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)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The test substance was not mutagenic in the in vitro bacterial reverse mutation assay.

in vitro micronucleus test: The test item has the potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in human TK6 cells in the absence and the presence of metabolic activation.

HPRT locus assay

Under the experimental conditions of this study, the read across substance (Similar Substance 01) is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-02-14 to 1990-03-12

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

5 strains tested but not including E. coli

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

1984

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

1984

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Op. 604/89
- Expiration date of the lot/batch: December, 1991

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: in solvent: room temperature, protected from light
- Stability under test conditions: 2 hours stable in water and DMSO

Species / strain / cell type:

other: Salmonella typhimurium strain TA 1535, TA 1537, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from rat liver, Aroclor 1254 induced

Test concentrations with justification for top dose:

10.0; 100.0; 333.3; 1000; and 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: deionized water
- Justification for choice of solvent/vehicle: good solubility of the test substance in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 72 hours at 37 °C
- Expression time (cells in growth medium): 72 hours at 37 °C

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertant cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test article is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, TA 1538, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No appropriate statistical method is available.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no 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:

no 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1: Number of revertants per plate (mean of three plates), experiment 1

	strain TA 1535		strain TA 1537		Strain TA 1538		strain TA 98		strain TA 100
conc. [µg]	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
Negative control	6	7	5	5	19	18	18	23	73*	82
Solvent control	7	8	4	5	16	19	17	22	79	75
10	8	7	5	5	14	18	19	26	77	68
100	5	5	4	6	15	17	19	26	71	74
333.3	5	8	5	3	18	20	19	21	72	57
1000	6	6	4	6	16	18	17	27	69	53
5000	5	7	2	5	17	20	19	26	73	70
Positive control sodium azide (10 µg)	738		146		1475		760		656
Positive control 2-Aminoanthracene (10 µg)		200		37		78		1440		1236

*Unexpected low revertant rate

  

Table 2: Number of revertants per plate (mean of three plates), experiment 2

	strain TA 1535		strain TA 1537		Strain TA 1538		strain TA 98		strain TA 100
conc. [µg]	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA	-MA	+MA
Negative control	21	16	6	8	15	17	15	30	75	99
Solvent control	21	17	6	9	21	23	15	27	78	88
10	29	21	7	6	22	16	18	27	71	79
100	19	22	7	9	21	30	14	24	66	84
333.3	16	21	4	10	21	30	13	31	73	88
1000	18	20	5	10	22	32	15	32	67	94
5000	17	16	6	8	24	22	16	43	71	80
Positive control sodium azide (10 µg)	1335		217		1345		1522		1291
Positive control 2-Aminoanthracene (10 µg)		65		107		547		1667		573

 

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 September 2016 - 17 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

26 Sep 2014

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH): In vitro Mammalian Cell Micronucleus Test, No B.49; No L 193

Version / remarks:

06 July 2012

Deviations:

yes

Remarks:

Due to some differences between the guidelines at diverging points (e.g. dose selection) the recommendations of the most recent OECD Guideline were followed.

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany

Type of assay:

in vitro mammalian cell micronucleus test

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

Species / strain / cell type:

human lymphoblastoid cells (TK6)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The TK6 cell line had shown its suitability to detect aneugenic effects in the Micronucleus test in vitro either in the absence and presence of CytB.
- Cell cycle length, doubling time or proliferation index:
high proliferation rate (doubling time of about 12 - 14 hours)
- Normal (negative control) cell cycle time: about 12 - 14 hours

MEDIA USED
- Type and identity of media including CO2 concentration:
RPMI 1640 containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL), 1% (v/v) amphotericine B (250 μg/mL); Cells were grown with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity and subcultured twice weekly.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Cytokinesis block (if used):

cytochalasin B (CytB)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from phenobarbital i.p. and β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

The concentrations tested in this study were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test.
4 hours exposure, 24 hours harvest time, without S9 mix
0; 225; 450; 900; 1800; 3600; 7200 μg/mL
4 hours exposure, 24 hours harvest time, with S9 mix
0; 225; 450; 900; 1800; 3600; 7200 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: culture medium RPMI 1640
- Justification for choice of solvent/vehicle: In comparison to other commonly used vehicles (e.g. DMSO), culture medium was the most
suitable one. Therefore, culture medium was used as vehicle.

