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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative results in Salmonella typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535, with and without metabolic activation (OECD 471, GLP).
Negative results in mammalian chromosomal aberration test with cultured peripheral human lymphocytes (OECD 473, GLP).
Negative results in mammalian cell gene mutation tests using Chinese hamster lung fibroblasts (V79), with and without metabolic activation (OECD 476, GLP).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
HPRT locus
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM medium supplemented with 10% fetal bovine serum and 1% neomycin
Metabolic activation:
with and without
Metabolic activation system:
co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/ß-naphtoflavone
Test concentrations with justification for top dose:
Experiment I
Without S9 mix: 0.25, 0.5, 1, 2 and 4 µg/mL (4 h)
With S9 mix: 4, 8, 16, 32 and 48 µg/mL (4 h)
Experiment II
Without S9 mix: 4, 8, 16, 64 and 96 µg/mL (24 h)
With S9 mix: 4, 8, 16, 48 and 56 µg/mL (4 h)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethane sulfonate, 150 µg/mL in nutrient medium, without S9; 7,12-dimethylbenz(a)anthracene, 1.1 µg/mL in DMSO, with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
4 h exposure with and without S9 mix
4 h exposure with S9 mix and 24 h without S9 mix
- Expression time (cells in growth medium): The expression time in growth medium was 7 days. For the selection of mutant cells the complete medium was supplemented with 11 µg/mL thioguanine.
- Selection time (if incubation with a selection agent): 2 days (experiment I) or 3 days (experiment II) after sub-cultivation stock cultures were trypsinized.
- Fixation time (start of exposure up to fixation or harvest of cells): 9-10 days

SELECTION AGENT (mutation assays): 11 µg/mL thioguanine

STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: 5 replications each in two independent experiments

DETERMINATION OF CYTOTOXICITY: cloning efficiency and relative total growth
- Method: mitotic index
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value is < 0.05.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥4 µg/mL (experiment I, -S9), ≥ 48 µg/mL (experiment I,+ S9); 96 µg/mL (experiment II, -S9, 24 h), 56 µg/mL (experiment II, +S9, 4 h)
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: In the preliminary test, the pH of the culture medium was adjusted at concentrations of 1900 and 3800 µg/mL with 2 N NaOH. In the main experiments no pH adjustment was necessary.
- Precipitation: precipitation occurred at 237.5 µg/mL an above after 24 h without metabolic activation. Following 4 h treatment no precipitation was noted.

RANGE-FINDING/SCREENING STUDIES: Concentrations between 29.7 and 3800 µg/mL were used to evaluate toxicity in the presence (4 h) and absence (4 h and 24 h treatment) of metabolic activation. After 24 h treatment without metabolic activation the cell growth was completely inhibited at 118.8 mg/L and above. Following 4 htreatment with and without metabolic activation the cell growth was already completely inhibited at a concentration of 29.7 µg/mL. Therefore, the preliminary experiment was repeated with concentrations of 0.23 to 30.0 µg/mL with and without metabolic activation. In the repeated preliminary experiment strong cytotoxic effects occurred only in the absence of metabolic activation at 7.5 µg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA: All mutant frequencies were within the historical range of solvent controls. A single exception occurred in experiment I at 2.0 µg/mL. The induction factor (4.0) exceeded the threshold of three times the corresponding solvent control and the range of the historical solvent control data is exceeded (44.0 mutant colonies/10E6 cells compared to 0.6 – 32.4 mutant colonies/10E6 cells of the historical solvent control data). However, the induction factor of the parallel culture remained below the threshold and this event is therefore considered to be not reproducible and of no biological relevance.

Table 1. Results of experiment I without metabolic activation.

