Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
other information
Study period:
26. May 2000 - 16. Jun 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 471)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Di-n-butylformal
- Physical state: colorless liquid
- Analytical purity: 99.8 %
- Lot/batch No.: Batch 905
- Storage condition of test material: Room temperature (N2 conditions)

Method

Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from rat livers (treated with 500 mg Aroclor 1254)
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from rat livers (treated with 500 mg Aroclor 1254)
Test concentrations with justification for top dose:
Standarp plate test: 20, 100, 500, 2500, 5000 µg/plate
Preincubation test I.: 0, 125, 250, 500, 1000, 2000 µg/plate
Preincubation test II.: 0, 18.75, 37.5, 75, 150, 300 µg/plate
Vehicle:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: limited solubility of the test substance in water
Controls
Negative controls:
yes
Remarks:
sterility control
Solvent controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S-9 mix: all strains 2-aminoanthracene; without S-9 mix: strains TA100, TA1535: N-methyl-N'-nitro-N-nitrosoguanidine; TA98: 4-nitro-o-phenylendiamine; TA1537: 9-aminoacridine; E.coli WP2 uvrA: 4-nitroquinoline-N-oxide
Details on test system and conditions:
METHOD OF APPLICATION: standard plate test

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3


METHOD OF APPLICATION: preincubation

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

NUMBER OF REPLICATIONS: 3
Evaluation criteria:
Doubling of the spontaneous mutation rate, dose-response relationship, reproducibility of the results.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Bacteriotoxic effects were observed in the SPT depending on strain and concentrations from 2500 µg/plate onward. In the PIT bacteriotoxicity was observed depending an strain and concentrations from 150 µg-300 µg/plate onward.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Bacteriotoxic effects were observed in the SPT depending on strain and concentrations from 2500 µg/plate onward. In the PIT bacteriotoxicity was observed depending an strain and concentrations from 150 µg-300 µg/plate onward.
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test substance precipitation was found.

Any other information on results incl. tables

Standart plate test (SPT):

 Dose (µg/plate)  TA1535     TA100     TA1537     TA98        E. coli WP2 uvrA                                                                        
   -S9  +S9  -S9  +S9  -S9  +S9 -S9  +S9 -S9  +S9                                                                         
 Acetone

26±2

21±1

132±10

133±19

10±1

17±2

35±2

37±3

37±2

30±2

 

 

 

 

 

 

                                                           

20 

23±3

18±3

130±6

134±9

13±2

10±2

30±3

33±6

30±4

27±1

 

 

 

 

 

 

                                                           

 100

18±3

23±3

147±31

133±29

11±6

9±4

27±5

35±3

25±5

23±5

 

 

 

 

 

 

                                                           

 500

21±3

23±7

119±14

137±21

11±1

8±2

30±4

32±3

20±3

24±2

 

 

 

 

 

 

                                                           

 2500

20±3

27±8x

74±21x

1337

5±1x

3±1x

4±4x

20±1x

22±4

23±3

 

 

 

 

 

 

                                                           

 5000

16±2x

21±6x

47±22x

127±8x

4±2x

-

-

12±2x

15±3

8±3

 

 

 

 

 

 

                                                           

 2 -AA

-

416±120

-

1479±178

 -

244±43

1065±117

 -

240±49

 

 

 

 

 

 

                                                           

 MNNG

1559±240

-

1298±145

-

-

 -

-

 -

-

-

 

 

 

 

 

 

                                                           

 AAC

-

-

-

-

620±16

-

-

-

-

-

 

 

 

 

 

 

                                                           

 NOPD

 -

-

-

-

-

-

944±32

-

 

 

 

 

 

 

                                                           

4NQO

 -

-

-

-

-

-

-

-

1162±110

-

 

 

 

 

 

 

                                                           

Mean ± SD

Preincubation-test (PIT) I:

 Dose (µg/plate)

 TA1535   

 TA100   

 TA1537   

 TA98   

 E. coli WP2

uvrA   

 

