(Z)-α-[[2-(tert-butoxy)-1,1-dimethyl-2-oxoethoxy]imino]-2-(tritylamino)thiazol-4-acetic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

the substance was found to be clearly non-mutagenic in all 5 tester strains used in a Bacterial reverse mutation (Ames) assay. The in vitro cytogenetic study conducted in human peripheral blood lymphocytes was negative. The mouse lymphoma assay was also clearly negative.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 January 2018 to 20 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Batch number: G317115
Expiry: 04 March 2021 (re-test)
Appearance: White to off-white powder
Storage conditions:
15 to 25°C, desiccated, protected from light
Purity: 96.29 % w/w
Weighing factor for formulation: 1.04g contains 1.000g, as free side chain
Date received: 01 December 2017

Species / strain / cell type:

other: human lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

The following test item concentrations were selected for metaphase analysis:
In the absence of S9 mix, 3-hour treatment (additional test): 20, 50 and 70 µg/mL.
In the presence of S9 mix, 3-hour treatment: 20, 30 and 40 µg/mL.
In the absence of S9 mix, 21-hour treatment (additional test): 10, 20 and 27.5 µg/mL.

The final concentrations of 1132 Side Chain to which cells were exposed initially are given below. Preliminary toxicity test: 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µg/mL

Main tests: -S9 mix (3 hours) 10, 20, 30, 40, 50, 60 and 70 µg/mL
-S9 mix (3 hours) 20, 30, 40, 50, 60, 70 and 80 µg/mL
+S9 mix (3 hours) 5, 10, 20, 30, 40, 50 and 60 µg/mL
-S9 mix (21 hours) 5, 10, 15, 20, 25, 30 and 35 µg/mL

-S9 mix (21 hours) additional) 10, 15, 20, 25, 27.5, 30, 32.5 and 35 µg/mL

Vehicle / solvent:

DMSO was used as the vehicle control.

Untreated negative controls:

yes

Remarks:

VEHICLE CONTROL

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

VEHICLE CONTROL

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: In preliminary and main tests the cells were treated for 3 and 21 hours in the absence of S9 mix and for 3 hours in the presence of S9 mix. In the additional main test the cells were treated for 3 and 21 hours in the absence of S9 mix.
- Expression time (cells in growth medium): Cultures were grown for 48 hours before treatment

-Harvesting and Fixation
Two hours before the cells were harvested, mitotic activity was arrested by addition of Colcemid® to each culture at a final concentration of 0.1 µg/mL. After 2 hours incubation, each cell suspension was transferred to a centrifuge tube and centrifuged for 5 minutes at 500g. The supernatant was removed and the cell pellets were treated with a hypotonic solution (0.075M KCl) for a 10 minute period at between 34 to 39°C. The suspensions were centrifuged at 500g for 5 minutes, supernatant removed and the cell pellets fixed by addition of ice-cold fixative (methanol:glacial acetic acid (3:1 v/v)). Following further centrifugation the supernatant was removed and replaced with fixative; this was repeated twice until the fixative was clear.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid®

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Slide Preparation
The fixed pellets were re-suspended, then centrifuged at 500g for 5 minutes and re-suspended in a small volume of fixative. A few drops of the cell suspensions were dropped onto precleaned microscope slides and allowed to air dry.One slide was prepared per culture. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell suspensions in fixative were stored at 2 to 8°C until slide analysis was completed.

NUMBER OF CELLS EVALUATED: The prepared slides were examined by light microscopy and the incidence of mitotic cells per 1000 cells assessed.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 150 cells examined

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Microscopic Examination
The prepared slides were examined by light microscopy and the incidence of mitotic cells per 1000 cells assessed. Slides were assessed for mitotic index (except when clear evidence of overt toxicity was observed, or in cultures where there were no signs of cytotoxicity).

- Determination of polyploidy and endoreplication: he incidence of polyploid and endoreduplicated cells (i.e. the ploidy status) were each recorded as a percentage of the 150 metaphases analyzed per slide, independently from the analysis for chromosome aberrations.