Concentration
[µg/mL]

Cloning efficiency relative [%] (Survival)

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

Cloning efficiency relative [%]

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

 

Culture I

Culture II

0 (Acetone)

100

100

11

100

100

12,9

EMS 150

95,8

75,0

209,9

96,5

104,9

117,0

0,25

98,6

88,0

5,1

92,5

73,7

7,5

0,5

99,2

96,6

11,7

97,6

97,0

17,4

1

100

112,5

7,3

97,8

78,8

14,8

2

97,2

108,9

44,0

97,2

104,8

11,2

4

91,4

10,4

0,0

89,7

8,3

4,0

6

-*

-*

8

-*

-

*: culture was not continued due to strong toxic effects.

**: culture was not continued since a minimum of only four analysable concentrations is required.

Table 2. Results of experiment I with metabolic activation.

Concentration
[µg/mL]

Cloning efficiency relative [%] (Survival)

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

Cloning efficiency relative [%]

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

 

Culture I

Culture II

0 (Acetone)

100

100

29,1

100

100

20,7

DMBA 1.1

80,7

80,2

665,9

86,6

95,5

411,3

2

96,0

116,6

**

98,0

105,4

**

4

92,7

98,2

28,1

95,0

100,3

10,5

8

88,5

110,7

5,2

89,4

118,5

22,5

16

90,6

141,1

11,6

94,5

100,3

18,4

32

87,3

95,0

13,0

85,3

88,8

23,6

48

41,3

40,7

29,8

12,1

12,5

16,8

64

-*

-*

*: culture was not continued due to strong toxic effects.

**: culture was not continued since a minimum of only four analysable concentrations is required.

Table 3. Results of experiment II without metabolic activation.

Concentration
[µg/mL]

Cloning efficiency relative [%] (Survival)

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

Cloning efficiency relative [%]

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

 

Without S9 mix

Without S9 mix

0 (Acetone)

100

100

14,6

100

100

13,5

EMS 150

81,0

133,5

224,7

110,0

129,3

260,1

2

102,3

98,9

**

109,7

107,6

**

4

102,8

136,3

9,7

111,8

82,0

11,1

8

103,1

115,3

17,9

111,3

93,5

16,3

16

101,9

114,3

17,3

100,9

104,9

17,8

64

91,2

164,5

11,8

102,6

60,1

9,5

96

24,1

32,9

15,0

25,5

23,9

22,1

128

-

-*

*: culture was not continued due to strong toxic effects.

**: culture was not continued since a minimum of only four analysable concentrations is required.

Table 4. Results of experiment II with metabolic activation.

Concentration
[µg/mL]

Cloning efficiency relative [%] (Survival)

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

Cloning efficiency relative [%]

Cell density in % of control [%]

Mutants per 1E+06 surviving cells

 

With S9 mix

With S9 mix

0 (Acetone)

100

100

10,5

100

100

10,2

DMBA 1.1

81,0

68,4

863,3

28,0

73,7

743,3

2

102,3

62,0

**

91,0

92,4

**

4

102,8

63,2

5,4

91,7

90,7

11,2

8

103,1

91,2

8,4

93,1

97,6

4,4

16

101,9

101,5

15,4

90,4

100,6

8,6

48

91,2

43,5

17,4

0

70,9

10,0

56

24,1

3,2

7,1

0

4,3

4,1

64

-*

-*

*: culture was not continued due to strong toxic effects.

**: culture was not continued since a minimum of only four analysable concentrations is required.

Conclusions:
Interpretation of results:
negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 May - 25 May 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Rheinland-Pfalz, Mainz, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
other: TA 97a, TA 98, TA 100, TA 102, TA 1535
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
First experiment: 50, 150, 500, 1501 and 5004 µg/plate with and without metabolic activation
Second experiment: 1250, 2500 and 5001 µg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylene diamine (NPD; 80 µg/plate, -S9, TA98, TA97a and TA102); sodium azide (6 µg/plate, -S9, TA100 and TA1535), benzo-a-pyrene (BaP; 40 µg/plate, +S9, TA98), 2-Amino-anthracene (2-AA; 3 µg/plate, +S9, TA97a, TA100, TA102 and TA1535 )
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, first experiment); preincubation (second experiment)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 4 replications each in one independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: titre determination; the titre control should reach a concentration of at least 10E9 bacteria/mL (100 colonies/plate)

