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Toxicological information

Genetic toxicity: in vitro

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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
Study period:
1982-08-02 to 1982-09-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP, comparable to current guidelines with some deviations.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
No confirmation in an independent experiment. No E. coli strains were investigated.
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
No confirmation in an independent experiment. No E. coli strains were investigated.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material : Dimethylhydantoin
- Physical state: solid
- Substance type: white crystalline solid
- Analytical purity: not data
- Lot/batch No.: 447:34-2
- Storage condition of test material: room temperature

Method

Target gene:
All were histidine auxotrophs. The mutations for each strains were; hisG46 for strains TA1535 and TA100, hisC3076 for TA1537 and hisD3052 TA1538 and TA98.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
no data
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from male Sprague-Dawley rats liver microsomes
Test concentrations with justification for top dose:
100, 500, 2500, 5000 and 10000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s): deionised distilled water
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
75 µg without activation with strain TA 1537
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
1.0 µg in the presence of activation with TA 98, TA100 and TA 1538 strains and at 4.0 µg with the TA 1535 and TA 1537 strains
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-nitrofluorene
Remarks:
10 µg without activation, with strains TA98 and TA 1538
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 1,3-propane sultone
Remarks:
0.04 µL without activation, with strains TA 100 and TA 1535
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
distilled , deionised water
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation

DURATION
- Exposure duration: 20 minutes at 37 °C
- Selection time (if incubation with a selection agent): 48 hours at 37 °C


NUMBER OF REPLICATIONS: 3

SELECTION AGENT (mutation assays): L-Histidine and D-biotin
Evaluation criteria:
For a test article to be considered positive, it must cause at least a doubling in the average revertants per plate of at least one tester strain. This increase in the average number of revertants per plate must be accompanied by a dose response to increasing concentrations of the test article, and in those cases where the observed increase is less than three-fold, the response must be reproducible.
Statistics:
All platings were done in triplicate. For each triplicate plating, an average and standard deviation were calculated.

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 / choice of top concentrations:
no cytotoxicity
Remarks:
tested up to 10000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
tested up to 10000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity determination study of the test substance, DMH, was carried out for eight serial half-log dilutions, 2.9, 9.4, 30, 95, 305, 977, 3125 and 10000 µg, plated with a diluted TA100 culture on non-selective agar (viable counts) and with an undiluted TA100 culture on selective agar (revertants). A negative control with no test substance, DMH, and only 50 µl of H2O, was added to the plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

 Table 1. Results for Gene Mutation Assay with metabolic activation

Averaged Revertants per plate ± S.D (with rat liver microsomes)

Concentration µL/plate

 

TA98

 

TA100

 

TA1535

 

TA1537

 

TA1538

001

35 (4)

109 (13)

9 (4)

11 (4)

14 (3)

500

36 (2)

108 (26)

10 (3)

11 (2)

19 (6)

2500

40 (6)

116 (27)

8 (2)

13 (7)

18 (3)

5000

33 (9)

80 (4)

8 (2)

11 (2)

16 (1)

10000

38 (5)

96 (5)

7 (1)

8 (2)

18 (1)

Solvent control (H2O)

35 (7)

107 (31)

10 (2)

9 (6)

20 (6)

Positive control

349 (34)

398 (31)

212 (5)

296 (12)

444 (28)

Table 2. Results for Gene Mutation Assay without metabolic activation

Averaged Revertants per plate ± S.D (without rat liver microsomes)

Concentration µL/plate

 

TA98

 

TA100

 

TA1535

 

TA1537

 

TA1538

100

18 (4)

111 (23)

31 (2)

7 (1)

9 (5)

500

18 (7)

131 (11)

36 (2)

7 (2)

6 (3)

2500

17 (6)

117 (31)

31 (7)

6 (2)

7 (3)

5000

22 (2)

110 (24)

35 (4)

7 (1)

10 (3)

10000

19 (6)

107 (20)

30 (6)

6 (1)

6 (4)

Solvent control(H2O)

21 (4)

127 (41)

24 (4)

5 (2)

10 (7)

Positive control

874 (29)

2100 (122)

2273 (115)

420 (217)

1657 (29)

Applicant's summary and conclusion

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

The test material was found not to cause any positive response in any of the Salmonella strains tested, with or without metabolic activation.