Evaluation criteria:

Acceptance Criteria:The concurrent vehicle control was considered acceptable for addition to the laboratories historical vehicle control database (lie below or close to the upper control limit). Where concurrent vehicle control data fell outside the 95% confidence limit it may be acceptable for inclusion in the historical control distribution as long as the data are not extreme outliers and there is evidence that the test system is ‘under control’ and there is evidence of no technical or human failure.Concurrent positive controls induced a response that were compatible with the laboratories historical positive control database and produced statistically significant increases compared with the concurrent vehicle control. Assessment Criteria:Providing that all of the acceptance criteria have been met, the test item was considered to be clearly positive if, in any of the experimental conditions examined: At least one of the test concentrations exhibited a statistically significant increase compared with the concurrent vehicle control.The increase was dose-related when evaluated with an appropriate trend test.Any of the results are outside the distribution of the historical vehicle control data (above the upper 95% confidence limit).If all of these criteria were met, the test item was considered able to induce chromosome breaks and/or gain or loss in the test system. Providing that all of the acceptance criteria have been met, a negative response was claimed if, in all of the experimental conditions examined: None of the test concentrations exhibited a statistically significant increase compared with the concurrent vehicle control. There was no concentration-related increase when evaluated with an appropriate trend test.All results are inside the distribution of the historical vehicle control data (within the 95% confidence limits). If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.

Statistics:

The following computerized system was used for statistical analysis: SAFEStat (SAS statistical applications for end users) Chromosome Aberrations application which was developed in SAS (SAS INSTITUTE 2002)

Key result

Species / strain:

lymphocytes: Human Lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

It is concluded that the test item 1132 Side Chain has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Executive summary:

A study was performed to assess the ability of 1132 Side Chain to cause structural chromosome aberrations in human lymphocytes cultured in vitro.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test item both in the absence and presence of exogenous metabolic activation (S9 mix).  Vehicle and positive control cultures were also included where appropriate.  Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage. The study consisted of a preliminary toxicity test, a main test and an additional main test.  In preliminary and main tests the cells were treated for 3 and 21 hours in the absence of S9 mix and for 3 hours in the presence of S9 mix. In the additional main test the cells were treated for 3 and 21 hours in the absence of S9 mix.  The mitotic index was assessed for all cultures to determine cytotoxicity. Based on the data from the preliminary toxicity test, test item concentrations were selected for the main test.

In the main test, justification for the highest analyzed concentration was based on cytotoxicity. The following test item concentrations were selected for metaphase analysis: In the absence of S9 mix, 3-hour treatment (additional test):  20, 50 and 70 µg/mL. In the presence of S9 mix, 3-hour treatment:  20, 30 and 40 µg/mL. In the absence of S9 mix, 21-hour treatment (additional test):  10, 20 and 27.5 µg/mL. Under the conditions described above, 1132 Side Chain caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations (excluding gaps), at any analyzed concentration, when compared with the vehicle control. There was no evidence of a linear dose-concentration relationship.  The mean proportion of cells with chromosomal aberrations (excluding gaps) for the vehicle and test item treated

cultures were within or close to the laboratory historical control range. No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, under any treatment condition, when compared with the vehicle control.  Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix. It is concluded that 1132 Side Chain has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system, under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02-05-2018 to 25-07-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: 1132 Side Chain CAS: 68672-66-2
Physical state/Appearance: White powder
Batch Number: G317115
Purity: 96.29%
Expiry Date: 04 September 2020
Storage Conditions: Room temperature in the dark over silica gel

Target gene:

S. typhimurium: Histidine locus
E.coli: Tryptophan operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: rfa-, uvrB-, pKM101 in TA 98 and TA100

Metabolic activation:

with and without

Metabolic activation system:

S9 Microsomal fractions (CD Sprague-Dawley), male, phenobarbital/B-Naptha flavone induced

Test concentrations with justification for top dose:

1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, The highest concentration tested was one that allowed maximum exposure up to 5000 µg per
plate of a formulation that was freely soluble.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene with S9; 1 µg/plate for TA100 2 µg/plate for TA1535 and TA1537 10 µg/plate for WP2uvrA

Details on test system and experimental conditions:

The negative (untreated) controls were performed to assess the spontaneous revertant colony
rate. The solvent and negative controls were performed in triplicate.
The positive control items used demonstrated a direct and indirect acting mutagenic effect
depending on the presence or absence of metabolic activation. The positive controls were
performed in triplicate. The sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (tested in
singular prior to Experiment 1)

S9-Mix and Agar
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for theduration of the test.
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL

A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented,
top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the
sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each
experiment.

Media
Top agar was prepared using 0.6% Bacto agar (lot number 6270923 08/2021) and 0.5%
sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan
solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were
purchased from SGL Ltd (lot numbers 47504 06/2018 and 47623 07/2018).