Evaluation criteria:
The test material may be considered as mutagen if at least one of the used strain (with or without metabolic activation) shows an increase of the revertant colonies over 2 times of the negative control (induction rate I ≥ 2) and/or a concentration-dependent relationship is apparent.
Statistics:
Mean values and standard deviation were calculated.
Species / strain:
other: TA97a, TA98, TA100, TA102 and TA1535
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:
COMPARISON WITH HISTORICAL CONTROL DATA: The mutation rates of the positive controls were lower than the stated rates of Prof. Ames. However, the rates were within the range of the historical control data of the Laus GmbH. The rates of the spontaneous revertants were partly lower than the stated rates of Prof. Ames. However, the rates were within the ranges of the historical control data of the Laus GmbH.

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA102

TA98

TA97a

 

Water

154 ± 20.2

16

 ±8.4

n.d.

n.d.

n.d.

DMSO

132 ± 25.4

16 ± 5.8

153 ± 18.2

12 ± 2.1

120 ± 18.1

50

126 ± 19

12 ± 6

165 ±15

8 ±1

108 ± 3

150

155 ± 9

10 ± 3

161 ±45

15 ± 4

89 ± 10

500

135± 22

6 ± 5

146 ± 21

9 ± 4

128 ±10

1501

140±12

9 ± 1

161 ± 13

7 ± 2

127 ±32

5004

158 ± 22

14 ± 4

82 ± 55

9 ± 5

121 ± 54

Positive controls, –S9

Name

SA

SA

4NPD

4NPD

4NPD

Concentrations

(μg/plate)

6

6

80

80

80

Mean No. of colonies/plate

(average of 3 ± SD)

1001

1001

1001

1001

1001

 

Water

n.d.

n.d.

n.d.

n.d.

n.d.

+

DMSO

140 ± 17.4

13 ± 3.4

145 ± 37.3

15 ±1.4

120 ± 18.1

+

50

125 ± 7

12 ± 6

158 ± 42

10 ± 3

115 ± 12

+

150

146± 13

12 ± 4

105 ± 61

8 ± 1

143 ± 18

+

500

166 ± 30

13 ± 5

152 ± 21

8 ± 2

149 ± 36

+

1500

143 ± 43

11 ± 6

141 ± 27

8 ± 2

151 ± 24

+

5000

0 148 ± 28

11 ± 3

166 ± 28

8 ± 2

144 ± 28

Positive controls, +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

3

3

3

40

3

Mean No. of colonies/plate

(average of 3 ± SD)

1001

1001

1001

150 ± 7

1001

1001 = more than 1000 colonies

4NPD = 4-Nitro-1,2-phenylendiamin

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

Table 2. Results of experiment 2 (pre-incubation).

With or without S9-Mix

Test substance concentration

(μg/plate)

Mean number of revertant colonies per plate

(average of 3 plates ± Standard deviation)

Base-pair substitution type

Frameshift type

TA 100

TA1535

TA102

TA98

TA97a

 

Water

137 ± 30

12 ± 1

n.d.

n.d.

n.d.

DMSO

180 ± 9.5

12 ± 2.4

147 ± 29.6

9 ± 2.9

161 ± 65.9

1250

122 ± 32

9 ± 2

126 ± 9

9 ± 1

125 ± 22

2500

95 ± 15

11 ± 3

132 ± 46

6 ± 1

138 ± 27

5001

113 ± 12

11 ± 4

106 ± 48

7 ± 2

129 ± 15

Positive controls. –S9

Name

SA

SA

4NPD

4NPD

4NPD

Concentrations

(μg/plate)

6

6

80

80

80

Mean No. of colonies/plate

(average of 3 ± SD)

1001

1001

1001

1001

1001

 

Water

n.d.

n.d.

n.d.

n.d.

n.d.

