Reaction mass of 2-[4-[(Hexahydro-2,4,6-trioxo-5-pyrimidyl) azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1) and Lithium 2-[4-[(hexahydro-2,4,6-trioxopyrimidin-5-yl)azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-05 to 2017-06-29

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 July 2016

Deviations:

yes

Remarks:

The test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Regulation (EC) No 440/2008 of 30 May 2008: In vitro Mammalian Cell Micronucleus Test, No B.49; No L 193

Version / remarks:

Commission Regulation (EC) No 640/2012 of 06 July 2012

Deviations:

no

Principles of method if other than guideline:

The following alterations from the guidelines were performed:
A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-[4-[(Hexahydro-2,4,6-trioxo-5-pyrimidyl)azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1) and Lithium 2-[4-[(hexahydro-2,4,6-trioxopyrimidin-5-yl)azo]phenyl]-6-methylbenzothiazole-7-sulphonate

EC Number:

938-398-1

Molecular formula:

C24H28N6O9S2.C18H12LiN5O6S2

IUPAC Name:

Reaction mass of 2-[4-[(Hexahydro-2,4,6-trioxo-5-pyrimidyl)azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1) and Lithium 2-[4-[(hexahydro-2,4,6-trioxopyrimidin-5-yl)azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Specific details on test material used for the study:

Batch no.: 0013479406

Method

Species / strain

Species / strain / cell type:

hepatocytes: huamn

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Human lymphocytes
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
- Sex, age and number of blood donors: Experiment I: male donor (27 years old)
Experiment II: male donor (23 years old)
- Whole blood were used: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

With regard to the content (87.0 g/100 g) of the test item, 2299 μg/mL were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 14.9 to 2299 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity.
In the pre-test for toxicity, no precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using a Cytokinesis-block proliferation index (CBPI) as an indicator for toxicity, no cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 2299 μg/mL were chosen as top treatment concentration for Experiment II.

Vehicle / solvent:

- Vehicle used: Water
- Justification for choice of vehicle:
The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

culture medium with 10.0% deionised water

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcin: without metabolic activation (continuous treatment)

Details on test system and experimental conditions:

METHOD OF APPLICATION: culture flasks

DURATION
- Exposure duration
Experiment I:
4 hours (with and without S9-mix)
Eperiment II
4 hours (with S9 mix)
20 hours (without S9 mix)

The cells were prepared 40 hours after start of treatment with the test item

- Fixation time (start of exposure up to fixation or harvest of cells): 40 hrs

Cytokinesis blocker: cytochalasin B

NUMBER OF REPLICATIONS: In each experimental group two parallel cultures were analysed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide. The cells were stained with Giemsa.
Fixative agent: ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively)

NUMBER OF CELLS EVALUATED:
At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
The frequency of micronucleated cells was reported as % micronucleated cells.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

Rationale for test conditions:

The micronucleus assay will be considered acceptable if it meets the following criteria:
a) The rate of micronuclei in the solvent controls falls within the historical laboratory control data range.
b) The rate of micronuclei in the positive controls is statistically significant increased.
c) The quality of the slides must allow the evaluation of a sufficient number of analyzable cells.

Evaluation criteria:

A test item can be classified as non-clastogenic and non-aneugenic if:
− the number of micronucleated cells in all evaluated dose groups is in the range of the historical laboratory control data and
− no statistically significant or concentration-related increase of the number of micronucleated cells is observed in comparison to the respective solvent control.

A test item can be classified as clastogenic and aneugenic if:
− the number of micronucleated cells is not in the range of the historical laboratory control data and
− either a concentration-related increase in three test groups or a statistically significant increase in the number of micronucleated cells is observed.

Statistics:

Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No

NUMBER OF CELLS WITH MICRONUCLEI: Please refer to the summary of results table

HISTORICAL CONTROL DATA
Percentage of micronucleated cells in human lymphocyte cultures (2015-2016)
Aqueous solvents: DMEM/Ham’s F12, Deionised water (10 % v/v) Organic solvents: DMSO (0.5 or 1.0 %), Acetone, Ethanol and THF (0.5 %)

Solvent Control without S9
Micronucleated cells in %

Pulse treatment (4/40)
No. of experiments: 78*
Mean: 0.60
95 % Ctrl limit: 0.08 – 1.12 0
1x SD: 0.26
Min – Max: 0.15 – 1.65

Continuous treatment (20/40)
No. of experiments: 79**
Mean: 0.57
95 % Ctrl limit: 0.12 – 1.03
1x SD: 0.23
Min – Max: 0.05 – 1.35

Solvent Control with S9
Micronucleated cells in %
Pulse treatment (4/40)
No. of experiments: 96*
Mean: 0.62
95 % Ctrl limit: 0.16 – 1.08
1x SD: 0.23
Min – Max: 0.15 – 1.30

Positive Control without S9
Micronucleated cells in %
Pulse treatment (4/40)
MMC
No. of experiments: 78
Mean: 12.48
95 % Ctrl limit: 1.44 – 23.52
1x SD: 5.52
Min – Max: 4.15 – 30.30

Continuous treatment (20/40)
Demecolcin
No. of experiments: 81
Mean: 3.72
95 % Ctrl limit: 1.43 – 6.01
1x SD: 1.15
Min – Max: 2.10 – 7.25

Positive Control with S9
Micronucleated cells in %
Pulse treatment (4/40)
CPA
No. of experiments: 165
Mean: 5.16
95 % Ctrl limit: 0.84 – 9.49
1x SD: 2.16
Min – Max: 2.10 – 11.90

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Any other information on results incl. tables

Table 1: Summary of results

Exp.	Preparation interval	Test item concentration in μg/mL	Proliferation index CBPI	Cytostasis in %*	Micronucleated cells in %**	95% Ctrl limit
Exposure period 4 hrs without S9 mix
I	40 hrs	Solvent control^1	2.00		0.65	0.08 - 1.12
		Positive control^2	1.67	33.3	17.95^S
		751	2.00	0.2	0.6
		1314	2.01	n.c	0.6
		2299	2.02	n.c.	0.75
Exposure period 20 hrs without S9 mix
II	40 hrs	Solvent control^1	2.02		0.55	0.12 - 1.03
		Positive control^3	1.59	42.3	4.95^S	1.43 - 6.01
		751	1.92	9.3	0.45
		1314	1.95	6.9	0.45
		2299	1.92	9.3	0.30
Exposure period 4 hrs with S9 mix
I	40 hrs	Solvent control^1	2.07		0.90	0.16 – 1.08
		Positive control^4	1.91	15.0	4.00^S	0.84 – 9.49
		751^#	2.08	n.c.	1.15
		1314	2.07	n.c.	0.70
		2299	2.06	0.8	0.45
II	40 hrs	Solvent control^1	2.17		0.90	0.16 – 1.08
		Positive control^5	1.86	26.5	12.50^S	0.84 – 9.49
		751^#	2.12	4.4	1.10
		1314^#	2.08	7.6	1.13
		2299^#	2.14	2.5	0.88

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

# The number of micronucleated cells was determined in a sample of 4000 binucleated cells

^SThe number of micronucleated cells is statistically significantly higher than corresponding control values

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

¹Deion. water 10.0 % (v/v)

²MMC 0.8 μg/mL

³Demecolcin 100 ng/mL

⁴CPA 17.5 μg/mL

⁵CPA 15.0 μg/mL