Benzoic acid, 2,3,4,5-tetrachloro-6-cyano-, methyl ester, reaction products with p-phenylenediamine and sodium methoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames tests: according to OECD 471, GLP, 5 strains, concentrations of 10-5000 µg/plate, with and without S9, negative

- Mouse lymphoma assay: according to OECD 476, GLP, concentrations of 3.2-1000 µg/mL, with and without S9, negative

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:

Apr - May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GYEMSZI National Institue of Pharmacy

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- In the study report the old CAS number of the test substance is given.
- Lot/batch No.of test material: D1003111P2
- Appearance: yellow powder
- Expiry date: 30 July 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dimethyl sulfoxide (DMSO) was used as vehicle to prepare the test item suspensions. The test item suspensions were freshly prepared at the beginning of the experiments.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension

Target gene:

his, trp

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (supplemented with cofactors) derived from phenobarbital (i.p.) and β-naphthoflavone (orally) induced rat liver

Test concentrations with justification for top dose:

Based on the results of the preliminary tests, the test item was suspended in DMSO in nominal concentrations of 50, 25, 10, 3.16, 1, 0.32 and 0.1 mg/mL to obtain seven dosing solutions (suspensions) for the test item doses. The maximum test concentration was 5000 µg test item/plate (with and without S9 mix).
Test Item concentrations tested: 5000, 2500, 1000, 316, 100, 31.6 and 10 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was used as vehicle to prepare the test item suspensions. All dilutions of test item were made in the testing laboratory using DMSO vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene (with S9 mix, 2 μg/plate, dissolved in DMSO, TA 98, TA 100, TA 1535 and TA 1537; and 50 µg/plate, dissolved in DMSO, E.coli WP2 uvrA); 4-nitro-o-phenylenediamine (without S9 mix, 4 μg/plate, dissolved in DMSO, TA 98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: about 48 hours

The tests (Initial and Confirmatory Mutation Tests) are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA 98 and TA 100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia WP2 uvrA culture demonstrate the deletion in the uvrA gene.
- The bacterial cultues demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titers is in the 10^9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analyzable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).
[A dose level is considered toxic if the reduced revertant colony numbers are observed as compared to the mean vehicle control value and the reduction shows a dose-dependent relationship, and / or the reduced revertant colony numbers are below the historical control data range and / or pinpoint colonies appear and / or reduced background lawn development occurs.]

Evaluation criteria:

The colony numbers on the control, positive control and the test plates were determined (counted manually), the mean values and appropriate standard deviations and mutation rates were calculated.
Mutation Rate = (Mean revertants at the test item (or control) treatments / Mean revertants of vehicle control)

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and / or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA 100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

precipitation at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Table 1: Summary Table of the Results of the Range Finding Test

Concentrations [µg/plate]	Salmonella typhimurium tester strains
	TA 98				TA 100
	-S9		+S9		-S9		+S9
Mean values of revertants per plate and Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated control	27.7	1.22	32.0	0.98	76.3	1.05	98.0	1.09
DMSO control	22.7	1.00	32.7	1.00	72.7	1.00	90.0	1.00
Ultrapure water control	-	-	-	-	79.3	1.00	-	-
5000	32.7	1.44	42.0	1.29	78.7	1.08	83.0	0.92
4000	35.0	1.54	40.7	1.24	75.7	1.04	78.7	0.87
2500	21.0	0.93	32.0	0.98	83.3	1.15	78.3	0.87
1250	19.7	0.87	32.3	0.99	79.0	1.09	83.0	0.92
625	23.7	1.04	39.3	1.20	79.0	1.09	85.7	0.95
125	25.7	1.13	27.3	0.84	78.7	1.08	89.7	1.00
25	28.3	1.25	34.0	1.04	80.0	1.10	81.0	0.90
5	27.7	1.22	27.0	0.83	80.3	1.11	82.0	0.91
NPD (4 µg)	191.3	8.44	-	-	-	-	-	-
SAZ (2 µg)	-	-	-	-	805.3	10.15	-	-
2AA (2 µg)	-	-	1093.3	33.47	-	-	1197.7	13.31

 

 

Table 2: Summary table of the results of the initial mutation test

Concentrations [µg/plate]	Salmonella typhimurium tester strains																Escherichia coli WP2 uvrA
	TA 98				TA 100				TA 1535				TA 1537
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated control	34.7	1.14	35.0	1.19	85.0	1.21	72.3	0.95	9.0	1.35	9.3	1.22	6.7	0.91	9.3	1.22	16.7	1.04	30.3	1.21
DMSO control	30.3	1.00	29.3	1.00	70.0	1.00	76.3	1.00	6.7	1.00	7.7	1.00	7.3	1.00	7.7	1.00	16.0	1.00	25.0	1.00
Ultrapure water control	-	-	-	-	78.0	1.00	-	-	6.0	1.00	-	-	-	-	-	-	19.0	1.00	-	-
5000	26.7	0.88	50.7	1.73	63.0	0.90	78.0	1.02	7.0	1.05	6.7	0.87	5.0	0.68	5.7	0.74	13.7	0.85	16.7	0.67
2500	25.7	0.85	45.0	1.53	84.7	1.21	85.0	1.11	6.7	1.00	9.3	1.22	8.0	1.09	7.3	0.96	11.7	0.73	16.0	0.64
1000	18.7	0.62	34.0	1.16	84.7	1.21	84.3	1.10	6.0	0.90	13.7	1.78	6.3	0.86	7.7	1.00	13.0	0.81	21.3	0.85
316	30.0	0.99	35.7	1.22	76.3	1.09	81.7	1.07	4.7	0.70	7.3	0.96	9.0	1.23	6.0	0.78	20.7	1.29	22.7	0.91
100	36.7	1.21	36.0	1.23	66.7	0.95	91.3	1.20	8.3	1.25	8.0	1.04	8.3	1.14	7.3	0.96	13.7	0.85	18.3	0.73
31.6	30.3	1.00	39.0	1.33	78.0	1.11	89.3	1.17	6.3	0.95	7.7	1.00	6.0	0.82	8.7	1.13	17.7	1.10	24.7	0.99
10	27.7	0.91	40.3	1.38	75.7	1.08	80.0	1.05	7.3	1.10	8.3	1.09	5.7	0.77	7.3	0.96	13.0	0.81	20.0	0.80
NPD (4 µg)	166.3	5.48	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
SAZ (2 µg)	-	-	-	-	889.3	11.40	-	-	920.0	153.33	-	-	-	-	-	-	-	-	-	-
9 AA (50 µg)	-	-	-	-	-	-	-	-	-	-	-	-	333.3	45.45	-	-	-	-	-	-
MMS (2 µL)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	652.7	34.35	-	-
2 AA (2 µg)	-	-	910.7	31.05	-	-	1480.3	19.39	-	-	116.0	15.13	-	-	137.3	17.91	-	-	-	-
2 AA (50 µg)	-	-	-	-			-	-	-	-	-	-	-	-	-	-	-	-	288.3	11.53