Test System and Supporting Information
Bacteria
The five strains of bacteria used, and their mutations, are as follows:
Salmonella typhimurium
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

Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution

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:
• British Industrial Biological Research Association, on a nutrient agar plate, on17 August 1987
• Trinova Biochem GmbH on 27 June 2017
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 2104309 04/2022) 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.

Rationale for test conditions:

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), Green and Muriel (1976) and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per
plate in the vehicle and untreated controls (negative controls). Typical 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
These values will also be confirmed against current in-house historical control profiles to
further validate acceptability. Although the number of spontaneous revertants can be
expected to fall within the ranges, they may occasionally fall outside these. Combined
historical negative and solvent control ranges for 2016 and 2017 are presented in Appendix 1.
All tester strain cultures should be in the range of 0.9 to 9 x 10e9 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 (S9-mix). The
historical ranges of the positive control reference items for 2016 and 2017 are presented in
Appendix 1.
A minimum of five concentrations of test item is required, the highest usually being at the
toxic or maximum recommended dose limit (5000 µg/plate). If the test item is non-toxic to
the bacterial strains, it will, if possible, be tested at a maximum of 5000 µg/plate regardless of
solubility.
There should be no evidence of excessive contamination.

Evaluation criteria:

If the results of the experiments are clearly negative or positive, the study will be concluded as
such. Reproducibility of any apparent effect may be taken into account, particularly when the
results are considered weakly positive or when dose-dependent elevations in revertant colony
numbers, not satisfying the criteria for a positive response, are observed.
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. A fold increase greater than two times the concurrent solvent control for TA100,
TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if
accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

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.

Key result

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

Key result

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

Key result

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

Key result

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

Key result

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

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.  

The individual plate counts, the mean number of revertant colonies and the standard

deviations, for the test item, positive and vehicle controls, both with and without metabolic

activation (S9-mix), are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and

Table 5 for Experiment 2.  

A history profile of vehicle, untreated and positive control values (reference items) is

presented in Appendix 1.

Experiment 1 (plate incorporation) – Table 2 and Table 3

Controls

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and

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.

Results

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.  

There was no visible reduction in the growth of the bacterial background lawn at any dose

level, either in the presence or absence of metabolic activation (S9-mix), although small

decreases in TA1537 revertant colony were noted at the upper dose levels.

A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate in

both the presence and absence of metabolic activation (S9-mix).  This observation did not

prevent the scoring of revertant colonies.

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) – Table 4 and Table 5

Controls

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and

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.

Results

The maximum dose level of the test item in the second experiment was the same as for

Experiment 1 (5000 µg/plate).  

There was no visible reduction in the growth of the bacterial background lawn at any dose

level, either in the presence or absence of metabolic activation (S9-mix).

A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate in

both the presence and absence of metabolic activation (S9-mix).  This observation did not

prevent the scoring of revertant colonies.

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).

Conclusions:

1132 Side Chain CAS: 68672-66-2 was not mutagenic in the bacterial mutation assay, when
tested in either the presence or absence of S9-mix. The maximum concentration was 5000 µg
per plate, the maximum concentration in accordance with current guidelines in the presence
and absence of S9-mix respectively.

Executive summary:

The purpose of this study was to assess the potential of 1132 Side Chain CAS: 68672-66-2 to

induce gene mutations (base pair substitutions and frameshift mutations) in vitro in bacterial

strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and Escherichia coli

(WP2uvrA).

The test method was designed to be compatible with the guidelines for bacterial mutagenicity

testing published by the major Japanese Regulatory Authorities including METI, MHLW and

MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation

Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008,

the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012;

77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse

Mutation Test.

Study Design

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli

strain WP2uvrA were treated with the test item 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 (10% liver S9 in standard co-factors).

The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was

1.5 to 5000 µg/plate.  The experiment was repeated on a separate day (pre-incubation

method) using fresh cultures of the bacterial strains and fresh test item formulations.  The

dose range was amended following the results of Experiment 1 and was 5 to 5000 µg/plate.

Seven test item concentrations per bacterial strain were selected in Experiment 2 in order to

achieve both four non-toxic dose levels and the potential toxicity of the test item following

the change in test methodology.

Results

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.

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.  There was no visible reduction in the

growth of the bacterial background lawn at any dose level, either in the presence or absence

of metabolic activation (S9-mix), in the first mutation test (plate incorporation method),

although small decreases in TA1537 revertant colony were noted at the upper dose levels.

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was

employed in the second mutation test (pre-incubation method).  There was neither a visible

reduction in the growth of the bacterial background lawn at any dose level, either in the

presence or absence of metabolic activation (S9-mix) or any substantial decreases in revertant

colony frequency.  

