Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 August 2017 - 07 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Bernel Ester TCC (CAS number 16502-99-1), batch number ESTS185/17 (P7803), was a clear liquid. It was received on 30 June 2017 and stored at 15-25°C, protected from light. Purity was stated as 94.4% and the retest date was given as 23 January 2019, see Certificate of Analysis. The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor.

Method

Target gene:
Histidine locus
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline
Metabolic activation:
with and without
Metabolic activation system:
mammalian liver post-mitochondrial fraction (S-9)
Test concentrations with justification for top dose:
Preliminary solubility data indicated that Bernel Ester TCC was soluble in N-methyl-2-pyrrolidone (NMP) at concentrations equivalent to at least 100 mg/mL. A maximum concentration of 5000 μg/plate was selected for Mutation Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997). A maximum concentration of 5000 μg/plate was also selected for Mutation Experiment 2.
Mutation Experiment 1 (S-9 +-): 5, 16, 50, 160, 500, 1600, 5000.
Mutation Experiment 2 (S-9 +-): 5,16, 50, 160, 300, 625, 1250, 2500, 5000.
Vehicle / solvent:
NMP.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene
Details on test system and experimental conditions:
Test System
The test system was suitably labelled to clearly identify the study number, bacterial strain, test article concentration (where appropriate), positive and vehicle controls, absence or presence of S-9 mix.
Mutation Experiments
Bernel Ester TCC was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45±1°C:
• 0.1 mL bacterial culture
• 0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
• 0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted (see Colony Enumeration Section).
As the results of Mutation Experiment 1 were negative, treatments in the presence of S-9 in Mutation Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay.
Volume additions for the Mutation Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle N-Methyl-2-pyrrolidone (NMP) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise ccurred.
Toxicity Assessment
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.
Colony Enumeration
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or splits in agar or precipitation affected the accuracy of the automated counter.
Evaluation criteria:
Acceptance Criteria
The assay was considered valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges.
2. The positive control chemicals induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between ifferent strains, and an active S-9 preparation.
Evaluation Criteria
For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. Any observed response was reproducible under the same treatment conditions.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.
Statistics:
Treatment of Data
Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the laboratory’s historical control ranges (Sections 7.3 and 7.4). Data were considered acceptable if the vehicle control counts fell within the calculated historical control ranges and the positive control plate counts were comparable with the historical control ranges.
The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate). However, adequate interpretation of biological relevance was of critical importance.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
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 specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
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 specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS
Toxicity, Solubility and Concentration Selection
Details of all treatment solution concentrations and final Bernel Ester TCC concentrations are provided in the Test Article Section.
Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Bernel Ester TCC at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity in the form of a slight thinning of background bacterial lawn was observed at 5000 μg/plate in all strains in the absence and presence of S-9.
Mutation Experiment 2 treatments of all the tester strains were performed in the absence and presence of S-9. The maximum test concentration of 5000 μg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160-5000 μg/plate except strain TA98 which was also treated at 5, 16 and 50 μg/plate in the absence and presence of S-9 in order to investigate reproducibility of increases in revertant numbers observed in the absence of S-9 in Mutation Experiment 1. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no evidence of toxicity was observed.
Precipitation was observed on all Mutation Experiment 2 test plates at 2500 μg/plate and above in the absence and presence of S-9.
Data Acceptability and Validity
The individual mutagenicity plate counts were averaged to give mean values, which are presented in Section 8. From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges with the exception of one individual vehicle control count each in strain TA1535 in the absence of S-9 and strain TA98 in the presence of S-9, which were outside the 99% reference ranges for these strains. Since these were only marginally outside the 99% reference ranges and the means and remaining replicates fell within the reference ranges, these data were considered to be characteristic of the strains and accepted as valid. The positive control chemicals all induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle controls confirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.
Mutation
Following treatments of all the test strains in the absence and presence of S-9, only Mutation Experiment 1 treatments of strain TA98 in the absence of S-9 at 50 and 1600 μg/plate resulted in an increase in revertant numbers that ≥2-fold the concurrent control. These increases were small in magnitude with no indication of a concentration relationship. Accordingly these increases were considered to have been due to normal biological variability and not evidence of mutagenic activity (Table 8.1).
No other increases in revertant numbers were observed that were ≥1.5-fold (TA102), ≥2-fold (TA98 and TA100) or ≥3-fold (TA1535 and TA1537) the concurrent control. This study was considered therefore to have provided no clear evidence of any Bernel
Ester TCC mutagenic activity in this assay system.

Applicant's summary and conclusion

Conclusions:
It was concluded that Bernel Ester TCC did not induce mutation in five histidinerequiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).
Executive summary:

Bernel Ester TCC was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

All Bernel Ester TCC treatments in this study were performed using formulations prepared in N-methyl-2-pyrrolidone (NMP).

Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Bernel Ester TCC at 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus vehicle and positive controls. Following these treatments, evidence of toxicity was observed at 5000 μg/plate in all strains in the absence and presence of S-9.

Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 μg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160-5000 μg/plate except strain TA98 which was also treated at 5, 16 and 50 μg/plate in the absence and presence of S-9 in order to investigate reproducibility of increases in revertant numbers observed in the absence of S-9 in Mutation Experiment 1. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no evidence of toxicity was observed.

Precipitation was observed on all Mutation Experiment 2 test plates at 2500 μg/plate and above in the absence and presence of S-9.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Although some ≥2-fold increases in revertant numbers were observed following Bernel Ester TCC treatments of strain TA98 in the absence of S-9 at 50 and 1600 μg/plate in Mutation Experiment 1, these were not concentration-related or reproducible and were of insufficient magnitude to be considered as clear evidence of mutagenic activity in this assay system.

It was concluded that Bernel Ester TCC did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 μg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).