 

 

Table 3: Summary table of the results of the confirmatory mutation test

Concentrations [µg/plate]	Salmonella typhimurium tester strains																Escherichia coli WP2 uvrA
	TA 98				TA 100				TA 1535				TA 1537
	-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9		-S9		+S9
Mean values of revertants per plate Mutation rate (MR)	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR	Mean	MR
Untreated control	25.7	1.12	40.3	1.14	91.0	1.19	99.0	1.02	11.7	1.67	15.0	1.36	4.7	1.08	7.3	0.92	17.3	0.75	29.3	0.87
DMSO control	23.0	1.00	35.3	1.00	76.3	1.00	96.7	1.00	7.0	1.00	11.0	1.00	4.3	1.00	8.0	1.00	23.0	1.00	33.7	1.00
Ultrapure water control	-	-	-	-	83.7	1.00	-	-	8.7	1.00	-	-	-	-	-	-	21.7	1.00	-	-
5000	18.0	0.78	42.3	1.20	68.7	0.90	95.3	0.99	7.7	1.10	9.7	0.88	5.7	1.31	7.7	0.96	13.3	0.58	29.7	0.88
2500	12.7	0.55	35.3	1.00	66.7	0.87	98.7	1.02	6.0	0.86	9.0	0.82	6.7	1.54	5.0	0.63	21.3	0.93	29.7	0.88
1000	20.0	0.87	35.3	1.00	71.3	0.93	88.3	0.91	8.0	1.14	10.7	0.97	5.7	1.31	5.3	0.67	16.3	0.71	29.0	0.86
316	32.3	1.41	33.0	0.93	77.0	1.01	91.3	0.94	7.3	1.05	10.0	0.91	4.7	1.08	9.0	1.13	20.7	0.90	31.0	0.92
100	30.7	1.33	28.0	0.79	66.7	0.87	75.7	0.78	9.0	1.29	10.3	0.94	4.7	1.08	6.0	0.75	16.7	0.72	27.3	0.81
31.6	29.3	1.28	35.0	0.99	60.7	0.79	85.0	0.88	6.0	0.86	11.0	1.00	4.7	1.08	8.3	1.04	17.3	0.75	35.3	1.05
10	21.0	0.91	33.7	0.95	61.7	0.81	78.3	0.81	7.3	1.05	9.3	0.85	4.7	1.08	7.3	0.92	16.0	0.70	28.3	0.84
NPD (4 µg)	193.3	8.41	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
SAZ (2 µg)	-	-	-	-	1169.3	13.98	-	-	730.0	84.23	-	-	-	-	-	-	-	-	-	-
9 AA (50 µg)	-	-	-	-	-	-	-	-	-	-	-	-	525.0	121.15	-	-	-	-	-	-
MMS (2 µL)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	645.3	29.78	-	-
2 AA (2 µg)	-	-	913.3	25.85	-	-	828.3	8.57	-	-	121.3	11.03	-	-	173.7	21.71	-	-	-	-
2 AA (50 µg)	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	202.0	6.00

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of phenobarbital / ß-naphthoflavone-induced rats.

The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test).

Based on the results of the preliminary Range Finding Test the following concentrations of the test item were prepared and used in the Initial and Confirmatory Mutation Tests: 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate.

In the performed preliminary experiments no cytotoxicity of the test item was observed. At the concentration choice the guideline criterion for non-cytotoxic substances was taken into consideration where the recommended maximum test concentration is 5000 µg/plate. The test item was not soluble, and precipitated at the concentration range of 5000 - 1000 µg/plate. The obtained precipitate interfered the scoring, therefore the necessity of an additional microscopic analysis of the plates was considered.

In the Initial and Confirmatory Mutation Tests the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.

The revertant colony numbers of vehicle control plates with and without S9 Mix were within the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May - Jun 2012

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:

21st July 1997

GLP compliance:

yes (incl. QA statement)

Remarks:

GYEMSZI National Institute of Pharmacy

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- In the study report the old CAS number of the test substance is given.
- Batch No.of test material: D1003111P2
- Appearance: Yellow powder
- Expiry date: 30 July 2021

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Solubility and stability of the test substance in the vehicle: The solubility and behaviour of the test item and its solutions - suspensions in the applied test system was determined in the preliminary solubility test. The RPMI 5 Medium (the treatment medium) was taken into consideration as vehicle at the 3-hour and 24-hour treatments.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension

Target gene:

TK

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: mouse lymphoma L5178Y TK+/- cell line (clone 3.7.2c)
- doubling time: 9 - 10 hours
- Methods for maintenance in cell culture if applicable: Stocks of the mouse lymphoma L5178Y TK+/- cell line (1-mL portions) in culture medium supplemented with 7% (v/v) DMSO are stored in liquid nitrogen (about -196°C). Each batch used for mutagenicity testing was checked for mycoplasma contamination previously. For cell cultivation, deep-frozen stock cultures were thawed at 37°C in a water bath, and volumes of 1 mL were transferred into 25 cm2 flasks containing 10 mL RPMI-10 medium. After incubation for about one day, the cells were centrifuged at 1000 rpm (173 g) for 5 minutes. Subsequently, the medium was removed and the cells were resuspended in 20 mL RPMI-10 medium, transferred to 75 cm² flasks and incubated until use. The cells were subcultured twice weekly (routine passage in 75 cm2 flasks). All incubations occurred with 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity.
- Modal number of chromosomes: stable karyotype with a near diploid number of 40 ± 1 chromosomes

