Ammonium sodium 2-[4-[[1-[[(2-methoxy-5-methyl-4-sulphonatophenyl)amino]carbonyl]-2-oxopropyl]azo]phenyl]-6-methylbenzothiazole-7-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Micronucleus study

The test substance is considered neither to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

 

Bacterial Reverse Mutation Assay (Ames test)

The test substance did not induce gene mutations in the S. typhimurium strains.

HPRT 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.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-01-18 to 2016-06-29

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 440/2008, In vitro Mammalian Cell Micronucleus Test, No B.49

Version / remarks:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Batch identification: #13298465
Physical state, appearance: Solid, yellow to orange
Storage conditions: Room temperature
Expiry date: January 2019

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: Yes, V79 cell line has 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),

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
MEM (minimal essential medium with Earle's salts) 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
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix induced with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

156.3 μg/mL, 312.5 μg/mL, 625.0 μg/mL, 1250.0 μg/mL, 2500.0 μg/mL, 5000.0 μg/mL

Based on the observations and toxicity data of a previously performed pretest of a HPRT study (top concentration: 2400 μg/mL; data not shown) and with regard to the composition of the test substance 5000 μg/mL test substacne was used as top concentration in both experiments of this cytogenetic study.

Vehicle / solvent:

- Vehicle used: Culture medium (Minimal Essential Medium: MEM)
- Justification for choice of vehicle: Due to the good solubility of the test substance in water, culture medium (Minimal Essential Medium: MEM) was selected as vehicle.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Exposure time: Without S9-mix: Exp.1: 4 hrs; Exp. 2: 24 hrs // With S9-mix: Exp.1 and Exp. 2: 4 hrs
Recovery time : Without S9-mix: Exp.1: 20 hrs // With S9-mix: Exp.1: 20 hrs, Exp. 2: 40 hrs
Harvest time: Without S9-mix: Exp.1 and Exp. 2: 24 hrs // With S9-mix: Exp.1: 24 hrs, Exp. 2: 44 hrs

SPINDLE INHIBITOR: Actin polymerisation inhibitor cytochalasin B

STAIN: 4’,6-diamidino-2-phenylindole dihydrochloride and propidium iodide

NUMBER OF REPLICATIONS: 2 per concentration

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
5x10E4 cells per slide were centrifuged at 600 rpm for 7 minutes onto labeled slides using a Cytospin centrifuge. At least two slides per flask were prepared.
After drying, the slides were fixed in 90% (v/v) methanol for 10 minutes. Before scoring, the slides were stained with a mixture of 4’,6-diamidino-2-phenylindole dihydrochloride and propidium iodide at a concentration of 0.25 μg/mL each.

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 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 membrane were scored.

DETERMINATION OF CYTOTOXICITY
- Method: Relative total growth (CBPI), cell morphology (microscopically)

OTHER EXAMINATIONS:
pH value: was measured at least for the top concentration and for the vehicle control with and without S9 mix.
Osmolality: was measured at least for the top concentration and for the vehicle control with and without S9 mix
Solubility: Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically / microscopically).

Evaluation criteria:

Acceptance criteria
The in vitro micronucleus assay is considered valid if the following criteria are met:
• The quality of the slides allowed the evaluation of a sufficient number of analyzable cells both in the control groups (vehicle/positive) and in at least three exposed test groups.
• Sufficient cell proliferation was demonstrated in the vehicle control.
• The number of cells containing micronuclei in the vehicle control was within the range of our laboratory’s historical negative control data (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• The positive control substances both with and without S9 mix induced a distinct, statistically significant increase in the number of micronucleated cells in the expected range.

Assessment 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 statistical evaluation of the data was carried out using an appropriate statistical analysis. 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).
If the results of this test were statistically significant compared with the respective vehicle control (p ≤ 0.05), labels (S) have been printed in the tables.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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
- Water solubility: Good
- Precipitation: No

GENOTOXICITY
The single statistical significance observed in the 1st Experiment without metabolic activation in an intermediate concentration of 2500 μg/mL has to be regarded as biologically irrelevant under the circumstance that this value was clearly within the historical negative control data range.

