Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames test): S. typhimurium TA 100, TA 1535, TA 98, TA 1537, TA 102: negative with and without metabolic activation
Gene mutation in mammalian cells (OECD 476, Mouse Lymphoma Assay): negative with and without metabolic activation
Chromosome aberration in mammalian cells (OECD 473, Chromosome aberration in vitro): negative with and without metabolic activation

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 Mar - 17 Apr 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Landesinstitut für Arbeitsschutz und Produktsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and beta-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment: 3.16, 10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
Experiment 1 (standard plate test): 3.16, 10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
Experiment 2 (pre-incubation test): 3.16, 10, 31.6, 100, 316, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: sodium azide (TA 100, TA 1535); 4-nitro-o-phenylenediamine (TA 98; TA 1537); methylmethanesulfonate (TA 102); with S9: 2-aminoanthracene (all strains without TA102; TA 102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: diminution of the background lawn or a reduction in the number of revertants

METABOLIC ACTIVATION SYSTEM:
The quality of the S9 homogenate was routinely tested in S. typhimurium using 2-aminoanthracene and benzo[a]pyrene
Evaluation criteria:
A test item is considered mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
Biologically relevant means:
- number of reversion is at least twice the control (TA 98, TA 100, TA 102)
- number of reversion is at least thrice the control (TA 1535, TA 1537)
Statistics:
Mean and standard deviation (no. of revertant colonies)
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed in any tester strain

RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed in a range-finding assay (plate incorporation), tested up to 5000 µg/plate in TA 98 and TA 100.

COMPARISON WITH HISTORICAL CONTROL DATA:
The data were in accordance the historical control data.

Table 1: Test results of experiment 1 (standard plate test).

EXPERIMENT 1 (Standard Plate Test, SPT)

S9-Mix

Without

 

Test item (µg/plate)

TA 100

TA 1535

TA 98

TA 1537

TA 102

Water

142±34.9

32±2.5

28±2.5

8±4.0

270±20.0

Control (DMSO)

97±15.1

16±1.5

25±5.2

11±4.0

227±9.5

31.6

108±12.9

24±6.0

31±2.1

10±3.6

196±16.3

100

107±9.1

22±4.0

25±3.8

8±2.6

212±11.1

316

113±16.5

20±1.2

29±3.1

8±3.5

217±9.8

1000

103±3.2

19±3.8

21±8.7

10±3.2

234±19.5

2500

111±19.7

21±2.1

18±5.1

7±2.1

219±9.1

5000

103±11.0

16±1.7

24±6.1

10±3.2

225±4.9

NOPD (10 µg)

-

-

424±34.8

86±10.5

-

NaN3 (10 µg)

1006±78.6

962±52.5

-

-

-

MMS (1 µL)

-

-

-

-

946±306.0

 

 

 

 

 

 

S9-Mix

With

 

 

 

 

 

 

Test item (µg/plate)

TA 100

TA 1535

TA 98

TA 1537

TA 102

Water

129±12.5

25±1.5

34±2.1

13±2.6

309±31.2

NC (DMSO)

123±3.6

26±2.3

22±9.6

8±1.7

259±11.0

31.6

123±5.5

24±6.4

30±4.6

8±1.7

263±39.7

100

120±7.9

26±1.7

27±2.9

10±5.5

277±13.2

316

117±18.9

20±4.6

31±4.7

11±1.7

249±20.5

1000

119±9.1

23±2.6

27±4.6

10±2.3

241±21.8

2500

118±19.6

28±2.6

28±3.6

11±2.1

279±10.1

5000

108±0.7

20±4.0

33±10.6

8±3.8

275±13.3

2-AA (2.5 µg)

1808±119.8

166±17.5

3160±201.7

375±11.0

547±68.7

 

Table 2: Test results of experiment 2 (preincubation assay).