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation: 0.125 and 0.250 μg/mL

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation: 0.5 and 1.0 μg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 24 hours

SPINDLE INHIBITOR: cytochalasin B

STAIN:
Before scoring, the slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI; stock: 5 mg/mL) and propidium iodide (stock: 5 mg/mL; Sigma-Aldrich) in Fluoroshield™ (Sigma-Aldrich, Cat.No. F6182) at a concentration of 0.25 μg/mL each.

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
5x104 cells per slide were centrifuged at 600 rpm for 7 minutes onto labeled slides using a Cytospin centrifuge (Cellspin I, Tharmac, Waldsolms, Germany). At least two slides per flask were prepared. In the case of strongly reduced cell numbers no slides were prepared. After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes.

NUMBER OF CELLS EVALUATED: at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The analysis of micronuclei was carried out following the criteria of Countryman and Heddle:
− The diameter of the micronucleus is less than 1/3 of the main nucleus.
− The micronucleus and main nucleus retain the same color.
− The micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell.
− Only binucleated cells clearly surrounded by a RPMI 1640brane were scored.

DETERMINATION OF CYTOTOXICITY
- Method: cell count, proliferation index

Evaluation criteria:

A test substance is considered to be clearly positive if the following criteria are met:
- A statistically significant increase in the number of micronucleated cells was obtained.
- A dose-related increase in the number of cells containing micronuclei was observed.
- The number of micronucleated cells exceeded both the value of the concurrent vehicle control and the range of our laboratory’s historical negative control data (95% control limit)
A test substance is considered to be clearly negative if the following criterion is met:
- Neither a statistically significant nor dose-related increase in the number of cells containing micronuclei was observed under any experimental condition.
- The number of micronucleated cells in all treated test groups was close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit)

Statistics:

The proportion of cells containing micronuclei was calculated for each test group. A comparison of the micronucleus rates of each test group with the concurrent vehicle control group was carried out for the hypothesis of equal proportions (i.e. one-sided Fisher's exact test, BASF SE).

Key result

Species / strain:

human lymphoblastoid cells (TK6)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

in the presence of S9 mix at 3600 μg/mL and above

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Precipitation: None

HISTORICAL CONTROL DATA: see tables 2 to 4

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity- cell counts:
In the main experiment in the absence and presence of S9 mix no cytotoxicity indicated by reduced cell numbers of below 50% of control was observed up to the highest applied test substance concentrations. These values were calculated based on cell numbers determined at the end of each experiment. In the pretest the parameter cell count is a valuable indicator of test substance toxicity. However, in the main experiments due to the use of the cytokinesis block method it is a measure of cell proliferation only until addition of CytB to the cultures. But, it also gives an useful information on cell loss due to test substance exposure.

Cytotoxicity- Proliferation index
In this study, no relevantly reduced proliferative activity was observed after 4 hours exposure at 24 hours preparation interval in the absence and presence of S9 mix in the test groups scored for cytogenetic damage. The slides were not scorable for cytogenetic damage due to strong cytotoxicity and/or poor quality in the main experiment in the presence of S9 mix at 3600 μg/mL and above.

