Positive control

-

34.0

33.0

26.0

0.0

n.t.

99.0

P: precipitation determined at the end of exposure period (macroscopic); cytotoxicity expressed as relative values compared with the respective vehicle control; aberrant cells excluding/including gaps and including cells carrying exchanges; n.d.: not determined; n.s.: not scorable due to strong cytotoxicity and/or poor metaphase quality; n.t.: not tested; only sample of 100 metaphases evaluated in positive control due to strong clastogenicity

 

Table 2: Summary of the results with S9 mix

Exp.

Schedule

Test groups

P

Genotoxicity [%]

Cytotoxicity

Exposure/ preparation period

Aberrant cells

Polyploid cells

Cell number [%]

Mitotic index [%]

incl. gaps

excl. gaps

with exchanges

 

 

 

2

 

 

 

4/18 h

Medium

-

5.5

2.0

0.5

0.0

100.0

100.0

81.3 µg/mL

-

n.d.

n.d.

n.d.

n.d.

113.2

n.d.

162.5 µg/mL

-

n.d.

n.d.

n.d.

n.d.

108.8

n.d.

325.0 µg/mL

-

6.0

2.0

0.5

0.0

112.9

112.2

650.0 µg/mL

-

7.5

3.5

1.0

0.0

73.6

109.9

1300.0 µg/mL

-

6.0

3.0

1.0

0.0

28.4

57.3

Positive control

-

28.0

23.0

18.0

0.0

n.t.

72.3

 

 

 

 

 

3

 

 

 

18/18 h

Medium

-

4.0

2.5

1.5

0.0

100.0

100.0

81.3 µg/mL

-

n.d.

n.d.

n.d.

n.d.

91.6

n.d.

162.5 µg/mL

-

2.5

0.5

1.5

0.0

118.0

112.2

325.0 µg/mL

-

4.0

2.0

0.5

0.5

114.3

93.3

650.0 µg/mL

-

4.0

2.5

0.5

0.5

60.3

107.1

1300.0 µg/mL

-

n.s.

n.s.

n.s.

n.s.

12.7

n.s.

Positive control

-

33.0

32.0

19.0

0.0

n.t.

101.0

P: precipitation determined at the end of exposure period (macroscopic); cytotoxicity expressed as relative values compared with the respective vehicle control; aberrant cells excluding/including gaps and including cells carrying exchanges; n.d.: not determined; n.s.: not scorable due to strong cytotoxicity and/or poor metaphase quality; n.t.: not tested; only sample of 100 metaphases evaluated in positive control due to strong clastogenicity

 

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-06-22 - 2011-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study (GLP)
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
Adopted on July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
Adopted on Aug, 1998
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
1 part S9 (liver of 5 male Wistar rats pre-treated with 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally) fraction was mixed with 9 parts S9 supplement (cofactors)
Test concentrations with justification for top dose:
- First experiment: 40.6, 81.3, 162.5, 325.0, 650.0 and 1300.0 µg/mL (with and without S9 mix, 4 h exposure);
- Second experiment: 40.6, 81.3, 162.5, 325.0, 650.0 and 1300.0 µg/mL (without S9 mix, 24 h exposure); 250.0, 500.0, 1000.0 and 1300.0 µg/mL (with S9 mix, 4 h exposure)

- Doses were selected in this study based on the observations and the toxicity data of a previously performed dose finding test for an in vitro chromosome aberration assay.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium (Ham's F12)
- Justification for choice of solvent/vehicle: good solubility of the test substance in water.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9 mix: 300 μg/mL ethyl methanesulfonate (EMS; dissolved in Ham's F12 medium without FCS); with S9 mix: 20 μg/mL methylcholanthrene (MCA; dissolved in DMSO, diluted with medium without FCS).
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Day 1: seeding of the cells pretreated with "HAT" medium in 175 cm² flasks (1x10E6 cells in 20 mL) and in 25 cm² flasks (200 cells in 5 mL)

DURATION
- Exposure duration: 4 or 24 hours (day 2; after seeding)
- Expression time (cells in growth medium): 2-3 days; from day 1/2 (directly after treatment) to days 7-9 (second passage; first one on day 5)
- Selection time (if incubation with a selection agent): 6-7 days: from day 7-9 to day 16 (coupled with second passage)
- Fixation time (start of exposure up to fixation or harvest of cells): fixation at day 16 (drying, fixation, staining and counting of the selected colonies)

SELECTION AGENT (mutation assays): 6-thioguanine
STAIN: at the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

NUMBER OF REPLICATIONS: duplicates
The number of colonies in every flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized to the number of cells seeded (uncorrected MF), and corrected with the absolute cloning efficiency 2 (CE2).

