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
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 insoluble in sterile distilled water at 50 mg/mL but was fully soluble 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, 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 5 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.
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 mcg/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 mcg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Vehicle / solvent:
simethyl 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 for Mutagenicity: Experiment 1 – Plate Incorporation Method
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 mcg/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 mcg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
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 (see 3.3.2.2) 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 (with and without S9), TA1535 and TA98 (without S9): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 mcg/plate.
E.coli strain WP2uvrA (with and without S9) and Salmonella strains TA1535 and TA98 (with S9): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 mcg/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.
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. Due to a hardware failure, Ames study manager and sorcerer
system suffered an extended downtime, resulting in manual counts being performed on all of the plates produced for Experiment 2. The plates were viewed microscopically for evidence
of thinning (toxicity).
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. These data are not given in the report.
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.
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 selected as the OECD TG 471 recommended dose level of 5000 μg/plate. 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 150 μg/plate (TA100), 500 μg/plate (TA1535, TA98 and TA1537)
and 1500 μg/plate (WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were initially noted from 500 μg/plate (TA100 and TA1537) and 1500 μg/plate
(TA1535, 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 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).
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.
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 50 μg/plate
(TA100), 150 μg/plate (TA1535, TA98 and TA1537) and 1500 μg/plate (WP2uvrA). In the presence S9-mix, weakened bacterial background lawns were initially noted from
150 μg/plate (TA100 and TA1537), 500 μg/plate (TA98 and TA1535) and 1500 μg/plate (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 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).

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 Isophoronediamine-cresyl glycidyl ether adduct 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 Isophoronediamine-cresyl glycidyl ether adduct was concluded as 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 Isophoronediamine-cresyl glycidyl ether adduct 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 Isophoronediamine-cresyl glycidyl ether adduct was concluded as non-mutagenic.