+

DMSO

174 ± 31.7

13 ± 2.9

117 ± 36.8

11 ± 3.7

132 ± 35.3

+

1250

143 ± 18

9 ± 1

136 ± 17

7 ± 3

159 ± 11

+

2500

142 ± 27

9 ± 2

154 ± 12

7 ± 1

116 ± 6

+

5001

130 ± 13

10 ± 2

68 ± 56

7 ± 2

131 ± 13

Positive controls. +S9

Name

2AA

2AA

2AA

BP

2AA

Concentrations

(μg/plate)

3

3

3

40

3

Mean No. of colonies/plate

(average of 3 ± SD)

1001

1001

1001

1001

1001

1001 = more than 1000 colonies

4NPD = 4-Nitro-1,2-phenylendiamin

2AA = 2-Aminoanthracene

BP = Benzo(a)pyrene

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:
13 Jul 2009 - 19 Feb 2010
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
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Rheinland-Pfalz, Mainz, Germany
Type of assay:
other: in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: in whole blood treated with heparin
Details on mammalian cell type (if applicable):
RPMI 1640 medium supplemented with
- 15% (v/v) fetal calf serum (FCS)
- 1% (v/v) penicillin/streptomycin (10000 U /10000 µg/mL)
- 4.8 µg/mL Phytohaemagglutinin solution in H2O
During exposure to the test substance, RPMI medium was used without FCS supplementation.
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
Pre-Experiment I:
4h treatment: 55.3, 110.6, 221.3, 442.5, 885, 1770 and 3540 µg/mL, with and without metabolic activation.
Pre-Experiment II:
4h treatment: 0.78, 1.56, 3.125, 6.25, 12.5, 25 and 50 µg/mL, without metabolic activation.

Experiment I:
4h treatment: 10, 15, 20, 25, 30, 35 and 40 µg/mL, with and without metabolic activation.
Experiment II:
4h treatment: 10, 15, 20, 25, 30, 35 and 40 µg/mL, with metabolic activation.
22h treatment: 0.5, 1, 5, 10, 15, 20, 25, 30, 40 an 50 µg/mL, without metabolic activation.

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: cyclophosphamide, 35 µg/mL in 0.9% NaCl, +S9; methanesulphonate, 600 and 350 µg/mL (experiment I and II, respectively) in nutrient medium, –S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 and 22 h
- Fixation time (start of exposure up to fixation or harvest of cells): 22 h
SPINDLE INHIBITOR (cytogenetic assays): colchicine 0.1 pg/mL
STAIN (for cytogenetic assays): Giemsa 10% (v/v) in Soerensen buffer
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
A test item is classified as non-mutagenic if: the number of induced structural chromosome aberrations in all evaluated dose groups is below 5.0 % aberrant cells, excluding gaps. No significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if: the number of induced structural chromosome aberrations is above 5.0 % aberrant cells, excluding gaps and either a concentration-related or a significant increase in the number of cells with structural chromosome aberrations is observed.

Statistics:
Statistical significance was calculated by means of the Fisher's exact test.
Species / strain:
lymphocytes: in whole blood treated with heparin
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 30 µg/mL without and from 40 µg/mL with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: was observed from 221.3 µg/mL and above with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. The applied concentrations ranged from 55.3 µg/mL to 3540 µg/mL with and without metabolic activation (exposure time 4 h, pre-experiment I). In pre-experiment II concentrations from 0.78 to 50 µg/mL were used.

ADDITIONAL INFORMATION ON CYTOTOXICITY: All concentraitons of the pre-experiment I showed cytotoxicity.

Table 1. Results of cytogenicity experiments.