Table 1: Summary table

Exp.	Exposure/ Preparation interval	Test groups	S9 mix	Prec.*	Genotoxicity Micronucleated cells** [%]	Cytotoxicity   Proliferation       Cell count index cytost- asis (CBPI) [%]          [%]
1	4/24 hrs.	Negative Control	-	n.d.	0.7	0	100
		225 µg/mL	-	-	n.d.	n.d.	126.7
		450 µg/mL	-	-	n.d.	n.d.	128.5
		900 µg/mL	-	-	n.d.	n.d.	127.4
		1800 µg/mL	-	-	3.5s	-1.9	120.6
		3600 µg/mL	-	-	2.7s	11.0	128.1
		7200 µg/mL	-	-	3.8s	21.9	121.4
		Positive Control1	-	n.d.	6.3s	28.6	109.3
1	4/24 hrs.	Negative Control	+	n.d.	0.2	0	100
		225 µg/mL	+	-	n.d.	n.d.	112.0
		450 µg/mL	+	-	1.9s	12.1	115.3
		900 µg/mL	+	-	2.5s	27.5	88.9
		1800 µg/mL	+	-	2.7s	11.3	102.3
		3600 µg/mL	+	-	n.s.	n.s.	100.9
		7200 µg/mL	+	-	n.s.	n.s.	125.5
		Positive Control2	+	n.d.	6.0s	8.4	97.7

* Precipitation in culture medium at the end of exposure period (macroscopic)

** Relative number of binucleated cells with micronuclei per 2000 cells scored per test group

S Frequency statistically significant higher than corresponding control values

n.d. Not determined

n.s. Not scorable due to strong cytotoxicity and/or poor slide quality

1 MMC 0.125 μg/mL

2 CPP 0.5 μg/mL

Table 2: HISTORICAL NEGATIVE CONTROL DATA

Cytochalasin B Method
Period: June 2015 - December 2016

With and without S9 mix - all vehicles*
Micronucleated Cells [%]
Exposure / Sampling period	4h/24h, 24 h/ 24 h
Mean	0.7%
Minimum	0.3%
Maximum	1.4%
Standard Deviation	0.28%
95% Lower Control Limit	0.1%
95% Upper Control Limit	1.3%
No. of Experiments	43

* = culture medium, DMSO 1% (v/v)

 

Table 3: HISTORICAL NEGATIVE CONTROL DATA

Cytochalasin B Method
Period: June 2015 - December 2016

Without S9 mix - all vehicles*
Micronucleated Cells [%]
Exposure / Sampling period	4 h / 24 h & 24 h / 24 h
Mean	0.7%
Minimum	0.3%
Maximum	1.4%
Standard Deviation	0.29%
95% Lower Control Limit	0.1%
95% Upper Control Limit	1.3%
No. of Experiments	38
With S9 mix - all vehicles*
Micronucleated Cells [%]
Exposure / Sampling period	4 h / 24 h
Mean	0.9%
Minimum	0.6%
Maximum	1.3%
Standard Deviation	0.25%
95% Lower Control Limit	0.1%
95% Upper Control Limit	1.6%
No. of Experiments	5

* = culture medium, DMSO 1% (v/v)

 

Table 4: HISTORICAL POSITIVE CONTROL DATA

Cytochalasin B Method

Period: June 2015 - December 2016

Without S9 mix
Mitomycin C (MMC) 0.125 - 0.250 µg/mL
Micronucleated Cells [%]
Exposure / Sampling Period	4 h / 24 h
Mean	4.3%
Minimum	3.0%
Maximum	6.5%
Standard Deviation	1.68%
95% Lower Control Limit	0.0%
95% Upper Control Limit	10.3%
No. of Experiments	4
With S9 mix
Cyclophosphamide (CPP) 0.625 - 1.250 µg/mL
Micronucleated Cells [%]
Exposure / Sampling Period	4 h / 24 h
Mean	2.3%
Minimum	2.0%
Maximum	2.4%
Standard Deviation	0.22%
95% Lower Control Limit	1.2%
95% Upper Control Limit	3.3%
No. of Experiments	3

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-01 to 2016-11-08

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)

Version / remarks:

28 Jul 2015

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0013479406

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Cell cycle length, doubling time or proliferation index: 12 - 16 hours
- Modal number of chromosomes: 20 chromosomes

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
All media were supplemented with:
- 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL)
- 1% (v/v) amphotericine B (stock solution: 250 μg/mL)

Culture medium
Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS).
- Checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

in induced with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

The test substance was poorly soluble in culture medium at the end of 4 hours exposure period.
No cytotoxicity was observed in the pretest when tested up to the highest required concentration of 2350 μg/mL. Therefore, in the main experiments of this HPRT study concentrations at the border of solubility in culture medium were tested for gene mutations.