MEDIA USED
- Type and identity of media:
* RPMI-0 = RPMI 1640 medium including stable glutamine supplemented with: 1% (v/v) penicillin/streptomycin (10000 IU / 10000 μg/mL) and 1% (v/v) sodium pyruvate (10 mM)
* RPMI-5 = Treatment medium (with S9 mix): RPMI-0 supplemented with 5% (v/v) fetal calf serum (FCS)
* RPMI-10 = Treatment medium (without S9 mix) and subculturing cells: RPMI-0 is supplemented with 10% (v/v) fetal calf serum
* RPMI-20 = Cloning efficiency and selection medium: RPMI-0 supplemented with 20% (v/v) fetal calf serum
* "THMG" medium = Pretreatment medium A: RPMI-10 supplemented with thymidine 3.0 μg/mL, hypoxanthine 5.0 μg/mL, methotrexate 0.1 μg/mL and glycine 7.5 μg/mL
* "THG" medium = Pretreatment medium B: RPMI-10 supplemented with thymidine 3.0 μg/mL, hypoxanthine 5.0 μg/mL and glycine 7.5 μg/mL
* "TFT" medium = RPMI-20 supplemented with trifluorothymidine (TFT) 4.0 μg/mL

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 mix from phenobarbital (i.p.) and β-naphthoflavone (oral) induced rats

Test concentrations with justification for top dose:

The following concentrations were investigated in the Assay 1:
3-hour treatment (with and without S9 mix): 3.2; 10; 31.6; 100; 316 and 1000 µg/mL

The following concentrations were investigated in the Assay 2:
24-hour treatment (without S9 mix): 3.2; 10; 31.6; 100; 316 and 1000 µg/mL
3-hour treatment (with S9 mix): 3.2; 10; 31.6; 100; 316 and 1000 µg/mL

The concentrations applied in the Assay 1 and Assay 2 were chosen according to the solubility and cytotoxicity results of the pre-experiments. The test item was considered as relatively insoluble therefore it was investigated beyond its limit of solubility under culture conditions.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 5 Medium (treatment medium)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

PRELIMINARY SOLUBILITY AND TOXICITY TESTS
A preliminary solubility test was performed for selection of the appropriate vehicle and treatment concentrations to be used in the dose-range finding test. In the preliminary solubility test the test item direct addition and the use of the aqueous RPMI 5 medium as vehicle was considered. In the preliminary toxicity test a 3-hour treatment in the presence and absence of S9 mix and a 24-hour treatment in the absence of S9 mix were performed to determine the test item toxicity. The treatment procedure of the cell cultures was the same as described below for the main mutation assays.
In the preliminary toxicity test, single cultures were used (with exception of the RPMI 5 medium controls at the 3-hour treatment (+ S9) and at the 24-hour treatment (- S9)) and positive controls were not included. The concentrations of 5000, 2000, 1000, 316, 100, 31.6, 10 and 3.2 µg/mL were investigated at the 3-hour treatments in absence and also in presence of S9 mix and at the 24-hour treatment in absence of S9 mix. The investigation of the 5000 and 2000 µg/mL concentrations was possible, however these treatments were strongly precipitated at the end of the incubation periods. The colour and the thickness of the obtained suspension at > 1 mg/mL concentration highly disturbed the scoring. The treated cells were washed following treatment with RPMI 10 medium and resuspended in 10 mL RPMI 10 culture medium. Cell concentrations were adjusted to 8 cells/mL and, for each dose, 0.2 mL was plated into each well of a 96-well microtiter plate. The microtiter plates were incubated at 37 °C +/- 1 °C in a humidified incubator gassed with 5 % (v/v) CO2 in air for 9 - 10 days (at the RPMI 5 medium controls 15 - 16 days). Wells containing viable clones were identified by eye and counted. At the end of this test the harmonized relative survival of each culture was determined. The obtained cell growth data were used to choose the dose levels for the main assays. Six concentrations were selected for the main mutation experiments.

METHOD OF APPLICATION: in medium
- Cell density at seeding: 1 x 10^7 cells per 75 cm² flask (3-hour treatment) and 4 x 10^6 cells per 25 cm² flask (24-hour treatment)

DURATION
- Preparation of test cultures: For the experiments, 3-3 vials were thawed rapidly, cells were diluted in RPMI 10 medium and incubated at 37 °C +/- 0.5 °C in a humidified atmosphere containing approximately 5 % CO2 in air. Well growing cells, subcultures were established in an appropriate number of flasks.
- Exposure duration: 3-hour treatment and 24-hour treatment
- Expression time: 2 days
- Selection time: 2 weeks

SELECTION AGENT: trifluorothymidine (TFT)

DETERMINATION OF CYTOTOXICITY
- relative survival (% RS)
- relative suspension growth (% RSG)
- relative viability (% RV)
- relative total growth (% RTG)

Evaluation criteria:

The assay is considered valid if all of the following criteria are met:
1. The mutant frequency in the negative (vehicle) control cultures fall within the normal range (above 50 - 170 mutants per 10^6 viable cells).
2. The positive control chemicals induce a statistically significant increase in the mutant frequency.
3. The plating efficiency of the negative controls is within the range of 65 % to 120 % on Day 3 (at the end of the expression period).
4. At least four test concentrations are present, where the highest concentration produces 80 - 90 % toxicity, precipitation, or is 5 mg/mL, or the highest practical concentration.

The test item is considered to be mutagenic in this assay if all the following criteria are met:
1. The assay is valid;
2. Statistically significant (p < 0.05) increases in mutation frequency are observed in treated cultures compared to the corresponding vehicle control values at one or more concentrations;
3. The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
4. There is a significant dose-relationship as indicated by the adequate trend analysis;
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 10^6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value.