Test item

Concentration

Mitotic Index

 

Aberrant cells in %

 

 

in µg/mL

in %

with gaps

without gaps

with exchanges

 

Exposure period 4h, fixation time 22h, without S9 mix

DMSO

0.5% (v/v)

100.0

5

1

0

EMS

600

48.4

23

21.5*

2.5

Test substance

10

94.0

2.5

1.5

0

20

70.2

3

2

0.5

30

50.4

0.5

0

0

 

Exposure period 4h, fixation time 22h, with S9 mix

DMSO

0.5% (v/v)

100

2

2

0

CPA

35

77.2

21.5

16.5*

0.5

Test substance

15

82.6

2.5

2

0

30

39.1

2.5

1.5

0.5

40

18.8

3

2

0

 

Exposure period 22h, fixation time 22h, without S9 mix

DMSO

0.5% (v/v)

100.0

0

0

0

EMS

350

44.8

39.5

33.5*

4.5

Test substance

50

97.1

5.5

2

0

100

77.6

3.5

2

0

125

36.8

2

1

0

 

Exposure period 4h, fixation time 22h, with S9 mix

DMSO

0.5% (v/v)

100.0

1.5

1.5

0

CPA

35

70.1

33

22*

1

Test substance

25

94.8

3.5

2

0

35

69.7

3.5

3

0

40

29.5

1

1

0

*:Aberration frequency statistically significant higher than corresponding control values

CPA: Cyclophosphamide

EMS: Ethyl methanesulphonate

Conclusions:
Interpretation of results:
negative
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions. No E.coli strain or S. typhimurium TA102 is included.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
no E. coli or TA102 was included
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
First experiment: 20, 100, 500, 2500 and 5000 µg/plate with and without metabolic activation (plate incorporation)
Second and third experiment: 3, 6, 12, 25 and 50 µg/plate with metabolic activation (plate incorporation)
0.8, 4, 20, 100 and 500 µg/plate without metabolic activation (pre-incubation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N´-nitro-N-nitroso-guanidine (MNNG; 5 µg/plate in water, -S9, TA100 and TA 1535); 4-nitro-o-phenylendiamine (10 µg/plate in DMSO, -S9, TA 98); 9-aminoacridine chloride monohydrate (100 µg/plate in DMSO, -S9, TA1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (10 µg/plate in DMSO, +S9, all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, experiment I and II) and preincubation (experiment III)

DURATION
- Preincubation period: 20 min

NUMBER OF REPLICATIONS: triplicates of each dose and control

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn
Evaluation criteria:
The test material may be considered positive in this test system if the following criteria are met:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.
Statistics:
Mean values and standard deviations were calculated.
Species / strain:
other: TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 50 µg/plate onwards
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid

Table 1. Test results of experiment 1 (plate incorporation).

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA1535

TA98

TA1537

Solvent

135 ± 5

15 ± 4

28 ± 2

8 ± 3

20

95 ± 6

14 ± 2

27 ± 6

14 ± 4

100

13 ± 3

B

20 ± 7

3 ± 2

500

B

B

7 ± 1

6 ± 4

2500

B

B

2 ± 1

B

5000

B

B

B

B

Positive controls, –S9

Name

MNNG

MNNG

NPD

AAC

Concentrations (μg/plate)

5

5

10

100

Mean No. of colonies/plate (average of 3 ± SD)

1630 ± 270

1340 ± 61

614 ± 49

973 ± 188

+

Solvent

119 ± 8

17 ± 3

38 ± 5

9 ± 2

+

20

115 ± 6

12 ± 3

38 ± 7

14 ± 1

+

100

114 ± 1

13 ± 4

46 ± 10

10 ± 5

+

500

28 ± 18

2 ± 2

30 ± 8

4 ± 2

+

2500

B

B

21 ± 2

3 ± 0

+

5000

B

B

10 ± 7

B

Positive controls, –S9

Name

2AA

2AA

2AA

2AA

Concentrations (μg/plate)

10

10

10

10

Mean No. of colonies/plate (average of 3 ± SD)

1880 ± 101

128 ± 20

1617 ± 64

125 ± 5

B = Reduced background lawn

MNNG = N-methyl-N´-nitro-N-nitroso-guanidine

2AA = 2-Aminoanthracene

NPD = 4-nitro-o-phenylendiamine

AAC = 9-aminoacridine

Table 2. Test results of experiment 2 (plate incorporation).

