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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:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Version / remarks:
1997
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Test material is Halb- PMDETA
Batch number: Vers. 98/044-10
CAS number: 2212-32-0
Purity: 99.4% (GC)
Appearance: Yellowish liquid
Storage: Room termperature

Method

Target gene:
Histidine (S. typhimurium) and tryptophan (E. Coli) operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
1 volume of Aroclor 1254 induced rat liver S-9 fraction mixed with 9 volumes of cofactors mix (MgCl2, KCl, Glu-6-p, NADP, phosphate buffer, NaH2PO4, Na2HPO2.2H2O)
Test concentrations with justification for top dose:
0, 20, 100, 500, 2,500 and 5,000 µg/plate (both experiments)
Vehicle / solvent:
Due to the good solubility of the test substance in water, water was selected as the vehicle.
Controls
Untreated negative controls:
yes
Remarks:
(sterility control)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene, (2-AA) in the presence of S-9; N-methyl-N'-nitro-N -nitrosoguanidine (MNNG) and 4-nitro-o-phenylene-diamine (4-NOPD) in the absence of S-9
Remarks:
60 µg 2-AA/plate for E. Coli and 2.5 µg 2-AA/plate (remaining strains); 5 µg MNNG/plate for TA 1535 and TA 100; 10 µg 4-NOPD/plate for TA 98, 100 µg 9-AAC/plate for TA 1537, 5 µg 4-NQO/plate for E. Coli. All positive controls were dissolved in DMSO.
Details on test system and experimental conditions:
METHOD OF APPLICATION: - Experiment 1 (range finder): in agar (plate incorporation test); Experiment 2: pre-incubation test
DURATION- Preincubation period (Exp. 2 only): ca. 20 min- Exposure duration: 48 - 72 hours at 37°C
NUMBER OF REPLICATIONS: 2 experiments; triplicate cultures/experiment
DETERMINATION OF CYTOTOXICITY - Method: reduction in the number of revertants or a clearing of the bacterial background lawn, reduction of the titer.The titer is generally determined only in the experimental parts with S-9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
Evaluation criteria:
Acceptance criteria of study: The number of revertant colonies in the negative controls was within the normal range of the historical control data for each tester strain.- The sterility controls revealed no indication of bacterial contamination.- The positive control articles both with and without S-9 mix induced a significant increase in the number of revertant colonies within the range of the historical control data- The titer of viable bacteria was equal or greater than 1E+09 per milliliter.Evaluation criteria:Positive result when; A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.Negative result when; The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.
Statistics:
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduction in the number of revertants
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduction in the number of revertants
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: No precipitation of the test item was observed up to the highest concentration tested.

Any other information on results incl. tables

Results of Experiment I (plate incorporation assay)

 

[C] µg/plate

Revertants /plate (mean value of 3 plates ± standard deviation)

TA 1535

TA 100

TA 1537

TA 98

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Water

0

20 ± 1

20 ± 3

129 ± 4

139 ± 11

11 ± 2

12 ± 2

26 ± 3

39 ± 1

31 ± 6

33 ± 4

Test substance

20

16 ± 2

17 ± 3

123 ± 8

150 ± 18

11 +1

10 ± 1

24 ± 2

33 ± 2

27 ± 2

40 ± 8

100

17 ± 1

17 ± 3

145 ± 11

151 ± 19

8 ± 1

9 ± 3

20 ± 2

32 ± 2

25 ± 4

33 ± 6

500

15 ± 1

14 ± 2

141 ± 13

131 ± 6

8 ± 1

9 ± 1

24 ± 3

30 ± 2

27 ± 5

38 ± 2

2500

17 ± 1

16 ± 2

131 ± 9

121 ± 9

8 ± 1

6 ± 2

27 ± 5

30 ± 2

31 ± 2

29 ± 2

5000

13 ± 2

12 ± 2

147 ± 7

161 ± 3

7 ± 2

7 ± 1

28 ± 3

27 ± 6

32 ± 3

33 ± 3

 

MNNG

5.0

605 ± 78

/

642 ± 21

/

/

/

/

/

/

/

2 – AA

2.5

/

133 ± 16

/

1348 ± 69

 

109 ± 12

/

1073 ± 60

/

/

60.0

/

/

/

/

/

/

/

/

/

204 ± 10

9 – AAC

100.0

/

/

/

/

589 ± 34

/

/

/

/

/

NOPD

10.0

 

/

/

/

/

 

691 ± 9

/

/

/

4-NQO

2.5

/

/

/

/

/

/

/

/

943 ± 21

/

 2-aminoanthracene, (2-AA); N-methyl-N'-nitro-N -nitrosoguanidine (MNNG), 4-nitro-o-phenylene-diamine (4-NOPD), 9-aminoacridine 9 - AAC), 4-nitroquinol i ne-N-oxide (4-NQO)

 

Results of Experiment II (preincubation assay)

 

[C] µg/plate

Revertants /plate (mean value of 3 plates ± standard deviation)

TA 1535

TA 100

TA 1537

TA 98

WP2 uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Water

0

18 ± 3

18 ± 2

122 ± 9

149 ± 11

11 ± 2

10 ± 2

23 ± 5

26 ± 3

33 ± 4

36 ± 2

Test substance

20

18 ± 2

12 ± 3

125 ± 15

138 ± 19

11 ± 3

10 ± 2

26 ± 2

27 ± 2

28 ± 4

27 ± 1

100

18 ± 4

17 ± 3

104 ± 4

120 ± 5

11 ± 3

14 ± 5

19 ± 4

24 ± 6

27 ± 7

31 ± 4

500

16 ± 3

12 ± 2

101 ± 12

122 ± 20

8 ± 1

10 ± 3

23 ± 5

22 ± 2

26 ± 5

33 ± 1

2500

17 ± 3

13 ± 3

91 ± 10

115 ± 20

8 ± 2

11 ± 3

20 ± 4

22 ± 7

28 ± 5

32 ± 6

5000

14 ± 2

9 ± 1

90 ± 6

131 ± 10

6 ± 2

11 ± 2

21 ± 2

26 ± 4

20 ± 8

27 ± 3

 

MNNG

5.0

1978 ± 54

/

1591 ± 159

/

/

/

/

/

/

/

2 – AA

2.5

/

155 ± 28

/

841 ± 11

 

114 ± 9

/

764 ± 69

/

/

60.0

/

/

/

/

/

/

/

/

/

234 ± 15

9 – AAC

100.0

/

/

/

/

356 ± 58

/

/

/

/

/

NOPD

10.0

 

/

/

/

/

 

994 ± 76

/

/

/

4-NQO

2.5

/

/

/

/

/

/

/

/

979 ± 67

/

2-aminoanthracene, (2-AA); N-methyl-N'-nitro-N -nitrosoguanidine (MNNG), 4-nitro-o-phenylene-diamine (4-NOPD), 9-aminoacridine 9 - AAC), 4-nitroquinol i ne-N-oxide (4-NQO)

Applicant's summary and conclusion

Conclusions:
No evidence of mutagenicity was seen in any of the tester strains, in the absence and presence of metabolic activation.
Executive summary:

A bacterial reverse mutation test (Ames test) was conducted with Halb-PMDETA according to OECD 471 and EEC 92 -69 B14 and B13. Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvrA were tested in triplicate the absence and in the presence of metabolic activation at concentrations up to 5000 µg/plate in two independent experiments. Plate incorporation methodology was used in Test 1 and pre-incubation methodology was used in Test 2 . There was some evidence of toxicity in strains TA1535 and TA1537 in the form of a marked reduction in the number of revertant colonies. There was no precipitation of the test article or increases in the number of revertants when compared to the concurrent controls. The overall conclusion is that the test material

Halb-PMDETA is not mutagenic when tested up to 5000 µg/plate in the absence and presence of metabolic activation in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 uvrA.