1st experiment: 4 hour exposure with and without S9-mix:
18.8, 37.5, 75.0, 150.0, 300.0, 600.0 μg/mL

2nd experiment: 4 hour exposure with and without S9-mix:
4.7, 9.4 ,18.8, 37.5, 75.0, 150.0 μg/mL

Vehicle / solvent:

- Vehicles used: Culture medium (Ham's F12)
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, culture medium (Ham's F12) was selected as vehicle.

Untreated negative controls:

yes

Remarks:

with and without S9 mix, were treated with culture medium without test substance in parallel to the other treatment groups

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

- Cell density at seeding: 20x10E6 cells in 40 mL)

DURATION
- Test substance incubation with test medium: approx. 20 – 24 hours after seeding
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7-9 days
- Selection time: TG" medium: Ham's F12 medium containing stable glutamine and hypoxanthine) incubation for about 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): Day 7

SELECTION AGENT: Ham's F12 medium containing stable glutamine and hypoxanthine)

Fixation: Methanol

STAIN: Giesma

NUMBER OF REPLICATIONS: 2 (Cytotoxicity determination)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:

NUMBER OF CELLS EVALUATED: 200 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

- OTHER:
pH
The pH was measured at least for the top concentrations and for the negative controls with and without S9 mix.

Osmolality
Osmolality was measured in at least the top concentrations and the negative controls with and without S9 mix.

Solubility
Test substance precipitation was assessed immediately after dosing the test cultures and at the end of treatment.

Cell morphology
The test cultures of all test substance concentrations were examined microscopically for cell morphology and cellular attachment at the end of the exposure period, which is a further indication for cytotoxicity.

Evaluation criteria:

Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50 % (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (95 % control limit).
Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• The positive controls both with and without S9 mix should induce a distinct, statistically significant increase in mutant frequencies in the expected range

Assessment criteria

A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95 % control limit).
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit)

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report.
The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0.
In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables.
However, both, biological and statistical significance are considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity, in the 1st Experiment in the absence of S9 mix at the highest applied test substance concentration of 600 μg/mL, the survival (CE1) was clearly reduced to 17.1 % of control.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No effects
- Effects of osmolality: No effects
- Precipitation: In this study, in the absence and the presence of S9 mix, test substance precipitation in culture medium at the end of exposure period was observed at least at the highest applied test substance concentrations.

In detail, in both experiments in the absence of S9 mix test substance precipitation was observed macroscopically in culture medium at the end of treatment at 150.0 μg/mL and above.
In the 1st Experiment in the presence of S9 mix precipitation in culture medium at the end of treatment was observed macroscopically from the lowest applied concentration of 18.8 μg/mL onward. These raw data recordings were in disagreement with the data of the pretest, and it was assumed that these data did not reflect the real conditions. However, assessment of precipitation with the unaided eye in cell culture flasks in the presence of S9 mix is somehow difficult. In the 2nd Experiment in the presence of S9 mix precipitation in culture medium at the end of treatment was observed macroscopically at 150 μg/mL as expected. To clarify the solubility issue an additional solubility testing was performed. At this trial, test substance precipitates in culture medium 4 hours after start of treatment were observed macroscopically from 75 μg/mL onward either in the absence or presence of S9 mix.
Finally, based on the observations in the pretest, the 2nd Experiment and an additional solubility testing the border of solubility has been expected in the range of 75.0 to 293.8 μg/mL.
Thus, it has to be considered that the lowest applied concentrations in the 1st Experiment in the presence of S9 mix did not show precipitation, and, thus, the requirements of the current OECD Guideline 476 were fulfilled.