A test item precipitate (particulate in appearance) was noted at and above 1500 µg/plate in

both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This

observation did not prevent the scoring of revertant colonies.  

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) 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).  

Conclusion

1132 Side Chain CAS: 68672-66-2 was not mutagenic in the bacterial mutation assay, when

tested in either the presence or absence of S9-mix.  The maximum concentration was 5000 µg

per plate, the maximum concentration in accordance with current guidelines in the presence

and absence of S9-mix respectively.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-07-2018 to 07-08-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Identification: 1132 Side Chain CAS 68672-66-2
Physical state/Appearance: White powder
Batch Number: G317115
Purity: 96.29%
Expiry Date: 04 September 2020
Storage Conditions: Room temperature, in the dark, over silica gel

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 fraction, Male Sprague Dawley rats, Phenobarbitone/B-Napthaflavone induced.

Test concentrations with justification for top dose:

0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000 µg/mL; the test item had a molecular weight greater than 200, therefore the maximum recommended dose level was set at 2000 µg/mL.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the
MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were
originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and
were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were
routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM)
supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate
(1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at
37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and
were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor
horse serum (R10), and without serum (R0), are used during the course of the study. Master
stocks of cells were tested and found to be free of mycoplasma.

Microsomal Enzyme Fraction
Lot No. PB/βNF S9 29/03/18 was used in this study, and was pre-prepared in-house (outside
the confines of the study) following standard procedures. Prior to use, each batch of S9 is
tested for its capability to activate known mutagens in the Ames test and a certificate of S9
efficacy is presented in Appendix 2.
S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and
MgCl2 (8 mM) in R0.
20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary
Toxicity Test and Main Mutation Test.

Experimental Design and Study Conduct
Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but
significant rate. Before the stocks of cells were frozen they were cleansed of homozygous
(TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained
Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine
(22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e.
THMG without Methotrexate) before being returned to R10 medium.

Test Item Preparation
Following solubility checks performed in-house, the test item was accurately weighed and
formulated in DMSO culture media prior to serial dilutions being prepared. The test item had
a molecular weight greater than 200, therefore the maximum recommended dose level was
set at 2000 µg/mL. The purity of the test item was 94.905 % and was accounted for during
dose formulation. There was no marked change in pH when the test item was dosed into
media and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991).
No analysis was carried out to determine the homogeneity, concentration or stability of the
test item formulation. The test item was formulated within two hours of it being applied to
the test system. It is assumed that the formulation was stable for this duration. This is an
exception with regard to GLP and has been reflected in the GLP compliance statement.

Control Preparation
Vehicle and positive controls were used in parallel with the test item. Solvent (DMSO)
exposure groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) (Sigma
batch BCBV9352, purity treated as 100%, expiry 10.05.23) at 400 µg/mL and 150 µg/mL,
respectively, was used as the positive control in the 4-hour and 24-hour exposure groups in
the absence of metabolic activation. Cyclophosphamide (Acros Organics batch A0373263,
purity 97%, Expiry 22.02.19) at 1.5 µg/mL was used as the positive control in the presence of
metabolic activation. The positive controls were formulated in DMSO.

Test Procedure
Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 10e5 cells/mL, using a 4 hour
exposure period both with and without metabolic activation (S9), and at 1.5 x 10e5 cells/mL
using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity
test was 7.81 to 2000 µg/mL for all three of the exposure groups. Following the exposure
periods the cells were washed twice with R10, resuspended in R20 medium, counted and then
serially diluted to 2 x 10e5 cells/mL, unless the mean cell count was less than 3 x 10
cells/mL in which case all the cells were maintained.
The cultures were incubated at 37°C with 5% CO2 in air and sub-cultured after 24 hours by
counting and diluting to 2 x 10e5 cells/mL, unless the mean cell count was less than 3 x 10
cells/mL in which case all the cells were maintained. After a further 24 hours the cultures
were counted and then discarded. The cell counts were then used to calculate Suspension
Growth (SG) values. The SG values were then adjusted to account for immediate post
exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle
control performed to give a percentage Relative Suspension Growth (%RSG) value.

