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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline standard with acceptable restriction - S. typhimurium TA 102 or E. Coli to detect crosslinking/ oxidizing agents are not included in the panel of tested strains - no data on substance purity

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1982
Report Date:
1982

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(lacking of test strains TA 102 or E. Coli, positive control in assays with metabolic activation tested in one strain only, confirmatory 2nd test performed only in TA 1537 and TA 1535)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Batch No.: EN 24375
Specific details on test material used for the study:
- Batch No.: EN 24375

Method

Target gene:
Histidine operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats induced with Aroclor 1254 with following cofactors at a ratio of 3:7
Test concentrations with justification for top dose:
25, 75, 225, 675 and 2,025 µg/plate
Vehicle / solvent:
- Vehicle: acetone
- Justification for choice of vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria.
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: cyclophosphamide (CP; 250 µg/plate in phosphate buffer, +S9, TA 1535 only); Daunorubicin-HCl (Drb-Hcl; 5 and 10 µg/plate in phosphate buffer, -S9, TA 98); N-methyl-N-nitro-N-nitrosoguanidine (MNNG; 3 and 5 µg/plate in phosphate buffer, - S9, TA 1535)
Remarks:
9(5)aminoacridine hydrochloride monohydrate (9-AA; 50 and 100 µg/plate in DMSO, -S9, TA 1537), 4 - nitroquinoline - N-oxide, NOQ, 0.125 and 0.25 µg/plate, -S9, TA 100)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation test)

DURATION
- Exposure duration: 48 hours at 37° C (in the dark)

NUMBER OF REPLICATIONS: 2 experiments; triplicate cultures/experiment

DETERMINATION OF CYTOTOXICITY
A preliminary toxicity test was carried out on strain TA 100 without microsomal activation with the concentrations ranging from 0.0001 to 2,000 ug/plate. The protocol used was the same as in the mutagenicity test, however, only two plates per concentration were used.
Evaluation criteria:
The test substance is generally considered to be nonmutagenic if the colony count in relation to the negative control is not doubled at any concentration.
Statistics:
When the colonies were counted, the arithmetic mean was computed.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the concentrations of 225 µg/plate and above the substance precipitated in soft agar

Any other information on results incl. tables

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

 

[C] µg/plate

Revertants /plate (mean value of 3 plates)

Base-pair substitution type

Frameshift type

TA 1535

TA 100

TA 1537

TA 98

-

+

-

+

-

+

-

+

NC

0

8

18

102

/

4

/

35

/

Drb-Hcl

5

/

/

/

/

/

/

314

/

10

/

/

/

/

/

/

353

/

4-NOQ

0.125

/

/

298

/

/

/

/

/

0.25

/

/

689

/

/

/

/

/

MNNG

3

1157

/

/

/

/

/

/

/

5

1590

/

/

/

/

/

/

/

 9AA

50

/

/

/

/

68

/

/

/

100

/

/

/

/

976

/

/

/

CP

250

/

280

/

/

/

/

/

/

 

Test substance

0

17

11

5

93

29

8

104

49

25

16

12

6

100

23

6

101

50

75

13

19

8

103

30

5

117

50

225

18

21

12

98

33

6

104

55

675

21

22

9

96

32

6

107

50

2,025

17

20

4

103

33

5

111

64

NC = Negative control

CP = Cyclophosphamide

Drb-Hcl = Daunorubicin-HC1

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

9AA = 9-aminoacridine

NOQ = 4 - nitroquinoline - N-oxide

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

 

[C] µg/plate

Base-pair substitution type

Frameshift type

 

 

TA 1535

TA 1537

 

 

-

+

-

+

NC

0

7

10

6

/

MNNG

3

266

/

/

/

5

1195

/

/

/

9AA

50

/

/

25

/

100

/

/

389

/

CP

250

/

412

/

/

 

 

 

 

 

 

Test substance

0

11

13

8

5

25

13

10

6

4

75

12

12

5

6

225

8

12

5

6

675

14

11

3

6

2,025

14

9

6

4

NC = Negative control

CP = Cyclophosphamide

MNNG = N-methyl-N-nitro-N-nitrosoguanidine

Applicant's summary and conclusion

Conclusions:
negative with metabolic activation
negative without metabolic activation