CYTOKINESIS BLOCK: CytB blocks
- Distribution of mono-, bi- and multi-nucleated cells: 1x No. mononucleate cells, 2 x No. binucleate cells, 3 x No. multinucleate cells

HISTORICAL CONTROL DATA
- Positive historical control data:
Cyclophosphamide (CPP): range: 2.4 – 13.8% micronucleated cells; mean: 4.8; SD: 2.0; 95% Lower Control Limit: 0.8; 95% Upper Control Limit: 8.8
Ethyl methanesulfonate: range: 2.0-6.4 % micronucleated cells; mean: 2.9; SD:0.7; 95% Lower Control Limit: 1.5; 95% Upper Control Limit: 4.3
- Negative (vehicle) historical control data:
range: 0.1 - 1.5% micronucleated cells; mean 0.5; SD: 0.2; 95% Lower Control Limit: 0.0; 95% Upper Control Limit: 1.0

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI, Relative population doubling (RPD) for the time period before addition of CytB
no cytotoxicity indicated by reduced RPD of below 50% of control, no reduced proliferative activity, cell morphology/attachment was not adversely influenced (grade > 2) at any dose tested for the occurrence of micronuclei.

Table 1: Summary table - experimental parts without S9 mix

Exp.	Exposure/ Preparation interval	Test groups [μg/mL]	S9-mix	Genotoxicity Micronucleated cells** [%]	Proliferation index cytostasis (CBPI) [%]	Relative population doubling (RPD) [%]
1	4/24 hrs	Negative control	-	0.3	0.0	100.00
		156.3	-	n.d.	n.d.	116.7
		312.5	-	n.d.	n.d.	122.8
		625.0	-	n.d.	n.d.	123.0
		1250.0	-	0.4	3.0	119.5
		2500.0	-	1.0 (s)	3.8	115.3
		5000.0	-	0.3 (s)	5.7	122.8
		Positive control (1)	-	1.5 (s)	8.1	112.5
		Positive control (2)	-	2.4 (s)	9.0	112.8
2	24/24 hrs	Negative control	-	0.7	0.0	100.00
		312.5	-	n.d.	n.d.	103.3
		625.0	-	n.d.	n.d.	97.3
		1250.0	-	0.6	14.4	107.9
		2500.0	-	0.7	14.6	108.6
		5000.0	-	0.3	18.2	101.3
		Positive control (1)	-	2.9 (s)	24.0	101.2

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

S Frequency statistically significant higher than corresponding control values (p≤0.05)

n.d. Not determined

(1) EMS 500 μg/mL

(2) EMS 600 μg/mL

 

Table 2: Summary table - experimental parts with S9 mix

Exp.	Exposure/ Preparation interval	Test groups [μg/mL]	S9-mix	Genotoxicity Micronucleated cells** [%]	Proliferation index cytostasis (CBPI) [%]	Relative population doubling (RPD) [%]
1	4/24 hrs	Negative control	+	0.5	0.0	100.0
		156.3	+	n.d.	n.d.	99.1
		312.5	+	n.d.	n.d.	108.6
		625.0	+	n.d.	n.d.	106.8
		1250.0	+	0.4	0.3	104.2
		2500.0	+	0.5	3.4	108.8
		5000.0	+	0.4	0.1	111.6
		Positive control (1)	+	2.3 (s)	33.2	105.1
2	4/44 hrs	Negative control	+	0.5	0.0	100.0
		312.5	+	n.d.	n.d.	94.1
		625.0	+	n.d.	n.d.	90.1
		1250.0	+	0.6	- 0.6	88.3
		2500.0	+	0.7	- 3.1	93.1
		5000.0	+	0.7	0.9	87.1
		Positive control (1)	+	3.8 (s)	-11.0	86.6

 

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

(s) Frequency statistically significant higher than corresponding control values (p≤0.05)

n.d. Not determined

(1) Cyclophosphamide: CPP 0.5 μg/mL

 

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

only four strains tested

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted: 26 May 1983

Deviations:

yes

Remarks:

only four strains

Principles of method if other than guideline:

References:
Ames BN, Durson WE, Yamasaki E, and Lee FD (1970). Proc. Nat. Acad. Sci. USA, 70, 2285.
Ames BN, McCann J, and Yamasaki E (1975) Mutation Research 31, 347.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9 Mix; Microsomal Enzyme Fraction prepared from animals pre-treated with Arochlor 1254.