3.3.4.2 Main Mutation Test
Several days before starting the experiment, an exponentially growing stock culture of cells
was set up so as to provide an excess of cells on the morning of the experiment. The cells
were counted and processed to give 1 x 10e6 cells/mL in 10 mL aliquots in R10 medium in
sterile plastic universals for the 4-hour exposure groups in both the absence and presence of
metabolic activation, and 0.3 x 10e6 cells/mL in 10 mL cultures were established in 25 cm2
tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation.
The exposures were performed in duplicate (A + B), both with and without metabolic
activation (2% S9 final concentration) at eight dose levels of the test item (2 to 32 µg/mL
for all three exposure groups), vehicle and positive controls. To each universal
was added 2 mL of S9 mix if required, 0.2 mL of the exposure dilutions, (0.2 mL or 0.15 mL
for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10
was used for the 24 hour exposure group).
The exposure vessels were incubated at 37°C for 4 or 24 hours with continuous shaking using
an orbital shaker within an incubated hood.

Assessments
Measurement of Survival, Viability and Mutant Frequency
At the end of the exposure periods, the cells were washed twice using R10 medium then
resuspended in R20 medium at a cell density of 2 x 10e5 cells/mL. The cultures were
incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the expression
period of two days, by counting and dilution to 2 x 10e5 cells/mL, unless the mean cell count
was less than 3 x 10e5 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 10e4 cells/mL and plated for
mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL
5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to
10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that
gives an indication of post exposure toxicity during the expression period as a comparison to
the vehicle control, and when combined with the Viability (%V) data, a Relative Total
Growth (RTG) value.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to twelve days
incubation at 37°C with 5% CO2 in air. The number of positive wells (wells with colonies)
was recorded together with the total number of scorable wells (normally 96 per plate). The
numbers of small and large colonies seen in the TFT mutation plates were also recorded as
the additional information may contribute to an understanding of the mechanism of action of
the test item (Cole et al., 1990). Colonies are scored manually by eye using qualitative
judgment. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface
of the well and are generally no more than one or two cells thick. In general, all colonies less
than 25% of the average area of the large colonies are scored as small colonies. Small
colonies are normally observed to be more than two cells thick. To assist the scoring of the
TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution,
2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation
plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by
viable cells and metabolised to give a brown/black color, thus aiding the visualization of the
mutant colonies, particularly the small colonies.

Rationale for test conditions:

Results from the preliminary toxicity test were used to set the test item dose levels for the
mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL
or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or
variable composition (UVCB) the upper dose level may need to be higher and the
maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless
of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should
lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival.
This optimum upper level of toxicity was confirmed by an IWGT meeting in New
Orleans, USA (Moore et al., 2002).

Evaluation criteria:

Dose selection for the mutagenicity experiments was made using data from the preliminary
toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is
approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity).
Relative Total Growth (RTG) values are the primary factor used to designate the level of
toxicity achieved by the test item for any individual dose level. However, under certain
circumstances, %RSG values may also be taken into account when designating the level of
toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded
from the data analysis, as any response they give would be considered to have no biological
or toxicological relevance.
An approach for defining positive and negative responses is recommended to assure that the
increased MF is biologically relevant. In place of statistical analysis generally used for other
tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above
the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10e-6
, which
is based on the analysis of the distribution of the vehicle control MF data from participating
laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly
positive if, in any of the experimental conditions examined, the increase in MF above the
concurrent background exceeds the GEF and the increase is concentration related (e.g., using
a trend test). The test chemical is then regarded as positive in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly
negative if, in all experimental conditions examined there is no concentration related
response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is
then regarded as negative this test system.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in line with assay requirements

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 7.81 to 2000 µg/mL.

The results for the Relative Suspension Growth (%RSG) were as follows:

Dose µg/mL	% RSG (-S9) 4 hour exposure	% RSG (+S9) 4 hour exposure	% RSG (-S9) 24 hour exposure
0	100	100	100
7.81	112	75	66
15.63	41	35	50
31.25	1	4	3
62.5	0	1	0
125	0	0	0
250	0	0	0
500	0	0	0
1000	0	0	0
2000	0	0	0

There was evidence of marked dose-related reductions in the Relative Suspension Growth

(%RSG) of cells treated with the test item in all three of the exposure groups when compared

to the concurrent vehicle control groups.  Precipitate of the test item was observed at and

above 250 µg/mL in the 4-hour exposure group in the absence of metabolic activation, and at

and above 500 µg/mL in the 4-hour exposure group in the presence of metabolic activation

and the 24-hour exposure group in the absence of metabolic activation.  However, the

precipitate was observed at dose levels beyond the onset of marked test item-induced toxicity

in all three of the three exposure groups.  Therefore, the maximum dose level selected for the

main test was limited by test-item induced toxicity, as recommended by the OECD guideline.

