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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study is comparable to OECD Guideline 471 with acceptable restrictions (no details about the test substance solution [55.2% test substance] used; no data on pH; no analytical control of test solutions; no data about historical controls [but vehicle control within values published elsewhere]).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,6-dimethylcyclohexylamine
EC Number:
229-942-2
EC Name:
2,6-dimethylcyclohexylamine
Cas Number:
6850-63-1
Molecular formula:
C8H17N
IUPAC Name:
2,6-dimethylcyclohexan-1-amine
Details on test material:
2,6-Dimethylcyclohexylamine "roh"; purity 55.2%; colourless to yellowish liquid
no further data

Method

Target gene:
His- (S. typhimurium)
Try- (E.coli)
Species / strain
Species / strain / cell type:
other: Salmonella typhimurium TA 100, 98, 1535, 1537, E. coli WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
Liver S9-fraction prepared from livers of rats i.p. injected with 500 mg/kg bw Aroclor1254 5 days prior to sacrifice.
Test concentrations with justification for top dose:
3 independent experiments with and without metabolic activation
1) standard plate test: 0; 40; 200; 1,000; 5,000 and 10, 000 µg/plate
2) preincubation assay: 0; 20; 100; 500; 2,500 and 5,000 µg/plate
3) preincubation assay: 0; 62.5; 125; 250; 500 and 1,000 pg/plate
Vehicle / solvent:
DMSO (dimethylsulfoxide)
Controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see below
Details on test system and experimental conditions:
S9-mix prepared freshly prior to experiment.
3 plates per dose level.

Positive control with S-9 mix:
2.5 µg/plate 2-aminoanthracene (2-AA) dissolved in DMSO for the strains TA 100, TA 98, TA 1537 and TA 1535
and 60 µg/plate 2-aminoanthracene dissolved in DMSO for the strain E. coli WP2 uvrA

Positive control without S-9 mix:
5 µg/plate N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) dissolved in DMSO for the strains TA 100 and TA 1535
10 µg/plate 4-nitro-o-phenylendiamine (NOPD) dissolved in DMSO for the strain TA 98
100 µg/plate 9-aminoacridine chioride monohydrate (AAC) dissolved in DMS0 for the strain TA 1537
10 µg/plate N-ethyl-N'-nitro-N-nitrosoguanidin (ENNG) dissolved in DMS0 for the strain E. coli WP2 uvrA
Evaluation criteria:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results
Statistics:
Mean +- SD (standard deviation) was calculated.

Results and discussion

Test resultsopen allclose all
Species / strain:
bacteria, other: Salmonella typhimurium TA 100, 98, 1535, 1537, E. coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> or = 5000 µg/plate; see Table below
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
bacteria, other: Salmonella typhimurium TA 100, 98, 1535, 1537, E. coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
> or = 1000 µg/plate; see Table below
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No test substance precipitation was found.
No data about historical controls but vehicle controls within values published elsewhere.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
Remarks on result:
other: other: standard plate test
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenicity in the Ames test with and without metabolic activation

Mean number of revertants +- standard deviation

Dose in µg per plate

TA98

TA100

TA1535

TA1537

E. coli WP2uvrA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-

Standard plate test

DMSO

32+-3

49+-10

101+-20

106+-10

16+-2

23+-4

10+-2

8+-3

21+-4

42+-7

40

28+-4

40+-11

111+-6

118+-11

24+-6

16+-5

8+-4

8+-2

29+-2

46+-14

200

21+-8

44+-16

85+-11

120+-17

19+-3

19+-2

6+-2

5+-2

27+-4

53+-10

1000

28+-1

43+-6

78+-22

125+-3

26+-6

15+-6

9+-1

6+-2

33+-3

50+-4

5000

28+-3

34+-10

Toxic

145+-11

16+-7

20+-2

10+-3

4+-1

29+-6

36+-8

10000

Toxic

Toxic

Toxic

Toxic

Toxic

Toxic

Toxic

Toxic

Toxic

20+-4 toxic

Positive control

927+-164

658+-64

513+-7

800+-65

541+-20

119+-20

159+-48

75+-4

730+-46

235+-40

-

Preincubation assay (I)

DMSO

26+-3

35+-5

102+-3

109+-8

19+-2

18+-2

11+-2

9+-2

27+-6

40+-5

20

25+-2

33+-3

110+-6

100+-5

17+-1

16+-1

12+-4

9+-2

31+-4

32+-6

100

26+-5

37+-1

94+-8

100+-4

15+-1

14+-4

9+-2

7+-3

29+-3

22+-2

500

22+-6

26+-3

85+-13

101+-8

10+-0

13+-3

7+-3

7+-2

24+-3

18+-2

2500

Toxic

Toxic

Toxic

66+-2 toxic

Toxic

Toxic

Toxic

Toxic

Toxic

13+-2 toxic

5000

Toxic

Toxic

Toxic

Toxic

Toxic

Toxic

Toxic

Toxic

Toxic

Toxic

Positive control

1188+- 128

758+-101

1261+-54

713+-51

1129+- 146

132+-10

765+-23

119+-7

536+-51

194+-18

-

Preincubation assay (II)

DMSO

27+-7

44+-11

109+-11

99+-2

16+-1

14+-4

7+-1

12+-4

43+-9

33+-1

62.5

36+-7

37+-10

104+-4

103+-5

21+-8

14+-3

7+-1

13+-2

42+-4

29+-5

125

34+-6

45+-4

98+-4

100+-10

13+-3

13+-3

10+-2

11+-2

42+-11

31+-7

250

36+-3

40+-8

109+-28

122+-4

21+-2

17+-5

8+-2

9+-2

49+-2

29+-6

500

38+-2

35+-8

76+-12

109+-23

17+-2

18+-6

8+-1

7+-3

35+-12

29+-2

1000

29+-5

38+-4

72+-11

103+-8

8+-4 toxic

16+-3 toxic

9+-3

7+-3 toxic

41+-4

26+-4

Positive control

969+-89

814+-125

909+-82

853+-44

1284+-66

125+-16

861+-46

166+-23

606+-52

253+-33

MA: metabolic activation

Applicant's summary and conclusion

Conclusions:
No mutagenic activity was detected with and without metabolic activation even at cytotoxic concentrations in the Ames test using S.typhimurium TA98, 100, 1535, 1537 and E. coli WP2uvrA.
Executive summary:

The study is comparable to OECD Guideline 471 with acceptable restrictions (no details about the test substance solution used [55.2% test substance]; no data on pH; no analytical control of test solutions; no data about historical controls [but vehicle control within values published elsewhere]).

In the Ames test with S.typhimurium TA98, 100, 1535, 1537 and E. coli WP2uvrA the test substance was studied using two different protocols with and without addition of a metabolic activation system: the standard plate test (dose levels: 0; 40; 200; 1000; 5000 and 10000 µg/plate) and the preincubation method (0; 20; 100; 500; 2500 and 5000 µg/plate or in a 2nd trial 0; 62.5; 125; 250; 500 and 1000 µg/plate). Negative and positive controls were valid. In each test system cytotoxic concentrations were reached. There was no increase in revertants in any strain.

Conclusion: No mutagenic activity was detected with and without metabolic activation even at cytotoxic concentrations in the Ames test using S.typhimurium TA98, 100, 1535, 1537 and E. coli WP2uvrA.