RANGE-FINDING/SCREENING STUDIES:
In the pretest for toxicity based on the purity of the test substance 2350.0 μg/mL was used as top concentration both with and without S9 mix at 4-hour exposure time.
The pretest was performed following the method described for the main experiment. The cloning efficiency 1 (survival) was determined as a toxicity indicator for dose selection and various parameters were checked for at least some selected doses.
In the pretest the pH value was not influenced by the addition of the test substance preparation to the culture medium at the concentrations measured.
In addition, precipitation of the test substance was not observed macroscopically in the stock solution (Test group: 2350 μg/mL). By the end of treatment test substance precipitation occurred in culture medium at concentrations of 293.8 μg/mL and above in the absence and presence of S9 mix.
After 4 hours treatment in the absence and presence of S9 mix cytotoxicity was not observed as indicated by a reduced relative cloning efficiency of about or below 20 %.

HISTORICAL CONTROL DATA
- Positive historical control data:

Summary (all experimental conditions)
Period: January 2014 - December 2015

Without S9 mix: 300 - 400 μg/mL ethyl methanesulfonate (EMS)

Corrected Mutant Frequency*
Exposure period: 4 hrs
Mean: 138.82
Minimum: 42.47
Maximum: 360.93
Standard Deviation: 86.31
95% Lower Control Limit: 0.00
95% Upper Control Limit: 325.03
No. of Experiments: 19

With S9 mix: 1.25 μg/mL 7.12-Dimethylbenz[a]anthracene (DMBA)
Corrected Mutant Frequency*
Exposure period: 4 hrs
Mean: 98.14
Minimum: 21.52
Maximum: 189.14
Standard Deviation: 48.01
95% Lower Control Limit: 0.00
95% Upper Control Limit: 202.20
No. of Experiments: 18

* = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)


- Negative (solvent/vehicle) historical control data:
Summary (all vehicles. all experimental conditions)
Period: March 2016 – August 2016

Without and with S9 mix - all vehicles*
Corrected Mutant Frequency**
Exposure period: 4 hrs
Mean: 2.39
Minimum: 0.00
Maximum: 9.93
Standard Deviation: 2.33
95% Lower Control Limit: 0.00
95% Upper Control Limit: 7.19
No. of Experiments: 37

* = culture medium. water 10 % (v/v). DMSO 1 % (v/v). acetone 1 % (v/v)
** = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

Summary (all vehicles)
Period: March 2016 – August 2016

Without S9 mix - all vehicles*
Corrected Mutant Frequency**
Exposure period: 4 hrs
Mean: 2.53
Minimum: 0.00
Maximum: 6.48
Standard Deviation: 1.78
95% Lower Control Limit: 0.00
95% Upper Control Limit: 6.36
No. of Experiments: 19

With S9 mix - all vehicles*
Corrected Mutant Frequency**
Exposure period: 4 hrs
Mean: 2.25
Minimum: 0.00
Maximum: 9.93
Standard Deviation: 2.85
95% Lower Control Limit: 0.00
95% Upper Control Limit: 8.43
No. of Experiments: 18

* = culture medium. water 10 % (v/v). DMSO 1 % (v/v). acetone 1 % (v/v)
** = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

Table 1: Summary of results – experimental parts without S9 mix

 

Exp.	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	Cytotoxicity***
						CE1[%]	CE2[%]
1	4	Negative control	-	n.d.	0.66	100.0	100.0
		18.8	-	-	3.79	104.6	95.1
		37.5	-	-	2.86	102.7	103.3
		75.0	-	-	1.66	83.3	98.7
		150.0	-	+	1.52	78.7	86.6
		300.0	-	+	2.45	41.4	80.3
		600.0	-	+	2.13	17.1	92.5
		Positive control^1	-	n.d.	205.86^S	67.7	72.8
2	4	Negative control	-	n.d.	1.36	100.0	100.0
		4.7	-	-	n.c.	94.3	n.c.
		9.4	-	-	2.17	92.3	93.6
		18.8	-	-	0.67	116.7	101.7
		37.5	-	-	0.76	97.0	88.8
		75.0	-	-	4.26	74.0	87.5
		150.0	-	+	2.97	35.7	80.0
		Positive control^1	-	n.d.	111.88^S	67.0	102.7