Results, which only partially satisfied the criteria, should be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity should require careful interpretation when assessing their biological significance. Indeed, extreme caution should be exercised with positive results obtained at levels of survival lower than 10 %. There is no requirement for verification of a clear positive response. Equivocal or negative results need to be verified in a follow-up experiment. Modification of study parameters in the follow-up experiment should be considered.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- pH and osmolality values were within acceptable ranges.

MUTANT FREQUENCY
- Assay 1: The obtained mutation frequencies remained nearly in the same (vehicle control) range far below the threshold (GEF criterion) for positive call and statistically significant differences in comparison with the negative control were not obtained in any case (Dunnett’s Test, α=0.05).
- Assay 2, 24 h treatment: The calculated mutation frequencies of the treated concentrations (3.2-1000 μg/mL) were in the same range, did not show dose-related tendencies, and did not differ statistically significantly from the mutation frequency of the RPMI 5 Medium (vehicle) control (Dunnett’s Test, α = 0.05).
- Assay 2, 3 h treatment: The mutation frequencies in the treated concentrations did not differ statistically significantly from the mutation frequency of the vehicle control (Dunnett’s Test, α=0.05).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Assay 1: No toxicity was observed in the whole examined concentration range based on the harmonised relative survival (harmonised RS) and based on the relative total growth values (RTG). The 23 % inhibition obtained at the concentration of 1000 μg/mL based on the harmonised RS in absence of S9 Mix was evaluated as reflecting the biological variability of the test system. The viability data did not show any test item effect in the function of the increasing concentration in the whole concentration range.
- Assay 2, 24 h treatment: No toxicity was observed in the whole examined concentration range based on the harmonised relative survival (harmonised RS) values and 46 % inhibition was obtained at the concentration of 316 μg/mL and 63% at 1000 μg/mL based on the relative total growth values (RTG) indicating the inhibitory effect of the test item. The viability data did not show any test item effect in the function of the increasing concentration in the whole concentration range.
- Assay 2, 3 h treatment: Differently from the Assay 1 results slight cytotoxicity was obtained based on the harmonised relative survival (harmonised RS) values. 39 % was obtained at 1000 μg/mL, 30 % at the concentration of 316 μg/mL. Based on the relative total growth 21 % inhibition was obtained at 316 μg/mL and 43 % at 1000 μg/mL.
In conclusion the test item showed a slight inhibitory effect that manifested at the higher concentration levels in absence and also in presence of metabolic activation. The obtained corresponding harmonised RS and RTG value changes (were caused –supposedly- by not observable inhomogeneities of the treatment suspensions) remained mostly within a biological variability range of the applied system.

Table 1: Results of the preliminary toxicity test

Concentration [µg/mL]	Number of empty wells / total number of wells	Plating Efficiency (PE)	Harmonized Relative Survival [% RS]
Treatment period [hours]: 3
Without exogeneous metabolic activation (- S9 mix)
3.16 10 31.6 100 316 1000 2000 5000	29/192 31/192 36/192 29/192 26/192 29/192 30/192 32/192	1.2001 1.1426 1.0501 1.1847 1.2571 1.1817 1.1658 1.1199	100.00 117.88 94.79 114.78 98.10 104.33 87.12 68.13
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.16 10 31.6 100 316 1000 2000 5000	77/384 22/192 38/192 21/192 45/192 32/192 38/192 35/192 35/192	1.0062 1.3984 1.0203 1.4199 0.9107 1.1199 1.0133 1.0641 1.0641	100.00 138.39 123.75 121.39 83.22 97.15 80.65 77.53 63.19
Treatment period [hours]: 24
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.16 10 31.6 100 316 1000 2000 5000	66/384 40/192 50/192 58/192 37/192 47/192 53/192 29/192 46/192	1.1110 0.9875 0.8414 0.7485 1.0312 0.8831 0.8100 1.1817 0.9082	100.00 119.41 115.19 110.15 111.63 90.81 67.17 44.54 26.84

 

 

Table 2: Summarized results of the survival data of the first assay

Concentration [µg/mL]	Number of empty wells / total number of wells	Plating Efficiency (PE)	Harmonized Relative Survival [% RS]
Treatment period [hours]: 3
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.2 µg/mL)	56/384 68/384 57/384 69/384 48/384 65/384 57/384 140/384	1.2334 1.0886 1.1925 1.1187 1.3051 1.1159 1.2091 0.6347	100.00 88.51 84.30 89.79 94.24 82.40 77.01 43.09
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	65/384 57/384 54/384 51/384 56/384 56/384 56/384 306/384	1.1568 1.2198 1.2407 1.2871 1.2250 1.2306 1.2211 0.1447	100.00 105.62 105.18 102.48 100.43 105.53 93.65 9.78

 

 

Table 3: Summarized results of the survival data of the second assay

Concentration [µg/mL]	Number of empty wells / total number of wells	Plating Efficiency (PE)	Harmonized Relative Survival [% RS]
Treatment period [hours]: 24
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.1 µg/mL)	74/384 93/384 117/384 88/384 95/384 63/384 84/384 218/384	1.0352 0.9183 0.7563 0.9622 0.8927 1.1497 0.9571 0.3596	100.00 85.34 83.06 98.96 91.81 104.72 80.85 23.51
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	64/384 69/384 84/384 69/384 69/384 64/384 77/384 143/384	1.1279 1.0865 0.9510 1.0730 1.0962 1.1261 1.0349 0.6229	100.00 99.06 81.66 87.33 79.41 69.60 61.07 59.41

 

 

Table 4: Summarized results of the viability data of the first assay

Concentration [µg/mL]	Number of empty wells / total number of wells	Plating Efficiency (PE)	Relative Viability [% RV]
Treatment period [hours]: 3
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.2 µg/mL)	66/384 99/384 57/384 69/384 59/384 60/384 63/384 92/384	1.1182 0.8500 1.1957 1.0938 1.1762 1.1695 1.1460 0.9061	100.00 76.02 106.94 97.82 105.19 104.59 102.49 81.03
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	74/384 60/384 69/384 67/384 65/384 61/384 67/384 314/384	1.0494 1.1654 1.0826 1.1216 1.1115 1.1588 1.1016 0.1284	100.00 111.06 103.16 106.89 105.92 110.43 104.98 12.24

