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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:
29 Sep - 09 Oct 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Basic data given: TA102 or E. coli WP2 are not included in the testing. According to OECD 471, at least five strains of bacteria should be included in the testing. In this Ames test, only four S. typhimurium strains (TA1535, TA1537, TA98, TA100) were used. It is known that these strains have not the potential to detect certain types of mutagens, such as cross-linking agents or oxidising mutagens. In order to detect such mutagens it is required to include S. typhimurium TA102 or to add a DNA repair-proficient strain of E.coli (e.g. E.coli WP2 uvrA or E.coli WP2 uvrA (pKM101)).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
- only 4 strains tested
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
- only 4 strains tested
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Analytical purity: no data
- Batch No.: 14840
- Storage condition of test material: At room temperature in the dark.

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Strains are regularly checked for their histidine-requirement, crystal violet sensitivity, UV-sensitivity, ampicillin resistance (TA98 and TA100) and the frequency of spontaneous revertants.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat livers from adult males (Wistar or Sprague Dawley) intraperitoneally injected with a solution (20% w/v) of Aroclor 1254 (500 mg/kg bw) in corn oil
Test concentrations with justification for top dose:
with S9-mix: 100, 300, 1000, 3333, 5000
without S9-mix: 100, 300, 1000, 3333, 5000
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: milli-Q water (Millipore corp., Bedford, Mass., USA)
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9-mix: sodium azide (TA1535, 1 µg in saline), 9-aminoacridine (TA1537, 60 µg in saline, daunomycine (TA98, 2 µg in saline), methylmethanesulfonate (TA100, 650 µg in DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
milli-Q water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: with S9-mix: 2-aminoanthracene (TA1535, TA1537: 0.5 µg in DMSO; TA98, TA100: 5 µg in DMSO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 2 experiments with and without S9-mix, each experiment with 3 replicate plates

DETERMINATION OF CYTOTOXICITY
- Method: viability determination
Evaluation criteria:
When the number of reverse mutation colonies increased by almost twice the solvent control or more, and reproducibilityor dependence on the dose of the test substance was observed, the result was considered positive.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity determination of the test substance in TA100 was done. The percentage survival of the TA100 culture was determined by comparing the number of colonies on the solvent control plate with those on the plates containing the test substance. Based on these data, the AMPD was tested in the main test up to a concentration of 5000 µg/plate in the absence and presence of S9-mix

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain -specific positive control values fell within the laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

Any other information on results incl. tables

Table 1: Results of the main test

1st experiment

number of revertants/plate: mean value of vehicle/solvent control ± SD

number of revertants/plate: mean value of positive control ± SD

max. number of revertants/plate: mean value of test material ± SD [concentration in experiment (µg/plate) resulting in max. number of revertants/plate]

without S9-mix

TA 98

17 ± 1

1621 ± 85

25 ± 1 [100]

TA 100

94 ± 11

673 ± 51

95 ± 16 [5000]

TA 1535

12 ± 3

195 ± 20

10 ± 3 [100]

TA 1537

12 ± 3

463 ± 35

9 ± 3 [100]

with S9-mix

TA 98

22 ± 2

440 ± 26

27 ± 5 [3330]

TA 100

85 ± 3

725 ± 83

95 ± 6 [100]

TA 1535

11 ± 3

358 ± 17

12 ± 4 [100]

TA 1537

7 ± 1

931 ± 59

10 ± 4 [3330]

2nd experiment

number of revertants/plate: mean value of vehicle/solvent control ± SD

number of revertants/plate: mean value of positive control ± SD

max. number of revertants/plate: mean value of test material ± SD [concentration in experiment (µg/plate) resulting in max. number of revertants/plate]

without S9-mix

TA 98

30 ± 3

1033 ± 83

46 ± 4 [1000]

TA 100

127 ± 16

883 ± 15

152 ± 17 [3330]

TA 1535

20 ± 2

210 ± 45

18 ± 8 [1000]

TA 1537

8 ± 2

273 ± 55

8 ± 3 [5000]

with S9-mix

TA 98

36 ± 5

904 ± 5

42 ± 4 [333]

TA 100

121 ± 4

1059 ± 73

139 ± 14 [100]

TA 1535

15 ± 5

203 ± 22

14 ± 3 [333]

TA 1537

6 ± 1

748 ± 23

9 ± 4 [333]

 

 

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative