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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
Study period:
1987.
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 strains tested.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
The laboratories were supplied with the chemicals, which were coded by the NTP chemical repository (Radian Corp., Austin, TX), along with information on the physical characteristics of the chemicals, their solubility in different solvents, and safety and decontamination information. Supplier: Res.Organic/inorganic.

Method

Target gene:
histidine requiring gene
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535
Details on mammalian cell lines (if applicable):
All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix.
Species / strain:
S. typhimurium TA 98
Details on mammalian cell lines (if applicable):
All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix.
Species / strain:
S. typhimurium TA 100
Details on mammalian cell lines (if applicable):
All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix.
Species / strain:
S. typhimurium TA 97
Details on mammalian cell lines (if applicable):
All strains were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 19831. Prior to their use for mutagenicity assays, all cultures were grown overnight with shaking at 37°C in Oxoid broth, and their phenotypes were analyzed.
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix.
Test concentrations with justification for top dose:
100, 333, 1000, 3333 and 10000ug/plate.
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: The solvent of choice was distilled water, followed by dimethyl sulfoxide, 95% ethanol, and acetone. The laboratory made an independent assessment of the solvent to be used.
Controls
Negative controls:
no
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 2-aminoanthracene, 4-nitro-o-phenylenediamine
Details on test system and conditions:
METHOD OF APPLICATION: preincubation. The preincubation assay was performed as described previously [Haworth et al, 1983]: The test chemical, Salmonella culture, and S-9 mix or buffer were incubated at 37ºC, without shaking, for 20 min. The top agar was added, and the contents of the tubes were mixed and poured onto the surface of petri dishes that contained Vogel-Bonner medium [Vogel and Bonner, 1956 (preparation: in 670ml distilled water are dissolved, successively: 10g MgSO4·7H2O, 100g citric acid·H2O, 500g anhydrous K2HPO4, 175g NaNH4HPO4·4H2O, the final volume being 1L; this solution is diluted 50x)]. The histidine-revertant (his+) colonies arising on these plates were counted following 2 days incubation at 37ºC. The plates were hand-counted when a precipitate was present; otherwise automatic colony counters were used.
- Cell density at seeding (if applicable):

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; other: A toxicity assay was performed to determine the appropriate dose range, by using TA100 or the system developed by Waleh (1982), see 'attached background material'.
- Any supplementary information relevant to cytotoxicity: Toxic concentrations were those at which a decrease in the number of his-f colonies was seen or at which there was a clearing in the density of the background lawn. Experiments were repeated at least 1 week following the initial trial.

- OTHER: The S-9 fractions of Aroclor 1254-induced, male Sprague-Dawley rat and male Syrian hamster livers were prepared as described previously [Haworth et al, 1983].
Evaluation criteria:
Data was evaluated as described by Haworth et al., 1983. An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic (+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose related increase was judged insufficiently high to justify a call of " + W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen. A chemical was judged to be mutagenic (+), or weakly mutagenic (+W), if it produced a reproducible, dose-related increase in his+ revertants over the corresponding solvent controls in replicate trials. A chemical was considered to be questionable (?) if a reproducible increase of hist revertants did not meet the criteria for either a " + " or " + W," or if only single doses produced an increase in his+ revertants in repeat trials.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes

Any other information on results incl. tables

Table 1. Neohesperidin dihydrochalcone (lab: MIC, solvent: DMSO).

Dose

TA 100

TA 1535

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

μg/plate

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

0.000

139

9.8

71

0.9

112

6.7

82

3.8

100

9.0

45

3.9

10

1.5

14

2.4

10

2.3

19

1.0

100.000

136

10.8

72

3.3

104

2.3

75

1.9

93

3.8

48

7.5

10

1.8

16

2.5

8

1.8

13

1.2

333.000

127

2.5

75

3.5

95

4.8

89

0.7

115

11.9

33

4.4

11

1.9

11

3.0

12

1.7

12

1.7

1000.000

128

5.2

84

4.6

92

8.1

84

4.5

104

6.1

40

4.9

11

1.2

15

2.2

8

3.2

15

2.7

3333.000

119

2.5

74

3.7

99

1.5

76

6.8

99

2.0

43

4.8

12

1.0

10

0.9

13

2.2

13

2.1

10000.000

119

2.8

71

2.3

106

5.9

78

3.5

88

1.2

41

5.9

10

1.2

9

1.2

11

1.0

17

4.1

POS

1238

19.9

1232

46.7

460

25.5

1077

23.8

421

8.0

884

33.4

149

14.5

168

8.7

128

10.5

106

0.6

 

Dose

TA 97

TA 98

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

NA

(-)

10% HLI

(-)

30% HLI

(-)

10% RLI

(-)

30% RLI

μg/plate

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

MEAN

SEM

0.000

96

3.7

99

5.9

142

6.7

101

5.8

133

6.1

16

3.0

34

5.8

30

3.0

32

2.8

44

2.7

100.000

85

5.7

89

0.6

124

8.8

116

5.2

152

6.4

18

2.2

31

0.7

35

0.3

32

2.3

39

4.7

333.000

88

7.5

85

4.1

130

4.2

91

8.4

138

11.9

17

2.3

37

2.9

30

0.6

27

1.2

41

0.7

1000.000

91

2.4

94

5.2

125

3.1

102

5.0

134

9.2

20

2.3

27

4.9

33

5.5

26

2.7

37

5.0

3333.000

82

2.0

88

2.7

129

3.7

110

9.4

134

10.3

23

1.7

30

4.1

39

0.9

36

1.9

41

4.1

10000.000

97

4.0

88

6.0

146

9.3

96

4.8

147

9.2

19

0.0

28

2.3

34

3.3

31

0.6

42

2.0

POS

797

8.4

634

17.4

378

13.0

558

18.0

290

12.6

1931

52.0

1212

25.4

418

16.1

814

37.6

366

20.2

* NA: without metabolic activation, HLI: Aroclor 1254- induced hamster liver homogenate; RLI: Aroclor 1254 -induced rat liver homogenate; (-): negative.

Applicant's summary and conclusion

Conclusions:
The test item was found to be non-mutagenic.
Executive summary:

The ability of the test item to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Haworth, similar to OECD 417. Four histidine dependent strains of Salmonella typhimurium (TA1535, TA97, TA98, TA100) were exposed to 0, 100, 333, 1000, 3333, and 10000 μg/plate of test item in the presence and absence of S9 metabolic activation (10 and 30% Aroclor 1254- induced hamster liver homogenate and rat liver homogenates), by the preincubation method. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.