 

 

Table 5: Summarized results of the viability data of the second assay

Concentration [µg/mL]	Number of empty wells / total number of wells	Plating Efficiency (PE)	Relative Viability [% RV]
Treatment period [hours]: 24
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.1 µg/mL)	60/384 65/384 57/384 59/384 81/384 57/384 64/384 114/384	1.1784 1.1604 1.2133 1.1749 0.9798 1.2194 1.1704 0.7752	100.00 98.47 102.96 99.70 83.14 103.48 99.32 65.78
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	59/384 56/384 54/384 51/384 56/384 53/384 58/384 117/384	1.1773 1.2153 1.2701 1.2848 1.2163 1.2433 1.1965 0.7440	100.00 103.23 107.88 109.13 103.31 105.60 101.63 63.19

 

 

Table 6: SG, RSG and RTG data of the first assay

Concentration [µg/mL]	Suspension growth (SG)	Relative Suspension Growth [% RSG]	Relative Total Growth [% RTG]
Treatment period [hours]: 3
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.2 µg/mL)	15.19 18.68 16.30 14.72 15.29 14.39 12.32 9.01	100.00 122.99 107.29 96.92 100.66 94.76 81.11 59.30	100.00 93.49 114.73 94.81 105.88 99.11 83.12 48.05
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	17.27 17.70 17.97 17.37 16.28 13.08 16.65 2.65	100.00 102.50 104.05 100.56 94.27 75.74 96.43 15.35	100.00 113.84 107.34 107.49 99.86 83.64 101.24 1.88

 

 

Table 7: SG, RSG and RTG data of the second assay

Concentration [µg/mL]	Suspension growth (SG)	Relative Suspension Growth [% RSG]	Relative Total Growth [% RTG]
Treatment period [hours]: 24
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.1 µg/mL)	32.07 25.64 25.64 30.74 31.65 16.85 11.99 5.31	100.00 79.97 79.96 95.88 98.72 52.56 37.38 16.56	100.00 78.75 82.33 95.59 82.08 54.39 37.12 10.89
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	14.92 14.47 13.76 13.23 14.55 11.23 8.34 9.22	100.00 96.98 92.17 88.62 97.48 75.26 55.88 61.78	100.00 100.11 99.43 96.71 100.70 79.47 56.79 39.04

 

 

Table 8: Summarized mutagenicity results of the first assay

Concentration [µg/mL]	Number of empty wells / total number of wells	Number of large colonies / total number of wells	Number of small colonies / total number of wells	Mutation frequency
Treatment period [hours]: 3
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.2 µg/mL)	625/768 671/768 635/768 641/768 619/768 634/768 640/768 241/768	114/768 53/768 108/768 78/768 106/768 80/768 91/768 338/768	29/768 44/768 25/768 49/768 43/768 54/768 37/768 189/768	92.45 79.78 79.86 82.89 91.96 82.13 79.98 654.29
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	631/768 636/768 645/768 639/768 650/768 644/768 626/768 487/768	95/768 92/768 69/768 92/768 86/768 90/768 102/768 118/768	42/768 40/768 54/768 37/768 32/768 34/768 40/768 163/768	93.73 81.12 80.78 82.36 75.28 76.17 93.11 1781.18

 

 

Table 9: Summarized mutagenicity results of the second assay

Concentration [µg/mL]	Number of empty wells / total number of wells	Number of large colonies / total number of wells	Number of small colonies / total number of wells	Mutation frequency
Treatment period [hours]: 24
Without exogeneous metabolic activation (- S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 NQO (0.1 µg/mL)	587/768 594/768 609/768 581/768 618/768 596/768 599/768 140/768	107/768 115/768 102/768 124/768 96/768 85/768 87/768 421/768	74/768 59/768 57/768 63/768 54/768 87/768 82/768 207/768	114.73 110.98 96.01 118.91 111.30 104.25 106.72 1113.90
Treatment period [hours]: 3
With exogeneous metabolic activation (+ S9 mix)
RPMI 5 Medium 3.2 10 31.6 100 316 1000 CP (5 µg/mL)	628/768 637/768 616/768 599/768 634/768 600/768 632/768 174/768	91/768 92/768 104/768 104/768 84/768 95/768 91/768 338/768	49/768 39/768 48/768 65/768 50/768 73/768 45/768 256/768	85.79 77.47 87.10 97.32 79.54 99.72 81.63 1010.51

Conclusions:

Under the conditions of this study, the test item did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test item to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (±S9 Mix) and for 24 hours without metabolic activation (-S9 Mix).

Based on the results of the preliminary Solubility and Toxicity Tests and regarding the practical difficulties with the test item handling, the test item was suspended and diluted in RPMI 5 Medium and the RPMI 5 Medium was parallel investigated as vehicle control.

The concentrations applied in the Assay 1 and Assay 2 were chosen according to the solubility and cytotoxicity results of the pre-experiments. The test item was considered as relatively insoluble therefore it was investigated beyond its limit of solubility under culture conditions. The following concentrations were investigated in the Assay 1 and Assay 2:

The following concentrations were investigated in the Assay 1:

3-hour treatment (±S9 Mix): 3.2; 10; 31.6; 100; 316 and 1000 μg/mL;

The following concentrations were investigated in the Assay 2:

24-hour treatment (-S9 Mix): 3.2; 10; 31.6; 100; 316 and 1000 μg/mL;

3-hour treatment (+S9 Mix): 3.2; 10; 31.6; 100; 316 and 1000 μg/mL.

In the performed mutation assays the cell cultures were treated with a range of the test item concentrations. After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2x105/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days) and diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

In the main assays, the relative harmonised survival the relative total growth of the cells, the viability (colony-forming ability at the end of the 2 day expression period following the treatment) and the potential mutagenicity (5-trifluorothymidine resistance) were determined.

The result of Assay 1 was considered to be negative, hence in the second Assay was performed with the originally planned concentration levels chosen based on the preliminary cytotoxicity test with the treatment periods of 3 (in presence of metabolic activation) and 24 hours (in absence of metabolic activation).

