Registration Dossier

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:
2018
Report Date:
2018

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
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:
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the OECD TG 471 maximum recommended dose level).
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, two bacterial strains (TA100 dosed in the absence and presence of metabolic activation (S9-mix) and TA1537
dosed in the absence of metabolic activation (S9-mix)) exhibited excessive toxicity and required a repeat experiment employing an amended test item dose
range of 0.05, 0.15, 0.5, 1.5, 5, 15, 50 and 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:
Test Item Preparation and Analysis
The test item was immiscible in sterile distilled water at 50 mg/mL and acetone at 100 mg/mL but was fully miscible in dimethyl sulphoxide and dimethyl formamide at
50 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was selected as the vehicle.
The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in pre-dried dimethyl sulphoxide by mixing on a vortex mixer and
sonication for 10 minutes at 40 °C. The Sponsor confirmed prior to starting the study that no correction for test item purity was required.
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.
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain
culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. 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.
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).
Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was as follows:
Salmonella strains TA100 and TA1537 (without S9): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, Salmonella strains TA100 and TA1537 (with S9) and TA1535 and TA98 (without S9): 0.05,
0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
E.coli strain WP2uvrA (with and without S9) and Salmonella strains TA1535 and TA98 (with S9): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both 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
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of 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 (see 3.3.3.2) 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.
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:
cytotoxicity
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:
cytotoxicity
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:
cytotoxicity
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:
cytotoxicity
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:
cytotoxicity
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. A couple of individual revertant counts for TA100 (negative controls) were marginally above the in-house vehicle and untreated
historical control maxima. These counts were 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. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the
normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the
sensitivity of the assay and the efficacy of the S9-mix were validated.
Experiment 1 (plate incorporation)
The maximum dose level of the test item in the first experiment was initially selected as the OECD TG 471 recommended dose level of 5000 μg/plate. However, two bacterial strains
(TA100 dosed in the absence and presence of metabolic activation (S9-mix) and TA1537 dosed in the absence of metabolic activation (S9-mix)) exhibited excessive toxicity and
required a repeat experiment where the toxic limit of the test item was employed as the maximum concentration.
The test item induced a toxic response in the first mutation test with weakened bacterial background lawns initially noted in the absence of S9-mix from 50 μg/plate (TA100 and
TA1537), 150 μg/plate (TA1535 and TA98) and 500 μg/plate (WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were initially noted from 50 μg/plate
(TA100), 150 μg/plate (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 metabolic activation (S9-mix). There were no biologically relevant 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 statistical value was noted in Experiment 1 (TA100 at 0.5 μg/plate in the presence of
S9-mix), however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.
Experiment 2 (pre-incubation)
Based on the results of Experiment 1, the toxic limit of the test item was employed as the maximum concentration in the second mutation test (pre-incubation method).
The test item induced a stronger toxic response in the second mutation test with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 μg/plate (TA100
and TA1537), 15 μg/plate (TA1535 and TA98) and 150 μg/plate (WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were initially noted from 50 μg/plate (TA100)
and 150 μg/plate (TA98, TA1537, WP2uvrA and TA1535).
No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).
There were no biologically relevant 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 2 (pre-incubation method). Minor statistical values were noted (TA98 at 0.15, 1.5 and 5 μg/plate in the presence of S9-mix), however
these responses were within the in-house historical vehicle/untreated control range for the strain and were, therefore considered of no biological relevance.

Applicant's summary and conclusion

Conclusions:
In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 3-aminopropyldiethylamine and 2-piperazin-1-ylethylamine did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test the material was concluded to be non-mutagenic.
Executive summary:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 3-aminopropyldiethylamine and 2-piperazin-1-ylethylamine  did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test the material was concluded to be non-mutagenic.