Test concentrations with justification for top dose:

20, 100, 500, 2500, 12500 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Good solubility of the test substance, readily soluble in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO alone

True negative controls:

no

Positive controls:

yes

Remarks:

Furthermore, 2-amino-anthracene which does not revert in the absence of metabolic activation was added at a dose of 10 µg/plate (activity check of liver enzyme preparation)

Positive control substance:

other: see remarks

Remarks:

M-Methyl-N-Nitro-N-Nitrosoguanidine (MNNG) 2 µg/plate (TA1535 & TA100); 9 Amino Acridine (9AA) 100 µg/plate (TA 1537); 4-Nitro-O-phenyldiamine (4NOPD) 10 µg/plate (TA 98).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Test tubes containing 9 mL portions of molten histidine deficient top agar at 45°C.
- Addition of 0.1 mL test solution or negative/positive control solution, 0.1 mL bacterial suspension, and optional, 0.5 mL S-9 Mix.
- Pouring of samples of 3 mL onto Vogel-Bonner agar plates (minimal medium).
- A solution containing 0.5 mM Histidine and 0.5 mM Biotin was added to top agar to enable that a background lawn of growth was possible in the test.
- Growth indicates that a reverse mutation has reverted the his- gene back to an active form.

DURATION
- Preincubation period: no
- Exposure duration: Incubation at 37°C °C for 48 hours

NUMBER OF REPLICATIONS: 3 (Petri dishes prepared per strain and per group)

CELLS EVALUATED: The bacterial colonies (his+ revertants) are counted.

DETERMINATION OF CYTOTOXICITY
- Method: bacterial growth (colony formation)

OTHER INFORMATION
Microsomal Enzyme Fraction: A commercial preparation was obtained from Uniscience Limited, 8 Jesus Lane, Cambridge, CBS 8BA, UK. This fraction is obtained from animals pre-treated with Arochlor 1254 (a mixture of polychlorinated biphenyls) and was stated to be from batch no. 03300 with a protein level of 40 mg/mL (Aryl hydrocarbon hydroxylase activity: 6.4 nmol hydroxy benzo(a)pyrene/20 minutes/mg protein).
Reference:
SPECIFICATIONS FOR RAT LIVER S-9 PRODUCT, AROCLOR 1254 INDUCED, CATOLOG NOS. 8360-01 AND 8360-03. Date prepared: 1984-04-25, Uniscience Limited, UK.

Evaluation criteria:

Positive result: Dose-related and statistically significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S-9 Mix.

Negative result: The number of induced revertants compared to spontaneous revertants should be less than two fold at all dose levels employed, the intervals of which should be between 3 and 5 fold and extend to the limits imposed by toxicity or solubility.

Statistics:

The arithmetic mean was calculated.

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:

not examined

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:

not examined

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:

not examined

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:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
No inhibition was observed at any of the concentrations of the test substance employed in this test and the main mutation study was set up using test substance at concentrations in the range 20 to 12500 µg/plate.


ADDITIONAL INFORMATION:
No significant increase in the number of revertant colonies was recorded for any of the strains at any of the dose levels employed in this study, either with or without metabolic activation.
All counts of revertant colonies were similar to those recorded for the negative control plates and were within the range expected for spontaneous reversion for each strain.
The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

 

Plate incorporation with S-9 Mix (colony number = mean values):

Dose (µg/per 0.1 mL [plate])	TA 98	TA 100	TA 1535	TA1537
0 (DMSO)	27	102	19	7
20	25	104	20	7
100	24	109	25	8
500	25	109	26	6
2500	26	103	24	8
12500	28	112	22	8
----------
Positive controls:
4-NOP (10 µg/plate)	850
MNNG (2 µg/plate)		> 1000	797
9-AA (100 µg/plate)				880
----------
9-AA (10 µg/plate)	> 1000	> 1000	487	617
Three plates per tester strain were evaluated.

 

 

Plate incorporation without S-9 Mix (colony number = mean values):

Dose (µg/per 0.1 mL [plate])	TA 98	TA 100	TA 1535	TA1537
0 (DMSO)	24	93	21	8
20	25	100	22	8
100	22	113	25	6
500	25	101	26	6
2500	26	107	28	6
12500	25	107	32	8
----------
Positive controls:
4-NOP (10 µg/plate)	917
MNNG (2 µg/plate)		> 1000	683
9-AA (100 µg/plate)				> 1000
----------
9-AA (10 µg/plate)	28	103	20	6
Three plates per tester strain were evaluated.

