Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Rheinland-Pfalz, Mainz, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N-methyl-N-oleoylglycine
EC Number:
701-177-3
Molecular formula:
C21H39NO3
IUPAC Name:
N-methyl-N-oleoylglycine

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium, 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
Controls
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.

Results and discussion

Test results
Species / strain:
S. typhimurium, 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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative