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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)

Data source

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

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid: viscous

Method

Target gene:
uvrB-
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix
Test concentrations with justification for top dose:
The test item was tested using the following method. Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were initially assayed
in triplicate against each tester strain, using the direct plate incorporation method. However, bacterial tester strains TA100 and TA1535 dosed in the absence of
S9-mix showed excessive toxicity after the first experiment (resulting in an insufficient number of non-toxic doses) and, therefore these particular strains had
to be repeated employing an amended test item dose range as follows:
Salmonella strains TA100 and TA1535 (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.

The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
Salmonella strains TA100, TA1535 and TA1537 (absence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
Vehicle / solvent:
Dimethyl sulphoxide
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
The five strains of bacteria used, and their mutations, are defined above.
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the
histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to
larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the
strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers
ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA
repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would
normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987
All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in
nutrient broth (Oxoid Limited; lot number 1712138 07/20) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity
with titres determined by viable count analysis on nutrient agar plates.

Experimental Design and Study Conduct
Test Item Preparation and Analysis
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house.
Dimethyl sulphoxide was therefore selected as the vehicle.
The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. No
correction was made for purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement
of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method

Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
Salmonella strains TA100, TA1535 and TA1537 (absence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.

Salmonella strains TA100, TA1535 and TA1537 (presence of S9) and TA98 (absence and presence of S9) and E.coli strain WP2uvrA (absence of S9):
0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
E.coli strain WP2uvrA (presence of S9):
0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.

Eight test item dose levels per bacterial tester strain were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item
following the change in test methodology from plate incorporation to pre-incubation.

Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer. 3.3.2.4 Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were
required, predominantly due to revertant colonies spreading slightly, thus distorting the actual
plate count.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation.
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 μg/plate.
Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates.
Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Acceptability Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
Combined historical negative and solvent control ranges for 2014 and 2015 are presented in Appendix 1.
All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix.
All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.
Evaluation criteria:
Evaluation Criteria
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and
Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response (Cariello and
Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met. Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

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:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and
the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

Experiment 2.
In the initial first experiment the maximum dose level of the test item was selected as the maximum recommended dose level of 5000 μg/plate. However, two of the Salmonella
strains showed excessive toxicity after the first experiment (resulting in an insufficient number of non-toxic doses) and had to be repeated employing the toxic limit of test item as
the maximum dose. In the first mutation test (plate incorporation method) the test item induced toxicity evident as visible reductions in the growth of the bacterial background lawns
of all of the tester strains, initially from 50 μg/plate in the absence of metabolic activation (S9-mix) and 150 μg/plate in the presence of S9-mix. Consequently the toxic limit of the test
item was employed as the maximum dose level in the second mutation test. The test item induced a similar toxic response after employing the pre-incubation method in the second
mutation test with weakened bacterial background lawns noted in the absence of S9-mix from 50 μg/plate (TA100, TA1535, TA98 and TA1537) and 150 μg/plate (WP2uvrA). In the
presence of S9-mix, weakened bacterial lawns were noted from 150 μg/plate (TA100, TA1535 and TA1537) and 500 μg/plate (TA98 and WP2uvrA). No test item precipitate was
observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without
metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the
bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases
in revertant colony frequency were observed in the first mutation test at 50 μg/plate (TA100) and 15 μg/plate (TA1535) in the presence of S9-mix only. These increases were considered
to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant
dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.3 times the concurrent vehicle controls.
The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies generally within the normal range. A single count for WP2uvrA (vehicle control dosed in the absence
of S9-mix after the second mutation test) was just below the minimum level. This count was still considered acceptable as the other vehicle and untreated control counts were within
expected range and the tester strain responded very well with the respective positive controls in both the presence and absence of S9-mix. All of the positive control chemicals used in the
test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were
validated.

Applicant's summary and conclusion

Conclusions:
Benzylated Polyamine was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with Benzylated Polyamine

using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. Benzylated Polyamine was considered to be non-mutagenic under the conditions of this test.