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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
% Total Organic Solids: 25.27%

Method

Target gene:
synthesis of the amino acid histidine
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:
S-9 mix
Test concentrations with justification for top dose:
50, 160, 500, 1600, and 5000 µg/plate. The highest dose level, 5000 µg/plate, was the maximum required by OECD 471.
Vehicle / solvent:
sterile saline (0.9% NaCl)
Controls
Untreated negative controls:
yes
Remarks:
sterile saline solution
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
cumene hydroperoxide
other: 2-aminoanthracene, TA 100, TA 98, TA 1537, and TA 1535, 2 µg/plate, with S-9 mix 2-aminoanthracene, TA 102, 4 µg/plate, with S-9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: "treat and plate" method - the bacteria were incubated with the test item, nutrient broth and buffer or S-9 mix for a treatment period of 3.5 hours and then the bacteria were washed to remove the test item and any histidine before mixing with top agar and plating on selective agar plates.
- Cell density at seeding (if applicable): 10E9 bacteria/mL

DURATION
- Preincubation period: 3.5 hours
- Exposure duration: three days at 37°C

NUMBER OF REPLICATIONS: each test point was done in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Rationale for test conditions:
‘Treat and plate’ treatment method in order to avoid the possibility that bio-available histidine in the test item might cause dose-related increases in the growth of the background lawn of non-revertant bacteria and the numbers of revertant colonies if the plate incorporation or pre-incubation treatment methods had been used.
Evaluation criteria:
The tests were considered to be valid as all of the following criteria were met:
• negative and positive control data were consistent with the historical control data for the testing 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.

The test item would have been considered to have shown no evidence of mutagenic activity if no increases in the mean number of revertant colonies which exceeded twice the negative control value were observed at any test point. The test item would have also been considered to have shown no evidence of mutagenic activity if moderately larger increases were observed that were not reproducible, not statistically significant, or which were sporadic (without a scientifically valid explanation for the dose-response relationship that involved a mutagenic effect of the test item).

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 resultsopen allclose all
Key result
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:
valid
Positive controls validity:
valid
Key result
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:
valid
Positive controls validity:
valid
Key result
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:
valid
Positive controls validity:
valid
Key result
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:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not valid
Untreated negative controls validity:
not valid
Positive controls validity:
not valid
Additional information on results:
Preliminary Test with TA 98: The test item was not toxic to the test bacteria: no marked reductions in the number of revertant colonies, or growth of the background lawn of non-revertant bacteria were observed. There was no evidence of mutagenic activity of the test item: no increases in the numbers of revertant colonies that exceeded two-fold the negative control values were observed. The highest dose level, 5000 µg/plate, was chosen for the main test based on these results.

No biologically significant increases in the number of revertant colonies were observed at any dose level of the test item in either main test. In the first main test, statistically significant increases in the number of colonies were observed in TA 1537 at the two highest dose levels with S-9 mix. In the second test, the number of ‘normal-sized colonies was similar to the negative control values at these dose levels, but many very small colonies were also observed and it was not possible to make an accurate count of the revertant colonies. In addition, there were very many minute colonies that could only be observed using a microscope, although they were larger than the colonies of the background lawn. These minute colonies were also observed at many other test points, including the negative control plates with S-9 mix of all five tester strains.

To determine whether the very small colonies were revertant colonies or not, an additional test in TA 1537 was performed. When the plates from the additional test were scored, very small colonies were again observed by eye on the plates at the two highest dose levels with S-9 mix. All of the ‘normal-sized’ colonies and 20 of the very small colonies from each plate at these dose levels were individually transferred to minimal glucose agar plates that had been supplemented with 0.5 mM D-biotin solution (200 μl/plate) using sterile plastic loops. Revertant colonies would grow on these plates, but non-revertant colonies would not. The plates were incubated at approximately 37 ºC for three days and then the numbers of colonies that had grown were counted. It was found that almost all of the normal-sized colonies were revertant colonies, while almost all of the very small colonies were not. The number of confirmed revertant colonies on these plates were similar to the negative control values. This additional test has shown conclusively that the number of confirmed revertant colonies in TA 1537 was not increased at the two highest dose levels with S-9 mix, because the extra, very small colonies seen at these test points were not revertant colonies. The increased numbers of colonies observed in Test 1 are suspected to be due to these very small non-revertant colonies, but this was not recognised when Test 1 was scored.
 
The nature of the very small colonies is unknown, but they could be derived from clumps of non-revertant bacteria that failed to separate when the bacterial pellets were resuspended after centrifugation at the end of the treatment period. They are not believed to be contaminant colonies because of their distribution in the tests.
The data presented for strain TA102 in Test 1 was obtained in a repeat test. The preceding tests with TA102 were not valid and are not presented in this report because the positive control treatments failed to produce acceptable increases in the numbers of revertant colonies, or the characteristics of the culture used were incorrect (not resistant to tetracycline).
The negative and positive control values were compatible with the historical control values for this laboratory. Two of the positive control values for strain TA 98 with S-9 mix in Test 2 and the values for TA 1537 with S-9 mix in Test 2 are just beneath the range for the historical control data. In addition, all of the positive control values for TA 1537 without S-9 mix in each test were above the historical range. The values are considered to be acceptable in each of these cases. The large increases in the number of revertant colonies produced by the positive control treatments demonstrated the sensitivity of the tests and the efficacy of the S-9 mix metabolic activation system.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this assay, the test substance has not shown any evidence of mutagenic activity in the Ames assay
Executive summary:

The objective of this assay was to assess the potential of the test substance to induce point mutations (frame-shift and base-pair) in five strains of Salmonella typhimurium: TA 98, TA 100, TA 102, TA 1535 and TA 1537. This assay was conducted in accordance with OECD Guideline No. 471.

Following a preliminary toxicity test in strain TA 98, the test substance 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 (100 μL/plate) of formulations of the test item dissolved in sterile saline (0.9 % NaCl) were added to the bacteria. All the dose levels are expressed in terms of the total protein content of the test item supplied, stated by the Sponsor to be 247.3 mg/mL. Negative control plates were treated with sterile saline (100 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: no marked reductions in the number of revertant colonies, or growth of the background lawn of non-revertant bacteria 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. Statistically significant increases in colony numbers were observed in TA1537 at the two highest dose levels with S-9 mix in the first main test. In the second main test and in an additional test, the numbers of ‘normal-sized’ colonies at these test points were similar to the negative control values. However, additional very small colonies were also present. Transferring the colonies from these plates from the additional test to minimal glucose agar plates supplemented with D-biotin confirmed that most of the normal-sized colonies were revertant, while most of the very small colonies were not. This additional test showed conclusively that the number of confirmed revertant colonies in TA 1537 was not increased at these test points, and thus that the test item is not mutagenic.

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 the test substance is not mutagenic in the Ames test.