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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

272-056-6

EC Name:

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

Cas Number:

68672-66-2

Molecular formula:

C32H33N3O5S

IUPAC Name:

(2Z)-2-({[1-(tert-butoxy)-2-methyl-1-oxopropan-2-yl]oxy}imino)-2-{2-[(triphenylmethyl)amino]-1,3-thiazol-4-yl}acetic acid

Test material form:

solid: particulate/powder

Details on test material:

white powder

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

Method

Species / strain

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.

Controls

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)

Results and discussion

Test results

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

Applicant's summary and conclusion

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.