 

*       Macroscopically visible precipitation in culture medium at the end of exposure period

**      Mutant frequency MF_corr.: mutant colonies per 10⁶cells corrected with the CE₂value

***    Cloning efficiency related to the respective vehicle control

^S       Mutant frequency statistically significant higher than corresponding control values

n.c.   Culture was not continued since a minimum of only four analysable concentrations is required

n.d.   Not determined

¹       EMS 400 μg/mL

²       DMBA 1.25 μg/mL

 

Table 1 continued: Summary of results – experimental parts with S9 mix

 

Exp.	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	Cytotoxicity***
						CE1[%]	CE2[%]
1	4	Negative control	+	n.d.	1.07	100.0	100.0
		18.8	+	+	1.43	113.2	99.3
		37.5	+	+	1.45	102.7	98.2
		75.0	+	+	1.04	92.6	102.8
		150.0	+	+	1.75	101.9	102.5
		300.0	+	+	5.28^S	74.8	94.3
		600.0	+	+	0.40	81.4	89.3
		Positive control^2	+	n.d.	221.50^S	82.9	76.2
2	4	Negative control	+	n.d.	3.51	100.0	100.0
		4.7	+	-	n.c.	112.2	n.c.
		9.4	+	-	2.39	106.3	110.1
		18.8	+	-	1.20	91.0	110.1
		37.5	+	-	2.18	106.9	100.4
		75.0	+	-	4.78	70.5	91.7
		150.0	+	+	1.46	78.1	90.4
		Positive control^2	+	n.d.	95.81^S	71.5	94.3

 

*       Macroscopically visible precipitation in culture medium at the end of exposure period

**      Mutant frequency MF_corr.: mutant colonies per 10⁶cells corrected with the CE₂value

***    Cloning efficiency related to the respective vehicle control

^S       Mutant frequency statistically significant higher than corresponding control values

n.c.   Culture was not continued since a minimum of only four analysable concentrations is required

n.d.   Not determined

¹       EMS 400 μg/mL

²       DMBA 1.25 μg/mL

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

in vivo micronucleus test: The test substance does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

29 Jul 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

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

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

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

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

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

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

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.

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.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

in vitro

Ames test

Key study

The test item was assessed for its potential to induce gene mutations according to the plate incorporation test using Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 10; 100; 333.3; 1000; and 5000 µg/plate. No toxic effects, normally evidenced by a reduction in the number of revertants, occurred in the test groups with and without metabolic activation in all strains used. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. A slight increase in the number of revertant colonies was observed in strain TA 98 at 5000 µg/plate in the presence of metabolic activation in experiment II. This effect is considered not to be relevant as it was not reproduced in the independent experiment. Up to the highest investigated dose, neither a significant and reproducible increase of the number of revertants was found in any strain as compared to the solvent control nor a concentration- dependent enhancement of the revertant number exists. The presence of liver microsomal activation did not influence these findings. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies. In conclusion the test article did not induce point mutations or frameshifts in the genome of the strains used.

Supporting study

In a second Ames test, the test matrerial was applied as a preparation containing 20.9 % test substance.

In addition, the preparation was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 0.2, 2, 20, 200 and 2000 µg or nL per petri dish both in the presence and absence of metabolic activation (rat liver S9 mix, Aroclor induced). As positive controls, 9-aminoacridine, daunomycine, N-methyl-N'-nitro-N-nitrosoguanidine, and 2-anthramine were used. As a result, no mutagenic effect was observed.

In vitro micronucleus test

The substance was assessed for its potential to induce micronuclei in TK6 cells in a study according OECD TG 487 (clastogenic or aneugenic activity). A single experiment was carried out with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested:

4 hours exposure, 24 hours harvest time, without S9 mix

0;225; 450; 900;1800; 3600; 7200 μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix

0; 225;450; 900; 1800;3600; 7200 μg/mL

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group.