As was seen previously in the preliminary toxicity test, there was evidence of marked dose

related toxicity following exposure to the test item in all three of the exposure groups (Tables

3, 6, and 9).  Based on the RTG and / or %RSG values observed, optimum levels of toxicity

were achieved in all three of the exposure groups.  There was evidence of very modest

reductions in viability (%V) in all three of the exposure groups, indicating that residual

toxicity had occurred (Tables 3, 6, and 9).  The concentrations of 28 and 32 µg/mL in the

4-hour exposure groups in both the absence and presence of metabolic activation, and

32 µg/mL in the 24-hour exposure in the absence of metabolic activation, were not plated out

for viability or 5-TFT resistance due to excessive toxicity.  The toxicity observed at

24 µg/mL in the 4-hour exposure groups in both the absence and presence of metabolic

activation exceeded the upper acceptable limit of 90%, therefore, these doses were excluded

from the statistical analysis (Tables 3 and 6).  Acceptable levels of toxicity were seen with

the positive control substances (Tables 3, 6, and 9).

The vehicle controls had mutant frequency values that were considered acceptable for the

L5178Y cell line at the TK +/- locus.  The positive controls produced marked increases in the

mutant frequency per viable cell achieving the acceptability criterion, indicating that the test

system was operating satisfactorily, and that the metabolic activation system was functional

(Tables 3, 6, and 9).

The test item did not induce any toxicologically significant increases in the mutant frequency

x 10e-6 per viable cell at any of the dose levels, in any of the three exposure groups.  The GEF

value was also not exceeded in any of the three exposure groups.

The numbers of small and large colonies and their analysis are presented in Tables 4, 7, and

10.

Conclusions:

The test item, 1132 Side Chain CAS 68672-66-2, did not induce any increases in the mutant
frequency in any of the three exposure groups at the TK +/- locus in L5178Y cells that
exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6. 1132 Side Chain CAS 6867266-2
was exposed up to dose levels that achieved optimum levels of toxicity in all three of the exposure groups.
Consequently 1132 Side Chain CAS 68672-66-2 gave a negative result in this assay.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential of

1132 Side Chain CAS 68672-66-2 to induce non-lethal gene mutations and chromosome

damage on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

The method was designed to be compatible with the OECD Guidelines for Testing of

Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine

Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No.

440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

Methods

One Main Mutation Test was performed.  In this main test, L5178Y TK +/- 3.7.2c mouse

lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item

at eight dose levels in duplicate, together with vehicle (dimethyl sulfoxide (DMSO)), and

positive controls using 4 hour exposure groups both in the absence and presence of metabolic

activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a

preliminary toxicity test at a concentration range of 7.81 to 2000 µg/mL.  The maximum dose

level selected for the main test was limited by test-item induced toxicity, as recommended by

the OECD guideline.  The dose levels plated for viability and expression of mutant colonies

were as follows:

Group	Concentration of 1132 Side Chain CAS 68672-66-2 (µg/mL) plated for viability and mutant frequency
4-hour without S9	2, 4, 8, 16, 20, 24
4-hour with S9 (2%)	2, 4, 8, 16, 20, 24
24-hour without S9	4, 8, 16, 20, 24, 28

Results

The vehicle control cultures had mutant frequency values that were acceptable for the

L5178Y cell line at the TK +/- locus.  The positive control substances induced marked

increases in the mutant frequency within the historical control data range, sufficient to

indicate the satisfactory performance of the test and of the activity of the metabolizing system

(Table 1).

The test item did not induce any toxicologically significant increases in any of the three

exposure groups and the GEF value was not exceeded at any test item concentration

(Table 1).

Conclusion

The test item, 1132 Side Chain CAS 68672-66-2, did not induce any increases in the mutant

frequency in any of the three exposure groups at the TK +/- locus in L5178Y cells that

exceeded the Global Evaluation Factor (GEF) of 126 x 10^-6.  1132 Side Chain CAS 6867266-2

was exposed up to dose levels that achieved optimum levels of toxicity in all three of the  exposure groups.

Consequently 1132 Side Chain CAS 68672-66-2 gave a negative result in  this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo testing of the test substance is not required, based upon the negative results obtained in the in vitro test battery.

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based upon the existing in vitro results, which were negative the battery of three studies, no further in vivo studies are required according to the Integrated Testing Strategy. The criteria to classify the substance for Germ Cell Mutagenicity, in accordance with 1272/2008/EC are not met.