2-[4-[[2-(cyanoimino)hexahydro-4,6-dioxo-5-pyrimidyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 March 2017 - 8 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2018

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Reference 3

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 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 Jul 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Type of assay:

other: micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-[4-[[2-(cyanoimino)hexahydro-4,6-dioxo-5-pyrimidyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1)

EC Number:

259-470-2

EC Name:

2-[4-[[2-(cyanoimino)hexahydro-4,6-dioxo-5-pyrimidyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1)

Cas Number:

55067-15-7

Molecular formula:

C19 H13 N7 O5 S2 . C6 H15 N O3

IUPAC Name:

2-[4-[[2-(cyanoimino)hexahydro-4,6-dioxo-5-pyrimidyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonic acid, compound with 2,2',2''-nitrilotris[ethanol] (1:1)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0013974305

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study was guaranteed until August 2019

Test animals

Species:

mouse

Strain:

other: Crl:NMRI

Details on species / strain selection:

The animals were selected according to the recommendations of the OECD and EU or on the basis of results published so far. Moreover, there has been up to now most experience and data for NMRI mice.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH
- Age at study initiation: 5 – 8 weeks
- Weight at study initiation: mean: 28.1 ± 1.46 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Polycarbonate cages, type II; single housing
- Diet: ad libitum, Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): Fully air-conditioned rooms with central air conditioning.
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle:
Using the vehicle deionized water a homogeneous suspension of the test substance was obtained. Therefore, deionized water was used as vehicle, which has been demonstrated to be suitable in the mouse micronucleus test and for which historical control data are available.
- Concentration of test material in vehicle: 25, 50 and 100 mg/mL
- Amount of vehicle: 20 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance to be administered per kg body weight was suspended in deionized water. To achieve the homogeneity of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and shaken thoroughly. All test substance formulations were prepared immediately before administration.

Duration of treatment / exposure:

24 hours (1st group) and 48 hours (2nd group)

Frequency of treatment:

once

Post exposure period:

none

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPP)
- Justification for choice of positive control(s): CPP is a well-established reference clastogen.
- Route of administration: oral (gavage)
- Final Concentration: 2 mg/mL

Examinations

Tissues and cell types examined:

femura prepared to obtain erythrocytes from bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Pretests were performed for dose selection.
The pretest was performed following the method described for the main experiment. The test substance was administered by gavage (orally) to NMRI mice of both sexes, three animals per dose and sex, in general. Then, the animals were examined for clinically evident signs of toxicity several times. About 48 hours after administration the surviving animals were sacrificed humanely by isoflurane anesthesia followed by cervical dislocation. In the pretest to determine the acute oral toxicity in males and females, the recommended highest dose of 2000 mg/kg body weight was tolerated by all animals without signs of toxicity. Feces discolored by the orange colored test substance was observed 4 hours and one day after administration. Besides, there was no indication for a gender specific toxicity. The usual application volume of 10 mL/kg body weight led to a non-applicable mass and therefore the volume was increased to 20 mL/kg body weight.

DETAILS OF SLIDE PREPARATION:
- The bone marrow suspension was mixed gently before transferring to the cellulose column. As soon as the suspension was fully soaked into the cellulose column 5 mL HBSS was added.
- The eluate containing erythrocytes was centrifuged at 300 x g for 5 minutes. The supernatant was removed gently and the precipitate was resuspended with 1 - 4 mL PBS
(Phosphate Buffered Saline with Ca and Mg) depending on the cell count.
- Labeled slides equipped with cell funnels were clamped into the rotor of the cytospin. Then, 200 μL cell suspension was transferred to each cell funnel and was centrifuged at 1 200 rpm (approx. 220 x g) for 7 minutes (at least two slides per animal).
- After drying the slides were stained with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After briefly rinsing in deionized water, the preparations were soaked in deionized water for about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL deionized water) for about 15 minutes.
- After rinsing twice in deionized water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
The slides for the evaluation of micronucleated erythrocytes were analyzed by use of the image analysis system Metafer4 MetaCyte (MetaSystems Hard & Software GmbH, Altlussheim, Germany). Polychromatic and normochromatic erythrocytes (PCE or NCE) that fulfill the criteria for scoring were automatically detected under the light microscope and were recorded by the system. Then, a trained scorer performed manually the analysis based on these preselected images on the screen. In case of low quality of images, e.g. artefacts of staining etc., the cells were assessed directly on the slides by light microscopy.
In general, 4000 polychromatic erythrocytes (PCE) were evaluated for the occurrence of micronuclei from each animal of every test group, so in total 20000 PCE were scored per test group. The normochromatic erythrocytes (= normocytes / NCE) were also scored. The following parameters were recorded:
• Number of polychromatic erythrocytes
• Number of polychromatic erythrocytes containing micronuclei
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the vehicle control group provides an index of a chromosome-breaking (clastogenic) effect or damage of the mitotic apparatus (aneugenic activity) of the test substance administered.
• Number of normochromatic erythrocytes
• Number of normochromatic erythrocytes containing micronuclei
The number of micronuclei in normochromatic erythrocytes at the early sacrifice interval shows the situation before test substance administration and may serve as a control value. A test substance induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice interval.
• Ratio of polychromatic to normochromatic erythrocytes
An alteration of this ratio indicates that the test substance actually reached the bone marrow, means the target determined for genotoxic effects. The microscopic analysis was run on coded slides.