 

 

VIABILITY AND SPONTANEOUS REVERSION TESTS

Strain                Salmonella typhimurium	Total Counts on Histidine deficient agar (x 10E7)	Minimal agar with overlay of Histidine/Biotin top agar
	1 mL of overnight nutrient broth
TA 98	21	26	28	28
TA 100	30	97	98	101
TA 1535	15	18	18	17
TA 1537	19	6	6	8

 

 

STERILITY CHECKS

Sample	Count
Top Agar + His/Biotin	0	0	0
Top Agar only	0	0	0
Top Agar + His/Biotin + S9	0	0	0

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-02-04 to 2016-06-07

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

Deviations:

yes

Remarks:

As follow-up on the revision of the OECD Guideline No. 476 minor changes in test procedure were implemented in this study (e.g. increased numbers of seeded cells and enzymatic dissociation of the cells at the end of exposure period).

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 of 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro gene mutation test in CHO cells (HPRT locus assay)

Specific details on test material used for the study:

Batch no.: #13298465

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix induced with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

Pretest for dose selection:
In the pretest for toxicity based on the purity 2400 μg/mL was used as top concentration both
with and without S9 mix at 4-hour exposure time.

Main test
1st Experiment
without S9 mix
0; 312.5; 625.0; 1250.0; 2500.0; 5000.0 μg/mL
with S9 mix
0; 312.5; 625.0; 1250.0; 2500.0; 5000.0 μg/mL
2nd Experiment
without S9 mix
0; 750.0; 1500.0; 3000.0; 5000.0 μg/mL
with S9 mix
0; 750.0; 1500.0; 3000.0; 5000.0 μg/mL

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

culture medium

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 logarithmically growing cells per flask (300 cm²)

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7-9 days
- Selection time: 6-7 days

SELECTION AGENT: TG medium

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 10E6 cells

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency (CE1 survival, CE2 viability)

OTHER EXAMINATIONS:
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/vehicle 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 groups 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:

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)

Statistics:

An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies.
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.
If the results of these tests were statistically significant compared with the respective vehicle control, labels (s, p ≤ 0.05).
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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

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

RANGE-FINDING/SCREENING STUDIES:
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 culture medium, no test substance precipitation occurred up to the highest applied concentration in the absence and presence of S9 mix.
After 4 hours treatment in the absence and presence of S9 mix, no cytotoxicity was observed as indicated by a reduced relative cloning efficiency of about or below 20% relative survival

HISTORICAL CONTROL DATA
- Positive historical control data: Yes, within the range
- Negative (vehicle) historical control data: Yes, within the range


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of about or below 20% of the respective negative control values were not observed in both experiments in the absence or presence of S9 mix, up to the highest applied concentrations.

CELL MORPHOLOGY
After 4 hours treatment either in the absence or presence of metabolic activation, the cell morphology and attachment of the cells was not adversely influenced (grade > 2) in any test group tested for gene mutations.

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

Exp.	Exposure period [h]	Test groups [μg/mL]	S9-mix	Prec.*	Genotoxicity** MFcorr. [per 10E6 cells]	Cytotoxicity*** CE1 [%]	Cytotoxicity*** CE2 [%]
1	4	Negative control	-	n.d.	2.25	100.0	100.0
		156.3	-	-	n.c.	92.8	n.c.
		312.5	-	-	1.91	98.4	103.4
		625.0	-	-	0.57	84.7	99.2
		1250.0	-	-	1.53	90.6	91.8
		2500.0	-	-	1.15	92.2	98.0
		5000.0	-	-	2.32	72.1	85.1
		Positive control (1)	-	n.d.	78.91 (s)	61.1	113.5
2	4	Negative control	-	n.d.	2.62	100.0	100.0
		375.0	-	-	n.c.	86.6	n.c.
		750.0	-	-	0.39	82.8	84.6
		1500.0	-	-	1.00	87.1	98.4
		3000.0	-	-	0.94	93.6	104.6
		5000.0	-	-	1.00	75.1	98.7
		Positive control (1)	-	n.d.	111.66 (S)	71.7	73.1

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

** Mutant frequency MFcorr.: mutant colonies per 10E6 cells corrected with the CE2 value

*** 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

 