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA1535

TA98

TA1537

Solvent

106 ± 8

21 ± 5

28 ± 2

9 ± 2

3

102 ± 8

18 ± 8

30 ± 4

9 ± 2

6

109 ± 5

20 ± 3

30 ± 3

15 ± 4

12

98 ± 4

20 ± 3

40 ± 3

9 ± 3

25

104 ± 7

17 ± 5

32 ± 5

12 ± 1

50

57 ± 7

10 ± 2

29 ± 4

7 ± 0

Positive controls, –S9

Name

MNNG

MNNG

NPD

AAC

Concentrations (μg/plate)

5

5

10

100

 

Mean No. of colonies/plate (average of 3 ± SD)

1977 ± 45

1843 ± 5

832 ± 44

483 ± 109

+

Solvent

110 ± 5

15 ± 1

43 ± 3

13 ± 2

+

0.8

94 ± 10

14 ± 2

43 ± 2

12 ± 2

+

4

101 ± 11

13 ± 2

46 ± 1

16 ± 3

+

20

101 ± 9

14 ± 3

43 ± 5

11 ± 3

+

100

95 ± 9

15 ± 3

49 ± 9

11 ± 3

+

500

22 ± 6

2 ± 1

41 ± 4

6 ± 1

Positive controls, –S9

Name

2AA

2AA

2-AA

2AA

Concentrations (μg/plate)

10

10

10

10

 

Mean No. of colonies/plate (average of 3 ± SD)

2050 ± 50

121 ± 9

1713 ± 90

127 ± 10

B = Reduced background lawn

MNNG = N-methyl-N´-nitro-N-nitroso-guanidine

2AA = 2-Aminoanthracene

NPD = 4-nitro-o-phenylendiamine

AAC = 9-aminoacridine

Table 3. Test results of experiment 3 (pre-incubation).

With or without S9-Mix

Test substance concentration

Mean number of revertant colonies per plate

(μg/plate)

(average of 3 plates ± Standard deviation)

 

Base-pair substitution type

Frameshift type

 

TA 100

TA1535

TA98

TA1537

Solvent

98 ± 8

23 ± 2

21 ± 7

11 ± 2

3

99 ± 10

22 ± 7

17 ± 4

13 ± 3

6

94 ± 6

24 ± 2

21 ± 3

14 ± 6

12

87 ± 20

25 ± 5

22 ± 3

9 ± 3

25

76 ± 12

26 ± 3

25 ± 4

11 ± 2

50

78 ± 10

24 ± 4

24 ± 5

12 ± 1

Positive controls, –S9

Name

MNNG

MNNG

NPD

AAC

Concentrations (μg/plate)

5

5

10

100

 

Mean No. of colonies/plate (average of 3 ± SD)

1340 ± 193

1370 ± 30

833 ± 32

450 ± 61

 ±

Solvent

110 ± 16

20 ± 2

37 ± 1

12 ± 2

 ±

0.8

116 ± 18

19 ± 1

35 ± 4

16 ± 5

 ±

4

118 ± 11

20 ± 6

37 ± 4

15 ± 2

 ±

20

95 ± 9

24 ± 1

41 ± 5

15 ± 2

 ±

100

116 ± 8

27 ± 3

39 ± 4

12 ± 4

 ±

500

76 ± 6

23 ± 4

35 ± 6

6 ± 2

Positive controls, –S9

Name

2AA

2AA

2AA

2AA

Concentrations (μg/plate)

10

10

10

10

 

Mean No. of colonies/plate (average of 3 ± SD)

1310 ± 272

240 ± 10

923 ± 223

333 ± 21

B = Reduced background lawn

MNNG = N-methyl-N´-nitro-N-nitroso-guanidine

2AA = 2-Aminoanthracene

NPD = 4-nitro-o-phenylendiamine

AAC = 9-aminoacridine

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

Genetic toxicity (mutagenicity) in bacteria in-vitro

Two bacterial gene mutation assays (Ames test) were performed with N-methyl-N-(C18-(unsaturated)alkanoyl)glycine following OECD guideline 471 and in compliance with GLP (LAUS, 2007; BASF, 1990).

