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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:
02 March 2010 - 31 May 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Principles of method if other than guideline:
The ‘treat and plate’ treatment method was used in each test to avoid the possibility that bio-available histidine in the test item might compromise the test.

GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
- Name of test material (as cited in study report): Alpha amylase [Trichoderma reesei LOH4AkAApaA (paA-1)] (GICC 03387)
- Substance type: UVCB
- Physical state: pale yellow liquid
- Lot/batch No.: 168109001
- Expiration date of the lot/batch: February 2012
- Stability under test conditions: The test material and dilutions (50% and 25%) were stable for at least 5 hours at room temperature. Further, the test material was stable for at least 7 days at 4 degrees of C and 90 days at minus 20 degrees of C
- Storage condition of test material: minus 20 degrees of C




Method

Target gene:
The study describes experiments performed to assess the effect of the test material alpha amylase in amino acid dependent strains of Salmonella typhimurium capable of detecting both induced frame-shift (TA1537 and TA98) and base-pair substitution mutations (TA1535, TA100 and TA102). The test system is a reverse mutation of amino acid dependent bacterial strains.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 from Aroclor 1254 induced SPF Wistar rats, manufactured at LAB Research and validated in separate test.
Test concentrations with justification for top dose:
Five dose levels (50, 160, 500, 1600, and 5000 μg/plate) were tested. All dose levels are expressed in terms of the total protein content of the test item supplied, being 46 mg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: sterile saline solution (0.9 % NaCl)
- Justification for choice of solvent/vehicle: The test substance is water-soluble and any human exposure will be in aqueous solutions.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Cumene hydroperoxide, sodium azide, 2-nitrofluorene, 9-aminoacridine, 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: All applications were done in medium before plating, i.e. a liquid culture assay (treat and plate assay).

DURATION
- Exposure duration: 3.5 hrs (liquid culture assay)
- Incubation time (selective incubation) : 3 days

DETERMINATION OF CYTOTOXICITY
- Method: Viable cell count

Evaluation criteria:
The numbers of revertant colonies at each treatment test point were compared to the corresponding negative control values for each set of triplicate plates.
The tests were considered to be valid as all the following criteria were met:
- negative and positive control data were consistent with the historical control data for this laboratory
- the positive control data showed marked increases over the concurrent negative control values
- the evaluation of the data was not restricted by loss of plates (e.g. through contamination).
The test item would have been considered to have shown evidence of mutagenic activity in this study if all of the following criteria had been met:
- increases in the numbers of revertant colonies were observed at one or more test points
- the mean number of revertant colonies at the test point showing the largest increase was more than twice the corresponding negative control value
- there was a credible scientific explanation for the observed dose-response relationship that involved a mutagenic effect of the test item
- the increases were reproducible between replicate plates and were observed in both main tests (when treatment conditions were the same)
- the increases were statistically significant
- the increases were not directly related to increased growth of the non-revertant bacteria
Statistics:
The numbers of revertant colonies at each treatment test point in the main tests were compared to the corresponding negative control values using the Analysis of Variance test. When this test showed statistically significant differences in the data, Dunnett’s test was used to determine the statistical significance of increases and decreases in the numbers of revertant colonies for each set of triplicate plates. The statistical analyses were performed with SAS® procedures (version 8.2) described in SAS/STAT® User’s Guide, SAS OnlineDoc®, 1999, SAS Institute Inc., Cary, North Carolina 27513, USA.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:

RANGE-FINDING/SCREENING STUDIES: The test item was not toxic to the test bacteria, either in the absence or presence of S-9 mix: no marked reductions in the number of revertant colonies or growth of the background lawn of non-revertant bacteria were observed, compared to the negative control plates.
On the basis of these results, 5000 ug test substance/plate was chosen as the highest dose level for the main tests.
COMPARISON WITH HISTORICAL CONTROL DATA: The negative and positive control values were compatible with the historical control values for this laboratory.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
It was concluded, that the results of this Ames test study gave no indication of mutagenic activity of alpha-amylase.
Executive summary:

Alpha-amylase was tested in two independent main tests. The ‘treat and plate’ treatment method was used in each test to avoid the possibility that bio-available histidine in the test item might compromise the test. Five dose levels (50, 160, 500, 1600, and 5000 μg/plate) were tested. Aliquots (200 μl/plate) of formulations of the test item mixed with sterile saline (0.9 % NaCl) were added to the bacteria. All dose levels are expressed in terms of the total protein content of the test item supplied, being 46 mg/ml. Negative control plates were treated with sterile saline (200 μl/plate). The treatments were performed both with and without a metabolic activation system (S-9 mix).

The test item was not toxic to the test bacteria at any dose level tested, either in the absence or presence of S-9 mix: no reductions in the growth of the background lawn of non-revertant bacteria or the number of revertant colonies were observed.

No biologically significant increases in the number of revertant colonies were observed at any dose level of the test item in either main test. Small, statistically significant increases in the number of revertant colonies were observed in the first main test in TA 1537 treated at 1600 and 5000 μg/plate in the presence of S-9 mix. These increases were not considered to be biologically significant and they did not meet the stated criteria for a mutagenic effect of the test item because they were too small (just 1.53 and 1.61-fold higher than the corresponding negative control values, respectively) and they were not reproduced in the second main test. No other statistically significant increases in the number of revertant colonies were observed in any test strain after treatment with the test item at any dose level either with or without S-9 mix.

The results obtained with the negative and positive controls demonstrated the sensitivity of the tests and the efficacy of the S-9 mix metabolic activation system.

Based on the results obtained in this study, it is concluded that alpha-amylase is not mutagenic in the Ames test.