The performed Assays fulfilled the validity criteria in connection with the negative control and positive control treatments as well as in connection with the number of analyzable concentration levels (at least four). The test item concentrations were chosen based on the test item solubility. In the examined concentration range noticeable cytotoxicity did not occur at the 3-hour and 24-hour treatments.

In the performed assays the obtained mutation frequencies (in absence and also in presence of exogenous metabolic activation) did not show dose-related tendencies, remained far below the relevant GEF thresholds for positive call and remained in the validity criterion range of the negative vehicle control cultures. The mutation frequencies were not statistically significantly different (Dunnett’s Test, α = 0.05) from that of the corresponding vehicle control throughout the study (Sections 10.2 and 10.3).

Under the conditions of this study, the test item did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- Micronucleus assay: according to OECD 474, GLP, mouse, doses of 500-2000 mg/kg bw/d, negative

- UDS test: according to OECD 486, GLP, rat, doses of 1000-2000 mg/kg bw/d, negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

- Physical state: Solid, orange
- Analytical purity: 97.8%
- Lot/batch No.: 13277HM8
- Storage condition of test material: Room temperature

Species:

mouse

Strain:

NMRI

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
- Mean weight at study initiation: 29.5 g
- Assigned to test groups randomly: yes, under following basis: appropriate computer program
- Housing: Makrolon cages, type M II; single housing
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water (e.g. ad libitum): Drinking water from bottles was available ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Using the vehicle corn oil a homogeneous suspension of the test substance was obtained. Therefore, corn oil was used as vehicle, which has been demonstrated to be suitable in the in vivo Micronucleus test and for which historical control data are available.
- Concentration of test material in vehicle: 50, 100 or 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
To achieve the homogeneity of the test substance in the vehicle, the test substance preparation was stirred with a spatula and shaken thoroughly. All test substance formulations were prepared immediately before administration.

Duration of treatment / exposure:

24 or 48 hrs

Frequency of treatment:

once

Dose / conc.:

500 mg/kg bw/day

Remarks:

Basis:
analytical conc.
Depending on the dose, about 88% - 104% of the theoretical values were determined analytically. Only the recovery rate of the low dose group (88%) was slightly lower than expected (90 - 110%).

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Basis:
analytical conc.
Depending on the dose, about 88% - 104% of the theoretical values were determined analytically. Only the recovery rate of the low dose group (88%) was slightly lower than expected (90 - 110%).

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Basis:
analytical conc.
Depending on the dose, about 88% - 104% of the theoretical values were determined analytically. Only the recovery rate of the low dose group (88%) was slightly lower than expected (90 - 110%).

No. of animals per sex per dose:

5 males per treatment

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive controls, both dissolved in deionised water, were administered to male animals once orally (cyclophosphamide, CPP) or intraperitoneally (vincristine sulphate, VCR) each in a volume of 10 mL/kg body weight. The animals were sacrificed after 24 hours.

Tissues and cell types examined:

The bone marrow was prepared according to the method described by Schmid (1976, 1977) and Salamone et al. (1980).
- The animals were anesthetized with isoflurane and afterwards sacrificed by cervical dislocation. Then the two femora were prepared by dissection and removing all soft tissues.
- After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with foetal calf serum (FCS) which was preheated up to 37°C (about 2 mL/femur).
- The suspension was mixed thoroughly with a pipette and centrifuged at 300 x g for 5 minutes. The supernatant was removed and the precipitate was resuspended in about 50 μL fresh FCS.
- One drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges. The preparations were dried in the air and subsequently stained.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In a pretest for the determination of the acute oral toxicity, 2 000 mg/kg body weight recommended as the highest dose according to the OECD Guideline was tolerated by all animals (male and female) without clinical signs of toxicity. Additionally, discoloured faeces were observed from 4 hours after test substance administration until sacrifice of the animals.
On account of the results of the pretest, 2 000 mg/kg body weight was selected as the highest dose in the present cytogenetic study. 1 000 mg/kg and 500 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES:
sacrifice interval 24 h
vehicle control, 500 mg/kg bw TS, 1000 mg/kg bw TS, 2000 mg/kg bw TS, 20 mg/kg bw CPP, 0.15 mg/kg bw VCR

sacrifice interval 48 h
vehicle control, 2000 mg/kg bw TS

DETAILS OF SLIDE PREPARATION:
- The slides were stained with eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
- After briefly rinsing in deionised water, the preparations were soaked in deionised water for about 2 - 3 minutes.
- Subsequently, the slides were stained with Giemsa solution (15 mL Giemsa plus 185 mL deionised water) for about 15 minutes.
- After rinsing twice in deionised water and clarifying in xylene, the preparations were mounted in Corbit-Balsam.

Evaluation criteria:

A finding is considered positive if the following criteria are met:
• Statistically significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent vehicle control value and the range of the historical vehicle control data.

A test substance is considered negative if the following criteria are met:
• The number of cells 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.

Statistics:

The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there are statistically significant differences between the untreated control group and the treated dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Statistical significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01
However, both biological relevance and statistical significance were considered together.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The single oral administration of the vehicle corn oil in a volume of 10 mL/kg body weight led to 1.5‰ polychromatic erythrocytes containing micronuclei after the 24-hour sacrifice interval or to 1.6‰ after the 48-hour sacrifice interval, respectively.
After the single administration of the highest dose of 2 000 mg/kg body weight, 1.9‰ polychromatic erythrocytes containing micronuclei were found after 24 hours and 1.5‰ after 48 hours.
In the two lower dose groups, rates of micronuclei of 1.1‰ (1 000 mg/kg group) and 2.1‰ (500 mg/kg group) were detected after a sacrifice interval of 24 hours in each case.
The positive control substance for clastogenicity, cyclophosphamide, led to a statistically significant increase (23.2‰) in the number of polychromatic erythrocytes containing exclusively small micronuclei, as expected.
Vincristine sulphate, a spindle poison, produced a statistically significant increase (45.0‰) in the number of polychromatic erythrocytes containing micronuclei. A significant portion increase, 13.0‰ was attributable to large micronuclei.
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the vehicle control group or in the various dose groups at any of the sacrifice intervals.
No dose-dependent inhibition of erythropoiesis induced by the treatment of mice with the test substance was detected. The ratio of polychromatic to normochromatic erythrocytes was in the range of the vehicle control values in all dose groups.