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, viability; before the experiment (CE) after exposure (CE1; ca. 200 cells per dose group were seeded in 25 cm² flasks in duplicate using 5 mL Ham's F12 medium incl. 10% FCS. After an attachment period of 20 – 24 hours, the cells were treated with the vehicle, test substance or positive control for 4 hours or 24 hours. The exposure periods were completed by rinsing several times with HBSS. Then the flasks were topped up with 5 mL Ham's F12 medium incl. 10% FCS.) and after expression (CE2; 200 cells were separated during the transfer into selection medium and seeded in two flasks containing 5 mL Ham's F12 medium incl. 10% FCS each). After seeding of the cells the flasks were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted. The absolute and relative cloning efficiencies (%) were calculated for each test group.

OTHER:
- Changes in the pH were recorded; the pH was measured, at least for the two top doses and for the vehicle controls with and without S9 mix.;
- Osmolarity was measured, at least for the top dose and for the vehicle controls with and without S9 mix;
- Test substance precipitation was checked immediately after treatment of the test cultures and at the end of treatment;
- The test cultures of all test groups were examined microscopically at the end of exposure period with regard to cell morphology, which allows conclusions to be drawn about the attachment of the cells.
Evaluation criteria:
- The HPRT assay is considered valid if the following criteria are met: (1) the absolute cloning efficiencies of the negative/vehicle controls is not less than 50%; (2) the background mutant frequency in the negative/vehicle controls is within the historical negative control data range of 0 – 15.95 mutants per 10E6 clonable cells; (3) the positive controls both with and without S9 mix must induce distinctly increased mutant frequencies; (4) at least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions are tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.
- A finding is assessed as positive if the following criteria are met: (1) increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range; (2) evidence of reproducibility of any increase in mutant frequencies; (3) a statistically significant increase in mutant frequencies and the evidence of a dose-response relationship. Isolated increases of mutant frequencies above historical negative control range (i.e. 15 mutants per 10E6 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 substance is considered non-mutagenic if the corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.
Statistics:
Due to the clearly negative findings, a statistical evaluation was not carried out.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
no relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolizing system.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no cytotoxicity indicated by reduced relative cloning efficiency of about or below 20% relative survival was observed up to the highest required concentration.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was not influenced by test substance treatment.
- Effects of osmolality: osmolarity was not influenced by test substance treatment.
- Precipitation: no precipitation of the test substance was observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutation frequencies of the vehicle control groups were within our historical negative control data range including all vehicles used in our laboratory and, thus, fulfilled the acceptance criteria of this study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9 mix, after 4 and 24 hours treatment the morphology and attachment of the cells was adversely influenced from 650 μg/mL onward. In both experiments in the presence of metabolic activation there were no adverse observations on cell morphology (cell attachment).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 1: Summary of results

Exp.

no.

Exposure period [h]

Test groups [µg/mL]

S9 mix

P

Genotoxicity

MFcorr

[per 10E6 cells]

Cytotoxicity [%]

CE1

CE2

 

 

 

 

1

 

 

 

 

4

Vehicle control

-

-

2.48

100.0

100.0

40.6

-

-

n.c.

98.3

n.c.

81.2

-

-

n.c.

103.5

n.c.

162.5

-

-

2.16

101.4

95.8

325.0

-

-

1.92

92.7

96.5

650.0

-

-

6.14

87.8

96.8

13000.0

-

-

0.36

80.0

88.4

Positive control

-

-

96.87

90.8

85.1

 

 

 

 

2

 

 

 

 

24

Vehicle control

-

-

1.02

100.0

100.0

40.6

-

-

n.c.

95.4

n.c.

81.3

-

-

n.c.

101.0

n.c.

162.5

-

-

7.20

93.8

97.4

325.0

-

-

4.35

96.9

105.4

650.0

-

-

0.00

97.5

99.8

1300.0

-

-

1.54

88.8

105.4

Positive control

-

-

356.41

75.6

79.7

 

 

 

 

1

 

 

 

 

4

Vehicle control

+

-

4.63

100.0

100.0

40.6

+

-

n.c.

103.1

n.c.

81.3

+

-

n.c.

115.2

n..c

162.5

+

-

0.65

140.7

94.8

325.0

+

-

1.86

132.9

99.4

650.0

+

-

2.38

131.5

92.3

1300.0

+

-

1.25

130.9

100.0

Positive control

+

-

47.70

110.1

91.5

 

 

 

 

2

 

 

 

 

4

Vehicle control

+

-

1.89

100.0

100.0

250.