EXPERIMENT 2 (preincubation assay)

S9-Mix

Without

 

Test item (µg/plate)

TA 100

TA 1535

TA 98

TA 1537

TA 102

Water

135±7.6

16±2.5

26±9.0

6±2.9

287±14.9

Control (DMSO)

126±9.5

12±3.2

18±1.0

5±2.3

221±2.5

31.6

92±6.7

17±3.1

21±3.5

8±3.5

238±14.2

100

116±25.4

13±3.2

19±6.1

9±3.5

254±4.9

316

98±12.1

12±4.7

21±1.5

5±1.5

229±12.2

1000

100±12.5

12±1.5

23±6.0

6±1.2

248±13.7

2500

101±5.1

14±5.1

22±4.0

5±5.1

258±33.2

5000

100±14.2

16±2.9

22±2.9

5±2.9

255±28.0

NOPD (10 µg)

-

-

470±35.9

61±30.3

-

NaN3 (10 µg)

1065±64.5

1152±105.1

-

-

-

MMS (1 µL)

-

-

-

-

1851±10.4

S9-Mix

With

 

 

 

 

 

 

Test item (µg/plate)

TA 100

TA 1535

TA 98

TA 1537

TA 102

Water

166±6.7

11±4.6

23±1.5

11±1.7

412±11.6

NC (DMSO)

130±0.0

10±1.2

32±2.0

5±3.1

323±6.1

31.6

146±13.7

14±4.6

33±4.5

5±2.0

296±15.0

100

146±7.8

11±3.6

28±7.8

5±1.0

303±44.8

316

121±20.6

17±4.6

25±5.7

4±0.6

310±12.1

1000

132±4.7

12±3.5

25±2.5

7±0.0

298±17.7

2500

149±9.5

10±2.3

34±2.5

6±2.1

309±26.6

5000

139±3.5

10±4.9

20±2.9

6±2.1

319±42.2

2-AA (2.5 µg)

2208±69.0

194±17.5

1949±348.5

186±30.4

671±23.7

 

Conclusions:
Interpretation of results:
negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Mar - 13 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Erlangen, Germany
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: cleansing medium: RPMI 1640 (supplemented with hypoxanthine, thymidine and glycine, and methotrexate);
complete culture medium: RPMI 1640 complete culture medium containing 10% horse serum, Penicillin/Streptomycin (per mL: 100 Units Pen./ 100 µg Strep.), 1mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 µg/mL amphotericin B
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital (80 mg/kg bw) and beta-naphthoflavone (100 mg/kg bw)
Test concentrations with justification for top dose:
Experiment I without and with metabolic activation:
0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM

Experiment II without and with metabolic activation:
0.8, 2.0, 4.0, 6.0, 7.0, 8.0, 9.0 and 10.0 mM
Vehicle / solvent:
- Vehicle/solvent used: RPMI medium + 5% horse serum (short-term exposure), RPMI medium + 7.5% horse serum (long-term exposure)
- Justification for choice of solvent/vehicle: solubility up to 10 mM
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Remarks:
+ S9 mix: benzo[a]pyrene in DMSO, final concentrations 2.5 µg/mL; - S9 mix: methylmethanesulfonate (MMS) in 0.9% NaCl, final concentrations 8 and 10 µg/mL; ethylmethanesulfonate (EMS) in RPMI, final concentration 200 and 300 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: pre-experiment (cytotoxicity): 4 h (±S9); experiment I: 4 h (±S9); experiment II: 4 h (+S9) and 24 h (-S9)
- Expression time (cells in growth medium): 48 h (incl. exposure period), cells were plated for determination of cloning efficiency and the mutation frequency in 96-well microtitre plates containing TFT selective medium. The microtitre plates were incubated 7 days (for viability determination) or 13- 14 days (for selection).
- Selection time (if incubation with a selection agent): 13-14 days


SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: two replicates in two independent experiments (main experiment)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, total and relative suspension growth, relative survival

OTHER EXAMINATIONS:
- Differentiation of colonies: small and large colonies were counted, as slow colony growth leading to the formation of small colonies is often correlated to extensive DNA damage
Evaluation criteria:
The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10E06 cells
- A dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations. According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result.

Acceptability:
The assay is considered acceptable if the following criteria were fulfilled (International Workshop on Genotoxicity Testing (IWGT)) (Moore et al., 2007):
- At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable.
- The cloning efficiency of the negative and/or solvent controls is in the range 65% -120%.
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 10E06 cells
- The cell number of the negative/solvent controls should undergo 8-32-fold increase during a 2-day growth period (short-term treatment) or 32-180-fold increase during a 3-day growth period (long-term treatment).
- The clastogenic positive controls (MMS, EMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 10E06 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 10E6 cells The RTG must be greater than 10%.
Key result
Species / strain:
mouse lymphoma L5178Y cells
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 applicable
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test substance was sufficiently soluble in medium.
- Precipitation: No precipitation was observed.