Table 1: Induction of Micronuclei in bone marrow cells

Test group	Sacrifice interval [hrs]	Animal No.	Micronuclei in PCE		Number of NCE ( c)
			total (a) [‰]	large (b) [‰]
Vehicle control:
corn oil	24	5	1.5	0.0	3914
Test substance
500 mg/kg bw.	24	5	2.1	0.1	4532
Test substance
1 000 mg/kg bw.	24	5	1.1	0.0	5094
Test substance
2 000 mg/kg bw.	24	5	1.9	0.0	4177
Positive control
cyclophosphamide
20 mg/kg bw.	24	5	23.2**	0.0	4778
Positive control
vincristine sulfate
0.15 mg/kg bw.	24	5	45.0**	13.0**	4304
Vehicle control
corn oil	48	5	1.6	0.0	4141
Test substance
2 000 mg/kg bw.	48	5	1.5	0.0	4033
PCE = polychromatic erythrocytes
NCE = normochromatic erythrocytes
bw. = body weight
a = sum of small and large micronuclei
b = large micronuclei (indication for spindle poison effect)
c = number of NCEs observed when scoring 10 000 PCEs
* = p ≤ 0.05
** = p ≤ 0.01

Conclusions:

Under the experimental conditions chosen here, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells of NMRI mice in vivo.

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

- Physical state: Solid, orange
- Analytical purity: 97.8%
- Storage condition of test material: Room temperature

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH: Crl:WI (Han)
- Age at study initiation: 8 – 10 weeks
- Mean weight at study initiation: 234.4 g (± 10.70 g)
- Assigned to test groups randomly: yes
- Fasting period before study: for at least 6 hours before administration
- Housing: Makrolon cages, type M III
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum
- Water (e.g. ad libitum): Drinking water from bottles will be available ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Using the vehicle corn oil a homogeneous suspension of the test substance was obtained. Therefore, corn oil was used as vehicle, which has been demonstrated to be suitable in the in vivo Micronucleus test and for which historical control data are available.
- Concentration of test material in vehicle: 50, 100 or 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
To achieve the homogeneity of the test substance in the vehicle, the test substance preparation was stirred with a spatula and shaken thoroughly. All test substance formulations were prepared immediately before administration.

Duration of treatment / exposure:

3 and 14 hours

Frequency of treatment:

once

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

Basis:
analytical conc.
Depending on the dose, about 94% – 104% of the theoretical values were determined analytically.

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

Basis:
analytical conc.
Depending on the dose, about 94% – 104% of the theoretical values were determined analytically.

No. of animals per sex per dose:

3 (Four animals were treated per test group, but only three animals were prepared. In case of a failing preparation the reserve animals would have been prepared)

Control animals:

yes, concurrent vehicle

Positive control(s):

50 mg/kg body weight 2-acetylaminofluorene (2-AAF), suspended in corn oil. The stability of 2-AAF is well-defined under the selected culture conditions, since it is a well established UDS-inducing substance.

Tissues and cell types examined:

About 3 hours and 14 hours after test substance administration, the animals were anesthetized and then the livers were perfused to obtain primary rat hepatocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In a pretest for the determination of the acute oral toxicity, 2000 mg/kg body weight recommended as the highest dose according to the OECD Guideline was tolerated by all animals (male and female) without clinical signs of toxicity. Additionally, discoloured faeces was observed from 4 hours after test substance administration until sacrifice of the animals.
On account of the results of the pretest, 2000 mg/kg body weight was selected as the highest dose in the present cytogenetic study and 1000 mg/kg body weight were administered as further doses.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling time 3 hrs and 14 hrs
Vehicle control, 1000 mg/kg bw, 2000 mg/kg bw, 50 mg/kg bw 2-AAF

Evaluation criteria:

A test substance is considered positive if an increase is demonstrated in both of the following:
• The mean net nuclear grain count (NNGC) must exceed zero at one of the dose groups.
• The mean net nuclear grain count (NNGC) clearly exceeds the value of the concurrent negative control group at one of the dose groups.
Statistical significance may give further evidence for a positive evaluation. However, both biological relevance and statistical significance should be considered together.

A dose-related increase of the percentage of cells in repair (NNGC ≥ 5) with values of ≥ 20%, and a dose-related increase in the mean number of net nuclear grain count (NNGC) of about zero is considered to be an indication for a marginal response which needs to be confirmed / clarified in a further experiment.

A test substance is considered negative if the following criteria are met:
• In all dose groups, the mean net nuclear grain counts (NNGC) are close to the values of the concurrent negative control groups and within the range of the historical negative control data.

Statistics:

Due to the clearly negative findings, a statistical evaluation was not carried out.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

(clinical examinations and cytotoxicity)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No relevant increase in the mean number of net nuclear grain counts (NNGC) was observed either 3 hours or 14 hours after single oral administration of the test substance.
In both parts of the study the mean net nuclear grain counts per animal of the test substance treated dose groups (-8.08 to -3.22) were close to the range of the respective vehicle control values (-7.31 to -4.15) and within or below the historical negative control data range (-7.92 to -1.96).
The rate of cells in repair (NNGC ≥ 5) per animal was in the range from 0% to 2%. The values were within the range of the respective vehicle control values (0% to 4%) and close to the range of the historical negative control data (0% to 1%).
Besides at 3-hour and 14-hour sampling time the positive control substance 2-AAF (50 mg/kg body weight, each) induced clearly increased DNA repair activity indicated by distinctly increased mean net nuclear grain counts per animal (6.67 to 11.05) and distinctly increased rates of cells in repair (57% to 80%). The values of both parameters were in the expected range of the historical positive control data.