+

-

2.65

112.6

90.1

500.0

+

-

1.45

109.6

99.5

1000.0

+

-

3.06

109.3

96.3

1300.0

+

-

0.89

108.9

97.2

Positive control

+

-

53.48

101.6

89.3

The vehicle control contains 1% acetone; Exp. no.: experiment number; P: precipitation in culture medium at the end of exposure period; Mutant frequency MFcorr.: mutant colonies per 10E6 cells corrected with CE2 value; Cloning efficiency related to the respective vehicle control; n.c.: culture was not continued due to strong toxicity, or since a minimum of only 4 analysable concentrations are required.

 

Conclusions:
Interpretation of results (migrated information):
negative
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Deviations:
yes
Remarks:
only 2-AA used as positive control substance in the presence of S9-mix
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His-/Trp- gene
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100; as well as E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction (1 volume) prepared from levers of at least 5 male Wistar rats pre-treated by 80 mg/kg b.w. phenobarbital (i.p.) and β-naphthoflavone (orally) for 24 hours, mixed with S9 supplement (cofactors; 9 volumes)
Test concentrations with justification for top dose:
- 1st Experiment: 0, 20, 100, 500, 2500 and 5000 μg/plate (TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA; standard plate test with and without S9 mix)
- 2nd Experiment: 0, 0.8, 4, 20, 100 and 200 μg/plate (TA 98; standard plate test with S9 mix)
- 3rd Experiment: 0, 20, 100, 500, 2 500 and 5000 μg/plate (TA 1535, TA 100, TA 1537, E. coli; with and without S9 mix); 0, 20, 100, 500, 2500 and 5000 μg/plate (TA 98; without S9 mix); 0, 0.8, 4, 20, 100 and 200 μg/plate (TA 98; with S9 mix); all preincubation tests
- 4th Experiment: 0, 10, 50, 250, 1 250 and 2500 μg/plate (TA 98; standard plate test with S9 mix)
- 5th Experiment : 0, 8, 40, 200, 1 000 and 2000 μg/plate (TA 1535, TA 100, TA 1537) ; 0, 0.4, 20, 100, 500 and 1000 μg/plate (TA 98); all preincubation tests with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: dissolution (test substance formulations were prepared immediately before administration); 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
Remarks:
DMSO
True negative controls:
no
Remarks:
the Ames test is regularly conducted in the testing facility
Positive controls:
yes
Positive control substance:
other: in µg/plate, all dissolved in DMSO; without S9-mix: 5µg MNNG for TA1535&TA100, 10 µg NOPD for TA98, 100µg AAC for TA1537, 5 µg 4-NQO for WP2 uvrA; with S9-mix: 2.5 (all TA strains) or 60 µg (WP2 uvrA) 2-AA.
Remarks:
2-AA (2-aminoanthracene), MNNG (N-methyl-N'-nitro-N-nitrosoguanidine), NOPD (4-nitro-o-phenylenediamine), AAC (9-aminoacridine), 4-NQO (4-nitroquinoline-N-oxide).
Details on test system and experimental conditions:
1) METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 – 72 hours in the dark at 37°C

SELECTION AGENT (mutation assays): histidine/tryptophan deprivation

NUMBER OF REPLICATIONS: triplicates

2) METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 - 72 hours in the dark at 37°C

SELECTION AGENT (mutation assays): histidine/tryptophan deprivation

NUMBER OF REPLICATIONS: triplicates

3)
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (the bacterial colonies are counted). Toxicity detected by a (1) decrease in the number of revertants, (2) clearing or diminution of the background lawn (= reduced his- or trp- background growth), or (3) reduction in the titer, is recorded for all test groups both with and without S9 mix in all experiments.
Titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments).
Evaluation criteria:
- Acceptance criteria: the experiment is considered valid if (1) the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain; (2) the sterility controls revealed no indication of bacterial contamination; (3) the positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above; and (4) the titer of viable bacteria was ≥10E8/mL.
- Assessment criteria: the test chemical is considered positive if a dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system. A test substance is generally considered non-mutagenic if the number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
Statistics:
The mean number of revertant colonies per plate and the standard deviations were calculated for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100; as well as E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
No increase in the number of his+ or trp+ revertants compared to the negative control values
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
A bacteriotoxic effect (slight decrease in the number of revertants, reduction in the titer) was observed in the standard plate test from 2500 μg/plate onward / in the preincubation assay from 1000 μg/plate, depending on the strain and test conditions.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no test substance precipitation was found with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of revertant colonies in the negative controls was within the range of the historical negative control data for each 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 or above.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A distinct decrease in the number of his+ revertants (and a reduction in the titer) was observed in the 1st Experiment from 100 μg/plate onward and in the 2nd Experiment from 0.8 μg/plate onward which was the lowest applied dose. In the 4th Experiment which was performed to corroborated the contradictary data, bacterotoxicity was observed from 1250 μg/plate onward, nearby the range observed with the other tester strains (2500 to 5000 μg/plate). This value (1250 µg/plate), therefore, has to be regarded as the most reliable one. It has to be assumed that in the 1st and 2nd Experiment any technical error occurred while using S9 mix. Accordingly, bacteriotoxicity occurred at 2500 μg/plate (1st Experiment) in the experimental part without metabolic activation using the tester strain TA 98.