COMPARISON WITH HISTORICAL CONTROL DATA: The solvent controls and positive controls were in the range of historical data of the test facility.

RANGE-FINDING/SCREENING STUDIES: No cytotoxicity was observed with or without S9-mix after treatment with up to 10 mM test substance for 4 h.

Table 1: Results of experiment 1.

Treatment

(mM)

4 hours (-S9)

Treatment

(mM)

4 hours (+S9)

 

RSG

[%]

RTG

[%]

CE

MF

IMF

 

RSG

[%]

RTG

[%]

CE

MF

IMF

0

100

100

96.5-116.0

80.7-93.5

-

0

100

100

74.8-80.5

112.1-116.4

-

0.1

94.9

87.6

98.0

104.4

17.3

0.1

107.7

122.0

88.0

112.8

-1.5

0.2

84.9

69.2

86.6

87.9

0.8

0.2

99.7

104.8

81.6

125.4

11.1

0.5

95.0

87.7

98.0

88.8

1.7

0.5

97.8

112.5

89.3

126.0

11.8

1.0

108.0

88.0

86.6

123.9

36.8

1.0

103.8

131.1

98.0

110.0

-4.2

2.5

99.3

83.4

89.3

120.1

33.0

2.5

96.0

123.2

99.6

116.0

1.7

5.0

97.9

94.8

102.9

106.1

19.0

5.0

101.8

126.6

96.5

91.7

-22.6

7.5

96.8

83.9

92.1

117.0

29.9

7.5

98.7

105.3

82.9

143.5

29.3

10

94.5

80.6

90.7

103.1

16.0

10

104.5

159.0

118.1

64.3

-50.0

 

 

 

 

EMS

 

 

 

 

 

B[a]P

 

 

 

 

 

300 µg/mL

80.0

62.3

82.9

594.2

507.1

2.5 µg/mL

66.2

65.7

77.0

554.4

440.1

MMS

 

 

 

 

 

 

 

 

 

 

 

10 µg/mL

65.4

46.7

75.9

387.6

300.5

 

 

 

 

 

 


  Table 2: Results of experiment 2.

Treatment

(mM)

24-Hours (-S9)

Treatment

(mM)

4-Hours (+S9)

 

RSG

[%]

RTG

[%]

CE

MF

IMF

 

RSG

[%]

RTG

[%]

CE

MF

IMF

0

100

100

93.5-95.0

51.9-68.4

-

0

100

100

81.6-84.1

70.7-95.3

-

0.8

108.5

98.3

85.4

62.9

2.8

0.8

105.7

112.2

88.0

41.5

-41.5

2.0

99.1

85.8

81.6

60.4

0.2

2.0

107.4

101.3

78.1

41.3

-41.7

4.0

91.2

81.4

94.1

98.0

37.8

4.0

102.0

115.1

93.5

45.1

-37.9

6.0

88.7

85.3

90.7

60.8

0.7

6.0

105.3

111.8

88.0

53.2

-29.9

7.0

83.1

86.4

98.0

82.1

21.9

7.0

104.2

105.8

84.1

68.3

-14.7

8.0

84.1

65.8

73.7

68.7

8.5

8.0

107.6

106.0

81.6

74.7

-8.3

9.0

81.9

76.4

88.0

60.9

0.7

9.0

107.9

118.1

90.7

57.7

-25.3

10

79.5

77.6

92.1

75.9

15.7

10

111.7

118.6

88.0

66.4

-16.7

 

 

 

 

EMS

 

 

 

 

 

B[a]P

 

 

 

 

 

300 µg/mL

 

27.9

57.9

1681.4

1621.3

2.5 µg/mL

65.0

52.3

66.7

655.7

572.7

MMS

 

 

 

 

 

 

 

 

 

 

 

10 µg/mL

 

34.3

54.7

971.3

911.2

 

 

 

 

 

 

RSG: relative suspension growth

RTG: relative total growth

CE: cloning efficiency

MF: mutant frequency

IMF: induced mutant frequency

Conclusions:
Interpretation of results:
negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Jul 2012 - 04 Oct 2013
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Erlangen, Germany
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:
Complete Culture Medium
MEM medium supplemented with:
10% foetal bovine serum (FBS), 100 U/100 µg/mL penicillin/streptomycin solution, 2 mM L-glutamine, 2.5µg/mL amphotericin, 25 µM HEPES

Treatment Medium (short-time exposure):
Complete culture medium without FBS.