The negative controls gave frequencies of micronucleated cells within our historical negative control data range for TK6 cells. Both positive control substances, mitomycin C (MMC) and cyclophosphamide (CPP), led to the expected increase in the number of cells containing micronuclei.

In this study, cytotoxicity indicated by clearly reduced cell count and/or proliferation index (cytostasis) was observed only in the experimental part with metabolic activation from 3600 μg/mL onward.

On the basis of the results of the present study, the test substance caused a clear increase in the number of cells containing micronuclei. Statistically significant and biologically relevant increased micronucleus rates were observed in the main experiment both without S9 mix and after adding a metabolizing system.

Thus, under the experimental conditions described, the test item is considered to have a chromosome-damaging (clastogenic) effect or to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in human TK6 cells in the absence and the presence of metabolic activation.

In vitro gene mutation test in CHO cells (HPRT Locus Assay)

An analogous substance (read-across source substance Similar Substance 01) was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro according to OECD 476 and GLP.

Two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested:

1st Experiment

without S9 mix

0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 μg/mL

with S9 mix

0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 μg/mL

2nd Experiment

without S9 mix

0; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 μg/mL

with S9 mix

0; 4.7; 9.4; 18.8; 37.5; 75.0; 150.0 μg/mL

 

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

Dose selection for genotoxicity testing was based on the solubility properties of the test substance in culture medium. The highest tested concentrations in both main experiments showed clear test substance precipitates in culture medium macroscopically at the end of exposure period.

In this study, in the absence and the presence of metabolic activation no cytotoxicity was observed up to the highest concentrations evaluated for gene mutations, except in the 1st Experiment in the absence of S9 mix.

Based on the results of the study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

in vivo

In vivo micronucleus test

The test substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice according OECD TG 474. For this purpose, the test substance, suspended in deionized water, was administered once orally to male animals at dose levels of 500 mg/kg, 1000 mg/kg and 2000 mg/kg body weight in a volume of 20 mL/kg body weight in each case.

Dose selection was based on the results of a preliminary range finding test in both sexes. No gender difference was observed and, therefore, only males were used in the main test. The animals were sacrificed and the bone marrow of the two femora was prepared 24 and 48 hours after administration in the highest dose group of 2000 mg/kg body weight and in the vehicle controls. In the test groups of 1000 mg/kg and 500 mg/kg body weight and in the positive control group, the 24-hour sacrifice interval was investigated only. After staining of the preparations, 4000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. The normocytes with and without micronuclei occurring per 4000 polychromatic erythrocytes were also recorded.

As vehicle control, male mice were administered merely the vehicle, deionized water, by the same route and in the same volume as the animals of the dose groups, which gave frequencies of micronucleated polychromatic erythrocytes within the historical vehicle control data range.

The positive control substance cyclophosphamide led to the expected increase in the rate of polychromatic erythrocytes containing micronuclei.

An inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected at doses of 2000 mg/kg body weight. Thus, confirming the bioavailability of the test substance.

According to the results of the present study, the single oral administration of the test item did not lead to any statistically significant or biologically relevant increase in the number of polychromatic erythrocytes containing micronuclei .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‰).

Thus, under the experimental conditions of this study, the test substance does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo.

Conclusion

No in vitro genotoxicity was detected in a reverse bacterial mutation Ames test with and without metabolic activation. In addition, in a HPRT locus assay performed with a structural analogue substance (Similar Substance 01) no mutagenic effect under in vitro conditions in CHO cells in the absence and the presence of metabolic activation was detected. On the other hand, the substance has the potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in human TK6 cells in the absence and the presence of metabolic activation. Therefore, an in vivo micronucleus test was performed and it was show that the test substance does not induce cytogenetic damage in bone marrow cells of NMRI mice in vivo. In conclusion, the test substance is not considered to be classified for genetic toxicity.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EC) No 2017/776.