In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from 1000 μg/plate onward. Besides with the tester strain TA 98 bacteriotoxicity (reduced his- background growth, slight decrease in the number of his+ revertants, reduction in the titer) was observed from 100 μg/plate onward without metabolic activation and from 500 μg/plate onward with metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
negative
Endpoint conclusion
Endpoint conclusion:
no study available

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

- In vitro gene mutation (bacteria)

2,4-Diaminomethylcyclohexane was evaluated for its mutagenic potential according to the OECD test guideline 471 (BASF SE, 2009), based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA were used and the doses ranged from 0.8μg/plate to 5000μg/plate (standard plate test [SPT] and preincubation test [PIT]), both with and without metabolic activation (liver S9 mix from induced rats). No precipitation of the test substance was found up to 5000 µg/plate, whereas a bacteriotoxic effect was occasionally observed from 100μg/plate onward. A relevant increase in the number of his+ or 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 Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

The study was conducted according to the OECD test guideline 471 (GLP, Val 1), without any deviations, and is therefore considered reliable to assess the gene mutation potential of the test substance.

 

- In vitro mammalian gene mutation

The potential of the registered substance to induce gene mutation was also evaluated in mammalian CHO cells according to the OECD test guideline 476 (BASF SE, 2011). The evaluated concentrations ranged from 40.6 to 1300 µg/mL (the highest required concentration of approx. 10 mM)) for 4 hours (both with and without metabolic activation), or 24 hours (without metabolic activation) exposure times, about 6-8 days expression phase, and about 1 week selection period. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The test substance was not cytotoxic at any concentration levels evaluated, and the negative controls gave mutant frequencies within the range expected for the CHO cell line. Positive control substances, EMS and MCA, led to the expected increase in the frequencies of forward mutations, with corrected mutant frequencies within the historical positive control data range. No relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolizing system. In both experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies (MFcorr.: 0.00 - 7.20 per 10E6 cells) were close to the respective vehicle control values (MFcorr.: 1.02 - 4.63 per 10E6 cells) and clearly within the range of the historical negative control data (without S9 mix: MFcorr.: 0.00 - 15.95 per 10E6 cells; with S9 mix: MFcorr.: 0.00 - 15.68 per 10E6 cells).

Therefore, because the test substance did not lead to a relevant increase in the number of mutant colonies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other under the experimental conditions chosen here, it is not a mutagenic substance in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.

The test was conducted according to the OECD TG 476 and clearly shows no evidence that the test substance could have a gene mutation potential in mammalian CHO cells. Taken together with the negative Ames test, a mutagenic potential for the test substance is not anticipated.

 

- In vitro mammalian chromosome aberration

The potential of the registered substance to induce structural chromosomal aberrations (clastogenic activity) and/or changes in the number of chromosomes (aneugenic activity) was assessed in V79 cells in vitro both in the absence and the presence of a metabolizing system (BASF SE, 2011), according to the OECD TG 473. Based in the therein prescribed maximum testing concentration (10 mM), 31.3 to 1300 μg/mL test substance concentration in medium was evaluated. A sample of 100 metaphases for each culture was then analyzed for chromosomal aberrations, except for the positive control cultures where only 50 metaphases were scored due to clearly increased aberration rates.

The negative controls gave frequencies of aberrations within the range expected for the V79 cell line and both positive control substances,and cyclophosphamide, led to the expected increase in the number of cells containing structural chromosomal aberrations. The test substance did not cause any biologically relevant increase in the number of structurally aberrant metaphases incl. and excl. gaps at both sampling times either without S9 mix or after adding a metabolizing system in two valid experiments performed independently of each other. No relevant increase in the frequency of cells containing numerical chromosome aberrations as demonstrated either.

Thus, under the experimental conditions chosen here, the conclusion is drawn that the test substance is not a chromosome-damaging (clastogenic) substance under in vitro conditions using V79 cells in the absence and the presence of metabolic activation.

The study was conducted according to the OECD TG 473 without any deviation and is clearly sufficient for assessment of in vitro chromosomal aberration potential of the test substance in mammalian cells. Taken together with the available negative bacterial and mammalian gene mutations assays and with the absence of any further indication for such effect, a genetic toxicity potential for the test substance is not anticipated.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 618/2012).