After Treatment Medium / Treatment Medium (long-time exposure):
Complete culture medium with 10% FBS.

Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital (80 mg/kg bw) and beta-naphthoflavone (100 mg/kg bw)
Test concentrations with justification for top dose:
Experiment I:
without metabolic activation: 0.16, 0.31, 0.63, 1.25, 2.5, 5.0 and 10.0 mM
with metabolic activation: 0.31, 0.63, 1.25, 2.5, 5.0 and 10.0 mM

Experiment II:
without metabolic activation: 0.75, 1.5, 2.5, 5.0, 7.5 and 10.0 mM
with metabolic activation: 1.5, 3.0, 5.0, 6.0 and 10.0 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Culture medium (MEM without FBS)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide, 0.83 µg/mL in nutrient medium, +S9; ethylmethanesulfonate, 400 (24 h treatment) and 900 µg/mL (4 h treatment) in nutrient medium; –S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9-mix) and 20 h (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.2 µg/mL) was added 17.5 h after start of the treatment
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 independent experiments

NUMBER OF CELLS EVALUATED: at least 200 well spread metaphases per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (number of mitotic cells in 1000 cells); rlative cell density (cells within the visual field at a 400-fold magnification)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
EVALUATION CRITERIA:
Criteria for determining a positive result:
- a concentration-related increase or reproducible increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 4.0% aberrant cells (without and with metabolic activation)).

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results. A statistical evaluation could be used as an aid for interpretation of the results.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group.

ACCEPTABILITY CRITERIA:
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
- the number of aberrations found in the negative and/or solvent controls falls within the range of historical laboratory control data: 0.0% - 4.0% (without and with metabolic activation)
- the positive control substance should produce biologically relevant increases in the number of cells with structural chromosome aberrations.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test substance was noted

RANGE-FINDING/SCREENING STUDIES: In a pre-experiment no cytotoxic effects were noted (decrease of relative mitotic index or relative cell density) were noted in all dose groups evaluated (up to 10 mM) in the presence and absence of metabolic activation.

COMPARISON WITH HISTORICAL CONTROL DATA: Data were provided for the negative and positive controls. All results were within the range of the historical controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxic effects (no biologically relevant decrease of the relative mitotic index, or the relative cell density below 70%) were noted without and with metabolic activation in all dose groups evaluated in experiment I and II.

Table 1:         Experiment I – Summary of Aberration Rates

 

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

[mM]

in %

with gaps

without gaps

Exposure period 4h, fixation time 20h, without S9 mix

C

-

100

3.5

1.5

EMS

900 µg/mL

96

12.5

10.5

Test substance

2.5

113

2.5

1.0

5

123

2.5

2.0

10

97

2.0

1.0

Exposure period 4h, fixation time 20h, with S9 mix

C

-

100

5.0

3.5

CPA

0.83 µg/mL

111

13.5

10.0

Test substance

2.5

111

3.5

2.0

5

109

8.5

5.0

10

113

3.5

2.0

 

 Table 2:         Experiment II – Summary of Aberration Rates

 

Test item

Concentration

Mitotic Index

Aberrant cells in %

 

[mM]

in %

with gaps

without gaps

Exposure period 20h, fixation time 20h, without S9 mix

C

-

100

2.0

1.5

EMS

400 µg/mL

99

10.5

9.0

Test substance

2.5

103

4.5

1.0

5

103

1.5

0.0

10

102

5.0

2.5

Exposure period 4h, fixation time 20h, with S9 mix

C

-

100

4.5

2.5

CPA

0.83 µg/mL

82

20.0

17.5

Test substance

3

98

3.0

1.5

6

81

3.0

1.5

10

96

5.0

2.5

200 cells evaluated for each concentration.

