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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:
2017-05-05 to 2017-06-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Method

Target gene:
his-
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
DNA polymerase A deficient
Metabolic activation:
with and without
Metabolic activation system:
S9 liver Mix
Test concentrations with justification for top dose:
31.6, 100, 316, 1000, 3160 and 5000 µg due to absence of cytotoxicity in a preliminary test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: purified water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Six concentrations ranging from 31.6 to 5000 µg alpha methyl glucoside/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.
- Cell density at seeding (if applicable): E008-E009 cells/mL

DURATION
- Preincubation period: 20 min
- Exposure duration: 48h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity is evidenced by a reduction in the number of spontaneous revertants by at least 50%, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures.


Rationale for test conditions:
as recommended in the guideline
Evaluation criteria:
The results of the negative and positive control cultures should be within the range of the historical data generated by the testing laboratory. The range of spontaneous reversion frequencies per plate is based on Kirkland (1990):
TA98: 20 - 60
TA100: 100 - 200
TA102: 240 - 320
TA1535: 10 - 35
TA1537: 3 - 20

Where concurrent negative or positive control data fall outside the range, they may be acceptable and considered for the inclusion into the historical control distribution as long as these data are not extreme outliers.

A test item is considered to show a positive response if
- the number of revertants is significantly increased (p ≤ 0.05, U-test according to MANN and WHITNEY) compared to the solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
Or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Results and discussion

Test resultsopen allclose all
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
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
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
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
Species / strain:
S. typhimurium TA 102
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

Any other information on results incl. tables

Historical control data:

positive controls

Strain S9-Mix

TA98

TA100

TA102

TA1535

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

 

2-nitro-fluorene

Benzo[a]

pyrene

sodium azide

2-amino-anthracene

Mito-mycin C

Benzo[a]

pyrene

sodium azide

2-amino-anthracene

9-amino-acidine

Benzo[a]

pyrene

Mean

151.2

150.4

952.9

948.8

1029.7

1024.3

135.6

135.4

76.7

77.6

SD

27.9

28.8

99.7

103.7

97.1

97.1

28.8

28.4

26.5

26.4

Min

91

96

677

703

781

781

51

49

28

31

Max

293

291

1213

1195

1637

1366

266

270

185

184

negative controls:

Strain S9-Mix

TA98

TA100

TA102

TA1535

TA1537

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean

30.3

32.1

145.1

145.2

277.3

279.0

19.7

19.9

6.7

6.7

SD

5.6

5.9

18.4

19.2

16.5

17.2

4.4

4.6

1.7

1.8

Min

20

20

107

101

245

203

10

10

2

3

Max

49

49

195

198

323

324

34

36

10

10

Applicant's summary and conclusion

Conclusions:
In the present test conducted according to OECD guideline 471, the Salmonella typhimurium strains TA 98, 100, 102, 1535, 1537 were incubated with the following concentrations of alpha methyl glucoside: 31.6, 100, 316, 1000, 3160 and 5000 µg /plate. No increases in revertant colonies were observed no cytotoxicity occurred. The test item is therefore considered non mutagenic under the present conditions.
Executive summary:

In a reverse gene mutation assay in bacteria (OECD guideline 471), strains TA 98, TA 100, TA 102, TA 1535 and TA 1537of S. typhimurium were exposed to alpha methyl glucoside (100% a.i.) in highly purified water at concentrations of (31.6, 100, 316, 1000, 3160 and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method and the preincubation method

Alpha methyl glucoside was tested up to limit concentration (5000 µg/plate)There was no increase in the number of revertants regardless of strain.  The positive controls induced the appropriate responses in the corresponding strains.  There was no evidence of induced mutant colonies over background.

 

This study is classified asacceptable.  This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.