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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:
not stated
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1997
Report Date:
1997
Reference Type:
publication
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
not stated
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Version / remarks:
not stated
Qualifier:
according to
Guideline:
other: “Methods of Testing New Chemical Substances” (Environmental Protection Bureau notification no. 237, Pharmaceutical Affairs Bureau notification no. 306, Basic Industries Bureau 1987 notification no. 303, dated 31 March, 1987
Version / remarks:
31 March 1987
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:
Purity of test material: 99.8 wt% (impurities: 0.06 wt% 1,4-acetoxyhydroxybutane-2, 0.07 wt% 2-(4-hydroxy-butyloxy)tetrahydrofuran)

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 (Kikkoman Co., Ltd., lot no: RAA-333, manufactured 8 September, 1995 and lot no: RAA-338, 15 December, 1995) extracted from oxygen-induced 7-week old Sprague-Dawley male rats administered phenobarbital (PB) and 5,6-benzoflavone (BF
Test concentrations with justification for top dose:
313~5000 μg/plate with a common ratio of 2, top dose according to guideline
Vehicle / solvent:
water for injection use
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: AF2: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; SA: Sodium azide; 9AA: 9-aminoacridine; 2AA: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 h
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

NUMBER OF REPLICATIONS:main test and repeatability test, 3 plates were used for both control groups and for each dose

DETERMINATION OF CYTOTOXICITY
not stated
Evaluation criteria:
If, for 1 or more of the 5 strains of bacteria used, under conditions without S9 mix and with S9 mix, the mean value of the revertant colony count on the surface of plates containing the test substance was 2 or more times that of the solvent control, and, if that increase could be repeated or dose-dependency was noted, then the test substance in this series of tests would be determined to be mutagenic (positive). However, if the dose showed that the mean value of the revertant colony count at the second time of testing was more than 2 times that of the solvent control, but the solvent control value was less than 10 and a dose-dependent increase in the revertant colony count was not noted, then the determination would be negative.
Statistics:
not performed

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:
not specified
Remarks:
negative in range finding test with same top dose
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:
not specified
Remarks:
negative in range finding test with same top dose
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:
not specified
Remarks:
negative in range finding test with same top dose
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:
not specified
Remarks:
negative in range finding test with same top dose
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Remarks:
negative in range finding test with same top dose
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Without the addition of S9 mix, the result in Test I for the 625 μg/plate showed an increase in revertant colony count which was more than twice that of the solvent control value. However, in the testing of TA1537 with the addition of S9 mix and in the tests with all the other bacteria, there was no increase in revertant colony count more than twice that of the solvent control. The main test was repeated using identical doses. As a result, no increase in revertant colony count more than twice that of the solvent control was noted.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study the test substance was not mutagenic to bacteria with and without metabolic activation.
Executive summary:

A bacterial mutagenicity test (according to OECD guidelines 471 and 472 and GLP-compliant) was performed in Salmonella typhimurium TA1535, TA1537, TA98 and TA100, and in Escherichia coli WP2 uvrA as preincubation test, with and without metabolic activation.

Tests with and without S9 mix were conducted twice, within the range of 313 -5000 μg/plate with a common ratio of 2.

For the test of TA1537, without the addition of S9 mix, the result in Test I for the 625 μg/plate showed an increase in revertant colony count which was more than twice that of the solvent control value. However, in the testing of TA1537 with the addition of S9 mix and in the tests with all the other bacteria, there was no increase in revertant colony count more than twice that of the solvent control.

For TA1537, since the results without S9 mix were different for Test I and Test II, the main test was repeated using identical doses. As a result, no increase in revertant colony count more than twice that of the solvent control was noted.

In all of the tests conducted, the positive control group showed an increase in revertant colony count for all of the test bacteria, which meant that, along with the values measured for the solvent control group, the revertant colony count was within the range of historical values, so the efficacy of this test series was confirmed.

It is concluded, based on the above results that the test material is not mutagenic (negative) for the test strains used.