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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1995-05-03 to 1995-06-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to an appropriate OECD test guideline, with acceptable restrictions. The restrictions were that the range of strains does not comply with the current guideline.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 strains used
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Umwelt, Raumordnung und Landwirtschaft des Landes Nordrhein-Westfalen
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
His operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa, uvrB (TA 98 & TA 100: pKM 101)
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:
50, 160, 500, 1000 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility properties and relative non-toxicity to bacteria
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without metabolic activation: TA 98: 2.5 µg/plate in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without metabolic activation: TA 100 & TA 1535: 2.5 µg/plate in aqua bidest
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without metabolic activation: TA 1537: 25 µg/plate in DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with metabolic activation: All strains: 2.5 µg/plate in DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION:in agar (plate incorporation)

DURATION

- Expression time (cells in growth medium): 72 hours

NUMBER OF REPLICATIONS: 3 plates for each test concentration

DETERMINATION OF CYTOTOXICITY
- Method: Background lawn assessment, revertant colony counts

Metabolic activation: Male Wistar rats recieved single i.p. injection of Aroclor 1254 ( 500 mg/kg body weight) in olive oil 5 days prior to the preparation of the S9 fraction. One part of S9 fraction was mixed with 9 parts of a cofactor solution resulting in the following mixture:
-10 % S9 fraction
-33 mM KCl
-8 mM MgCl2
-5 mM glucose-6-phosphate
-4 mM NADP, 100 mM Na2HPO4/NaH2PO4, pH 7.4.
0.5 ml of S9 mix were added to a total volume of 2.7 ml, giving a final concentration of approximately 1%
Evaluation criteria:
Solvent control data must be within range of historical data.

The mean of each positive control must exhibit at least a three fold increase in the number of revertants over the mean value of the respective vehicle control.

A minimum of four non-toxic dose levels are required to evaluate assay data.

For a test substance to be considered positive, it must cause at least a doubling in the mean revertants per plate of at least one tester strain.
The increase must be accompanied by a dose response towards increasing concentrations of the test article.

Cytotoxicity is defined by a reduction in the number of revertant colonies and / or a clearing of the background lawn.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 1537, TA 98 and TA 100
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
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
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:
COMPARISON WITH HISTORICAL CONTROL DATA: Results were within range of historical control data
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 2: Experiment 1 Preincubation Number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA1535

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

Negative control**

29

41

No

146

135

No

11

14

No

0*

27

54

No

135

114

No

10

11

No

50

26

42

No

125

127

No

9

17

No

160

26

44

No

122

140

No

15

23

No

500

26

40

No

133

155

No

23

43

No

1600

25

41

No

160

154

No

64

93

No

5000

28

27

No

179

178

No

112

141

No

Positive control

129

553

No

412

1952

No

337

184

No

*solvent control with DMSO

**negative control with water

Table 2: Experiment 1 Preincubation Number of revertants per plate (mean of 3 plates)

 

TA1537

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

Negative control**

23

14

No

0*

18

12

No

50

21

14

No

160

14

13

No

500

14

12

No

1600

15

12

No

5000

13

13

No

Positive control

54

250

No

*solvent control with DMSO

**negative control with water

Table 3: Experiment 2 Plate incorporation Number of revertants per plate (mean of 3 plates)

 

TA98

TA100

TA1535

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

MA

+ MA

Cytotoxic
(yes/no)

Negative control**

21

43

No

129

138

No

8

12

No

0*

22

47

No

123

129

No

12

10

No

50

21

41

No

114

127

No

11

9

No

160

29

41

No

126

139

No

16

21

No

500

24

47

No

143

135

No

27

38

No

1600

28

42

No

167

149

No

64

83

No

5000

32

31

No

193

184

No

108

131

No

Positive control

112

1984

No

428

1711

No

275

198

No

*solvent control with DMSO

**negative control with water

Table 3: Experiment 2 Plate incorporation Number of revertants per plate (mean of 3 plates)

 

TA1537

Conc.
(
µg/plate)

— MA

+ MA

Cytotoxic
(yes/no)

Negative control**

14

20

No

0*

11

16

No

50

12

21

No

160

12

18

No

500

8

19

No

1600

9

15

No

5000

9

16

No

Positive control

80

120

No

*solvent control with DMSO

**negative control with water

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive with and without metabolic activation

In a bacterial reverse mutation assay according to OECD 471 and to GLP, (3-chloropropyl)diethoxymethylsilane induced a mutagenic effect in Salmonella typhimurium strains: TA 1535 with and without metabolic activation. Appropriate solvent and positive controls were used and gave expected results. The test substance is considered to be a direct-acting mutagen, inducing base pair mutations.