Table 2: Summary of results - experimental parts with S9 mix

Exp.	Exposure period [h]	Test groups [μg/mL]	S9-mix	Prec.*	Genotoxicity** MFcorr. [per 10E6 cells]	Cytotoxicity*** CE1 [%]	Cytotoxicity*** CE2 [%]
1	4	Negative control	+	n.d.	2.00	100.0	100.0
		156.3	+	-	n.c	91.4	n.c.
		312.5	+	-	2.97	95.8	96.3
		625.0	+	-	2.99	90.0	114.6
		1250.0	+	-	1.85	96.4	108.0
		2500.0	+	-	1.26	87.5	113.7
		5000.0	+	-	0.59	88.4	96.3
		Positive control (2)	+	n.d.	48.47 (s)	68.4	74.9
2	4	Negative control	+	n.d.	2.34	100.0	100.0
		375.0	+	-	n.c.	97.4	n.c.
		750.0	+	-	0.75	101.4	89.0
		1500.0	+	-	3.66	128.6	91.3
		3000.0	+	-	3.15	113.9	95.7
		5000.0	+	-	1.57	112.7	85.3
		Positive control (2)	+	n.d.	61.81 (s)	132.7	84.9

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

** Mutant frequency MFcorr.: mutant colonies per 10E6 cells corrected with the CE2 value

*** 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

(2) DMBA 1.25 μg/mL

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Key study

Miconucleus study

In the GLP and OECD 487 guideline complaint study the test substance was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity).

Two independent experiments were carried out, both with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation).

 

Based on the observations and the toxicity data of a previously performed pretest, a HPRT study with concentrations up to 5000 μg/mL was conducted:

1st Experiment: 4 hours exposure, 24 hours harvest time, with/without S9 mix

2nd Experiment:

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

4 hours exposure, 44 hours harvest time, with S9 mix

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 the historical negative control data range for V79 cells. Both positive control substances, ethyl methanesulfonate (EMS 500 μg/mL or 600 μg/mL) and cyclophosphamide (CPP 0.5 μg/mL), led to the expected increase in the number of cells containing micronuclei.

In this study, no cytotoxicity indicated by reduced cell count (indicated by relative population doubling) or proliferation index (CBPI) was observed up to the highest applied test substance concentration.

On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system. The single statistically significant value observed at an intermediate concentration (2500 μg/mL) in the 1st Experiment in the absence of S9 mix has to be regarded as biologically irrelevant.

Thus, under the experimental conditions described, the test substance is considered neither to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Key study

Bacterial reverse mutation test (Ames test)

The test substance was tested for mutagenicity with the strains TA 98, TA 100, TA 1535 and TA 1537 Salmonella typhimurium (Ames Test). The mutagenicity studies were conducted in the absence and in the presence of a metabolizing system derived from rat liver homogenate. A dose range up to 12500 µg/plate was used in the pre-test. No inhibition was observed at any of the concentrations of the test substance employed in this test and the main mutation study was set up using test substance at concentrations in the range 20 to 12500 µg/plate. The test substance did not show in these bacterial test systems either with or without exogenous metabolic activation at the dose levels investigated a dose dependent increase in the number of revertants in any of the bacterial strains. All counts of revertant colonies were similar to those recorded for the negative control plates and were within the range expected for spontaneous reversion for each strain. The positive controls showed the expected results and the activity of the S9 fraction was found to be satisfactory. Based on these results it can be stated that the test substance is not mutagenic in these bacterial test systems.

According to the results of the present study, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here.

Key study

In vitro gene mutation test in CHO cells (HPRT locus assay)

In the GLP and OECD 476 guideline compliant study the test substance 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.

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; 312.5; 625.0; 1250.0; 2500.0; 5000.0 μg/mL

with S9 mix

0; 312.5; 625.0; 1250.0; 2500.0; 5000.0 μg/mL

2nd Experiment

without S9 mix

0; 750.0; 1500.0; 3000.0; 5000.0 μg/mL

with S9 mix

0; 750.0; 1500.0; 3000.0; 5000.0 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.

Finally, 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 increase in the frequencies of forward mutations.

 

In this study in the absence and the presence of metabolic activation, no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any increase in the mutant frequencies either without S9 mix and 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.

Justification for classification or non-classification

Classification, Labeling, 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. Based on the available in vitro genetic toxicity study (Ames test), micronucleus test, HPRT study it is concluded that the test substance is not considered to be classified for mutagenicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776