C:       Negative Control (Culture Medium)

EMS:   Positive Control (without metabolic activation: Ethylmethanesulfonate)

CPA:    Positive Control (with metabolic activation: Cyclophosphamide)

 

 

Conclusions:
Interpretation of results:
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Mutagenicity in bacteria

Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid was tested for its mutagenic properties in bacteria in a study according to OECD guideline 471 and in compliance with GLP (Scheib, 2012). S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were treated with the test substance diluted in DMSO up to 5000 µg/plate in 2 independent experiments (standard plate test and pre-incubation test) in the presence and absence of metabolic activation (S9-mix). No precipitation was observed up to 5000 µg/plate and no cytotoxicity was noted in any tester strain at any concentration up to 5000 µg/plate. No increase in the number of revertants was observed with the test substance in any strain at any concentration. The solvent control and the positive controls were valid. In conclusion, the test substance was not mutagenic in bacteria in the presence and absence of metabolic activation under the conditions of the study.

Mutagenicity in mammalian cells

The potential mutagenicity of Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line was assessed in a GLP-compliant study conducted according to OECD guideline 476 (Trenz, 2013). No cytotoxicity was observed with or without S9-mix after treatment with up to 10 mM test substance for 4 h in a range-finding assay. In the main experiment L5178Y TK +/- mouse lymphoma cells were treated with the test material with and without metabolic activation system (S9 mix) in two independent experiments. Vehicle (RPMI medium + horse serum) and positive controls were included. In the first experiment, cells were exposed to the test material for 4 h in the presence and absence of S9 mix at concentrations of 0.1, 0.2, 0.5, 1.0, 2.5, 5.0, 7.5, and 10 mM. In the second experiment, cells were treated for 4 h with metabolic activation and for 24 h without metabolic activation at concentrations of 0.8, 2, 4, 6, 7, 8, 9, and 10 mM.

No cytotoxic effects were noted in the presence and absence of S9-mix. The test material did not induce any toxicologically significant concentration-related increases in the mutant frequency at any concentration, either with or without metabolic activation. No precipitation was observed up to the maximum concentration of 10 mM. The vehicle controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency, demonstrating the sensitivity of the test system and the efficacy of the metabolic activation system.

The test material was thus considered to be non-mutagenic to L5178Y cells under the conditions of the test.

Clastogenicity in vitro

Clastogenic effects of Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid were assessed in a chromosome aberration test in V79 cells conducted according to OECD guideline 473 (Zeller, 2013). Chinese hamster fibroblasts (V79) were exposed to 0.16 - 10 mM or 0.31 - 10 mM test substance in the presence and in the absence of metabolic activation, respectively. For microscopic analysis 2.5, 5, and 10 mM (Experiment I and II without S9; Experiment I with S9) and 3, 6, and 10 mM (Experiment 2 with S9) were chosen.Positive and negative (medium) control cultures were included in each assay and historical control data were provided. No precipitation and no cytotoxicity was observed at any concentration.

In experiment I without metabolic activation the aberration rate of the negative control (1.5%) and all dose groups treated with the test item (2.5 mM and 10 mM 1% and 5 mM 2%) were within the historical control data of the testing facility (0.0% – 4.0%).

With metabolic activation, the aberration rates of the negative control (3.5%) and the treated dose groups 4 and 6 at 2.5 mM and 10 mM (2.0%) were within the historical controls. An increase of aberrant cells was noted at a concentration of 5.0 mM (5% chromosome aberration). Based on the increased single value in experiment I a clear assessment was not possible. To verify the observed effects an independent repetition of the experiment was performed (second experiment with and without metabolic activation).

In experiment II without metabolic activation the aberration rate of the negative control (1.5%) and all dose groups treated with the test item (2.5 mM (1.0%), 5.0 mM (0.0%) and 10 mM (2.5%)) were within the historical control data of the testing facility (0.0% – 4.0%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. Herewith the increase of the aberration rate in experiment I was not confirmed in experiment II.

With metabolic activation the aberration rates of the negative control (2.5%) and all dose groups treated with the test item (3.0 mM and 6.0 mM (1.5%), and 10.0 mM (2.5%)) were within the historical control data of the testing facility (0.0% – 4.0%). The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding negative control. In addition, no dose-response relationship was observed. Herewith the increase of the aberration rate in experiment I was not confirmed in experiment II.

EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

In conclusion, the test item is considered to be non-clastogenic under the conditions of the experiment. 

Justification for classification or non-classification

The available data on genetic toxicity of Reaction mass of 2-(3,4-dimethyl-1H-pyrazol-1-yl)succinic acid and 2-(4,5-dimethyl-1H-pyrazol-1-yl)succinic acid do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.