Evaluation criteria:

A finding is considered positive if all of the following criteria are met:
• A statistically significant and dose-related increase in the number of PCE containing micronuclei.
• The number of polychromatic erythrocytes containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data (95% control limit).
A test substance is considered negative if the following criteria are met:
• The number of polychromatic erythrocytes containing micronuclei in the dose groups is not
statistically significant increased above the concurrent vehicle control value and is within the range of the historical vehicle control data (95% control limit).
A scientific judgment is required in case of equivocal data.

Statistics:

For analysis of the rate of micronucleated polychromatic erythrocytes the Jonckheere-Terpstra test were used which is a non-parametric trend test. In addition, a pair-wise comparison of each test group with the control group was carried out using the Wilcoxon test (one-sided+). The statistical unit is the animal and, therefore, the rate per animal was used. The calculation was performed using R. The results were listed in the tables. If the results of these tests were significant, symbols (* for p ≤ 0.05; ** for p ≤ 0.01) were used in the tables. However, both biological relevance and statistical significance were considered together.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

inhibition of erythropoiesis induced by the treatment with the test item was detected from 2000 mg/kg body weight at 24-hour sacrifice interval

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals:
In the pretest the recommended highest dose of 2000 mg/kg body weight was tolerated by all animals without signs of toxicity. Feces discolored by the orange colored test substance was observed 4 hours and one day after administration. Besides, there was no indication for a gender specific toxicity.
- Evidence of cytotoxicity in tissue analyzed: no
- Harvest times: 48 hours
- High dose with and without activation: 2000 mg/kg bw


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No. The rate of micronuclei in all dose groups and at all sacrifice intervals (0.5 – 3.3‰) was within the range of the concurrent vehicle controls (1.0 – 3.3‰), and all values were within the range of the 95% control limit of our historical vehicle control data (1.3 – 4.4‰).
- Ratio of PCE/NCE: see table 1
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes

OTHER:
The concentration control analyses of all concentrations revealed that the values were in the expected range of the target concentrations, i.e. were always in a range of 90% - 110% of the nominal concentration.

Distinct organ toxicity indicated by an inhibition of erythropoiesis induced by the treatment with the test item was detected from 2000 mg/kg body weight at 24-hour sacrifice interval.
The ratio of PCE to NCE was clearly influenced after treatment with 2000 mg/kg body weight (67% of control, respectively) compared to the concurrent vehicle control value. Animal weights were not relevantly influenced by test substance exposure compared to the respective vehicle control animals.

Any other information on results incl. tables

Table 1: Summary table - Induction of Micronuclei in bone marrow cells

Test group	Sacrifice interval [hrs]	Animal No.	Micronuclei in PCE   Mean^a[‰]    range^b[abs]		PCE per 4000 erythrocytes^c       [abs]             [rel]
Vehicle control deionized water	24	5	2.3	7-13	2344	100%
Test substance 500 mg/kg bw.	24	5	1.6	2-9	2069	88%
Test substance 1000 mg/kg bw.	24	5	2.3	8-12	2239	96%
Test substance 2000 mg/kg bw.	24	5	1.9	3-10	1571	67%
Positive control cyclophosphamide 20 mg/kg bw.	24	5	11.3**	29-57	2644	113%
Vehicle control deionized water]	48	5	1.6	4-9	2133	100%
Test substance 2000 mg/kg bw.	48	5	2.1	3-13	2235	105%

PCE   = polychromatic erythrocytes

NCE   = normochromatic erythrocytes

bw.     = body weight

a = sum of small and large micronuclei

b = number of micronuclei per animal (sample size: 4000 PCE)

c = calculated number of PCE per 4000 erythrocytes (PCE + NCE) when scoring a sample of up to 20000 PCE per test group

*= p<0.05

**= p<0.01