Table 1: DNA repair activity

Test group	Sampling time [hrs]	Animal No.	NNGC		Cells in repair [%]
			mean	SD
Vehicle control:
corn oil	3	3	-5.59	1.50	2
Test substance
1 000 mg/kg bw.	3	3	-7.18	0.98	1
Test substance
2 000 mg/kg bw.	3	3	-5.10	0.68	1
Positive control
2-AAF
50 mg/kg bw.	3	3	8.63	2.22	70
Vehicle control:
corn oil	14	3	-4.82	1.13	3
Test substance
1 000 mg/kg bw.	14	3	-4.08	0.75	0
Test substance
2 000 mg/kg bw.	14	3	-4.98	0.77	0
Positive control
2-AAF
50 mg/kg bw.	14	3	8.00	1.16	65
NNGC = net nuclear grain count
SD = standard deviation
bw. = body weight

Conclusions:

Under the experimental conditions chosen here, the test substance does not induce unscheduled DNA synthesis in rat hepatocytes in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

in vitro:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (TA 98, TA 100, TA 1535 and TA 1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of phenobarbital / ß-naphthoflavone-induced rats. The study included a Preliminary Solubility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test). 

In the performed preliminary experiments no cytotoxicity of the test item was observed. At the concentration choice the guideline criterion for non-cytotoxic substances was taken into consideration where the recommended maximum test concentration is 5000 µg/plate. The test item was not soluble, and precipitated at the concentration range of 5000 - 1000 µg/plate. The obtained precipitate interfered the scoring, therefore the necessity of an additional microscopic analysis of the plates was considered. Based on the results of the preliminary Range Finding Test, the following concentrations of the test item were prepared and used in the Initial and Confirmatory Mutation Tests: 5000; 2500; 1000; 316; 100; 31.6 and 10 µg/plate.

In the Initial and Confirmatory Mutation Tests the test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. The revertant colony numbers of vehicle control plates with and without S9 Mix were within the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.

 

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test item to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (±S9 Mix) and for 24 hours without metabolic activation (-S9 Mix). Based on the results of the preliminary Solubility and Toxicity Tests and regarding the practical difficulties with the test item handling, the test item was suspended and diluted in RPMI 5 Medium and the RPMI 5 Medium was parallel investigated as vehicle control. The concentrations applied in the Assay 1 and Assay 2 were chosen according to the solubility and cytotoxicity results of the pre-experiments. The test item was considered as relatively insoluble therefore it was investigated beyond its limit of solubility under culture conditions. The following concentrations were investigated in the Assay 1 and Assay 2:

- Assay 1: 3-hour treatment (±S9 Mix): 3.2; 10; 31.6; 100; 316 and 1000 μg/mL;

- Assay 2: 3-hour treatment (+S9 Mix): 3.2; 10; 31.6; 100; 316 and 1000 μg/mL; 24-hour treatment (-S9 Mix): 3.2; 10; 31.6; 100; 316 and 1000 μg/mL

After the treatment the cell cultures were washed, re-suspended, the cell densities determined and adjusted to 2x10^5/mL. The cells were transferred to flasks for growth through the expression period (for approximately 2 days) and diluted to be plated for survival. At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability. In the main assays, the relative harmonised survival the relative total growth of the cells, the viability (colony-forming ability at the end of the 2 day expression period following the treatment) and the potential mutagenicity (5-trifluorothymidine resistance) were determined. The result of Assay 1 was considered to be negative, hence in the second Assay was performed with the originally planned concentration levels chosen based on the preliminary cytotoxicity test with the treatment periods of 3 (in presence of metabolic activation) and 24 hours (in absence of metabolic activation). The performed Assays fulfilled the validity criteria in connection with the negative control and positive control treatments as well as in connection with the number of analyzable concentration levels (at least four). In the examined concentration range noticeable cytotoxicity did not occur at the 3-hour and 24-hour treatments. In the performed assays the obtained mutation frequencies (in absence and also in presence of exogenous metabolic activation) did not show dose-related tendencies, remained far below the relevant GEF thresholds for positive call and remained in the validity criterion range of the negative vehicle control cultures. The mutation frequencies were not statistically significantly different from that of the corresponding vehicle control throughout the study. Under the conditions of this study, the test item did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used.

 

in vivo:

Next to the in vitro assays, two in vivo tests were conducted to examine the genotoxic potential of the test item.

At first, the substance was assessed for its potential to induce chromosomal damage (clastogenicity) or spindle poison effects (aneugenic activity) in NMRI mice using the micronucleus test method. For this purpose, the test substance, suspended in corn oil, 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 10 mL/kg body weight in each case. 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. 2000 polychromatic erythrocytes were evaluated per animal and investigated for micronuclei. No relevant inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected. According to the results of the present study, the single oral administration of the test substance did not lead to any relevant increase in the number of polychromatic erythrocytes containing either small or large micronuclei.

 

In the course of a second study, the compound was assessed for its potential to induce DNA repair synthesis (unscheduled DNA synthesis; UDS) in hepatocytes of Wistar rats in vivo at 3-hour and 14-hour sampling time. For this purpose, the test substance, suspended in corn oil, was administered once orally to male animals at dose levels of 1000 mg/kg and 2000 mg/kg body weight in a volume of 10 mL/kg body weight in each case. The animals were anesthetized and the hepatocytes were harvested by in situ liver perfusion 3 and 14 hours after administration of the test substance. After an attachment period of at least 2 hours the cells were incubated for 4 hours with radiolabeled thymidine in vitro. After washing the hepatocytes were cultivated overnight until fixation. After autoradiography and hematoxylin-eosin-staining three animals per test group with at least 100 cells per animal were scored for DNA repair activity (incorporation of radiolabeled thymidine). No signs of toxicity were observed after administration of 1000 mg/kg and 2000 mg/kg body weight at both sacrifice intervals. No reduced viability of hepatocytes as indication for test substance induced toxicity was observed. The single oral administration of the test item did not lead to an increase in the mean net nuclear grain counts at any dose level at both sacrifice intervals. The test substance did not induce DNA-damage leading to increased unscheduled DNA synthesis and it did not induce cytogenetic damage in bone marrow cells. Thus, the substance is not considered to be genotoxic in vivo.

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 1272/2008. No adverse findings on genotoxicity were observed in in vitro or in vivo studies. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.