In the first experiment, the strains Salmonella typhimurium TA97a, TA98, TA 100, TA 102 and TA 1535 were tested in two independent experiments according to the plate incorporation and preincubation procedure in the absence and presence of a metabolic activation system (Aroclor 1254-induced rat liver S9-mix). The experiments were conducted each in 4 replications at concentrations from 50 to 5004 µg/plate (vehicle: DMSO). No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed up to the highest dose tested. The included positive and negative controls showed the expected results. Under the conditions of the study, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.

A further experiment in the tester strains Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 is available (BASF, 1990). In concentrations from 0.8 to 5000 µg/plate with and without a metabolic activation system, no increase in the number of revertant colonies was observed in any of the strains tested. However, in concentrations from 50 µg/plate onwards cytotoxicity was observed. The included positive and negative controls showed the expected results. Thus, under the conditions of the study, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.

Genetic toxicity (cytogenicity) in mammalian cells in-vitro

An in vitro mammalian chromosome aberration test was conducted with N-methyl-N-(C18-(unsaturated)alkanoyl)glycine in accordance with OECD guideline 473 under GLP conditions (LAUS, 2011). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of fresh heparinised human whole blood cultures, incubated for 4 h with and without and for 22 h without a metabolic activation system (S9-mix from rats treated with Aroclor 1245). Concentrations of 10-40 µg/mL (4 h incubation) and 0.5-50 µg/mL (22 h incubation) of the test substance in the vehicle DMSO were applied. The negative as well as the positive controls showed the expected results. In the pre-experiment cytotoxicity was observed from 55.3 µg/mL with and without metabolic activation. In the main experiments a reduction in the mitotic index was observed from 30 µg/mL without and from 40 µg/mL with metabolic activation. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed. Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in-vitro

The in vitro mammalian cell gene mutation study of N-methyl-N-(C18-(unsaturated)alkanoyl)glycine was carried out according to OECD guideline 476 under GLP conditions (Harlan, 2010). Gene mutations in the HPRT locus were investigated in Chinese hamster lung fibroblasts (V79) in the presence and absence of a metabolic activation system (Phenobarbital/beta-naphtoflavone-induced rat liver, S9). V79 cells were incubated with the test material at 0.25, 0.5, 1, 2 and 4 µg/mL for 4 h in the absence and at 4, 8, 16, 32 and 48 µg/mL in the presence of a metabolic activation system. Concentrations of the second experiment without metabolic activation for an exposure time of 24 h ranged from 4 to 96 µg/mL, and concentrations with metabolic activation for an exposure period of 4 h ranged from 4 to 56 µg/mL. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data of the laboratory. Cytotoxicity was apparent at concentrations from 4 µg/mL and 48 µg/mL in experiment I without and with metabolic activation, respectively. In the second experiment, cytotoxicity was observed from 96 µg/mL and 56 µg/mL without and with metabolic activation, respectively. In the second experiment, precipitation was observed from 237.5 µg/mL after 24 h without metabolic activation. There was no significant increase in the number of forward mutations at the HPRT locus of V79 cells treated with the test material, neither in the presence nor in the absence of a metabolic activation system. Under the conditions of the study, N-methyl-N-(C18-(unsaturated)alkanoyl)glycine did not show gene mutation activity in this test performed in V79 cells in vitro.


Justification for selection of genetic toxicity endpoint
No study was selected since all available in vitro studies were negative.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on substance specific studies, the available data on genotoxicity do not meet the criteria for classification according to Regulation (EC) No 1272/2008 and are therefore conclusive but not sufficient for classification.