 

 

 

 

 

 

                                                 

 

 -S9

 +S9

 -S9

 +S9  -S9  +S9 -S9  +S9  -S9 +S9                                                                 
 Acetone

22±2

20±4

113±6

115±12

10±1

12±1

28±3

45±2

30±4

26±8

 

 

 

 

 

 

 

                                                 

125

18±5

14±4

126±8

111±5

10±3

10±2

30±3

36±1

31±3

23±2

 

 

 

 

 

 

 

                                                 

 250

3±2

18±3

107±9

90±23

10±1

8±1

29±6

32±6

26±2

22±4

 

 

 

 

 

 

 

                                                 

500 

x

x

x

x

x

x

x

x

x

x

 

 

 

 

 

 

 

                                                 

1000

x

x

x

x

x

x

x

x

x

x

 

 

 

 

 

 

 

                                                 

 2000

x

x

x

x

x

x

x

x

x

x

 

 

 

 

 

 

 

                                                 

2-AA

-

132±19

-

647±45

-

115±13

-

549±25

-

234±26

 

 

 

 

 

 

 

                                                 

 MNNG

729±56

-

818±55

-

-

-

-

-

-

-

 

 

 

 

 

 

 

                                                 

 AAC

-

-

-

-

334±16

-

-

-

-

-

 

 

 

 

 

 

 

                                                 

NOPD

-

-

-

-

-

-

799±46

-

-

-

 

 

 

 

 

 

 

                                                 
4NQO -  -  -

 - -  - - 708±70  -                                                                

Preincubation-test (PIT) II:

 Dose (µg/plate)

 TA1535   

 TA100   

 TA1537   

 TA98   

 E. coli WP2

uvrA   

 

 

 

 

 

 

 

                                                 

 

 -S9

 +S9

 -S9

 +S9  -S9  +S9 -S9  +S9  -S9 +S9                                                                 
 Acetone

21±4

20±3

102±2

115±13

11±3

11±2

28±3

35±3

33±6

34±3

 

 

 

 

 

 

 

                                                 

18.75

19±2

17±1

99±4

112±3

11±2

9±2

21±1

35±5

31±1

29±2

 

 

 

 

 

 

 

                                                 

37.5

17±3

16±2

99±8

100±8

10±2

10±2

23±3

33±7

30±3

24±2

 

 

 

 

 

 

 

                                                 

 75

17±1

15±4

84±9

98±15

6±4

7±2

22±2

23±2

23±3

21±4

 

 

 

 

 

 

 

                                                 

150

13±1

10±1

90±5

85±16

6±2

6±1

17±6

22±2

23±2

15±3

 

 

 

 

 

 

 

                                                 

300

6±1

7±3x

70±2

64±15

2±1

4±2x

11±2

15±3

15±4

9±2x

 

 

 

 

 

 

 

                                                 

2-AA

-

110±3

-

569±14

-

94±5

-

518±171

-

203±8

 

 

 

 

 

 

 

                                                 

MNNG

652±17

-

857±77

-

-

-

-

-

-

-

 

 

 

 

 

 

 

                                                 

AAC

-

-

-

-

404±19

-

-

-

-

-

 

 

 

 

 

 

 

                                                 

NOPD

-

-

-

-

-

-

945±50

-

-

-

 

 

 

 

 

 

 

                                                 
4NQO -  -  -

 - -  - - 527±24  -                                                                

Mean ± SD

X: reduced background growth

2-AA: 2-aminoanthracene

MNNG; N-methyl-N-nitro-N-nitrosoguanidine

4NQO: 4-nitroquinoline-N-oxide

NOPD: 4-nitro-o-phenylendiamine

AAC: 9-aminoacridine chloride monohydrate

According to the results of the study, the test substance is not mutagenic in the Ames test and in the Escherichia coli - reverse mutation assay under the experimental conditions chosen here. The positive control gave the expected values.

Applicant's summary and conclusion