2-Propenenitrile, reaction products with 2,2,4(or 2,4,4)-trimethyl-1,6-hexanediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay (AMES) according to OECD Guideline 471, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used.

In an in vitro mammalian cell chromosome aberration assay according to OECD guideline 473, the test item did not show clastogenic properties.

In a mammalian cell forward mutation assay (HPRT) according to OECD guideline 476, the test item did not show genotoxic properties.

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:

from 2017-09-19 to 2017-11-14

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:

1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998-08

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2 (R1): Genotoxicity testing and data interpretation for pharmaceuticals intended for human use

Version / remarks:

2012-06

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: D1608206

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

Target gene:

his/trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

uvrA

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver

Test concentrations with justification for top dose:

16.0, 50.0, 160.0, 500.0, 1600.0, 5000.0 µg/plate, recommended maximum test concentration

Vehicle / solvent:

- Solvent used: ultrapure water (ASTM Type 1)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine (NPD)

Remarks:

without S9 mix, 4 µg/plate for TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix, 2 µg/plate for TA100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix, 50 µg/plate for TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

witout S9 mix, 2 µL/plate for E.coli WP2 uvrA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Remarks:

with S9 mix, 2 µg/plate for all TA strains, 50 µg/plate for E.coli WP2 uvrA

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: colony and background lawn development

Evaluation criteria:

Evaluation of Experimental Data
The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined (counted manually, evaluated by unaided eye), the mean values, standard deviations and the mutation rates were calculated.

Mutation Rate = (Mean revertants at the test item (or control*) treatments) / mean revertants of vehicle control
* untreated, vehicle or positive 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 TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 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 was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.

Criteria for a Negative Response:
A test item is considered non-mutagenic in this bacterial reverse mutation assay 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.

Key result

Species / strain:

S. typhimurium TA 98

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

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:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

Positive controls validity:

valid

Table 1: Summary Table of the Results of the Initial Mutation Test (Plate Incorporation Test)

Concentrations (μg/plate)	Salmonella typhimuriumtester strains																Escherichia coli WP2 uvrA
	TA98				TA100				TA1535				TA1537
	-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	19.7	1.09	26.0	1.26	87.7	1.00	91.7	1.01	9.0	0.79	13.3	0.95	8.3	0.93	9.0	1.13	32.7	0.91	46.7	0.85
DMSO Control	16.0	1.00	21.3	1.00	–	–	94.3	1.00	–	–	14.7	1.00	6.0	1.00	7.3	1.00	–	–	43.7	1.00
Ultrapure Water Control	18.0	1.00	20.7	1.00	87.3	1.00	90.7	1.00	11.3	1.00	14.0	1.00	9.0	1.00	8.0	1.00	36.0	1.00	54.7	1.00
5000	21.3	1.19	24.7	1.19	83.3	0.95	107.0	1.18	10.7	0.94	12.3	0.88	9.3	1.04	9.3	1.17	49.7	1.38	48.7	0.89
1600	22.0	1.22	24.7	1.19	82.0	0.94	108.0	1.19	9.7	0.85	14.3	1.02	6.7	0.74	6.0	0.75	44.3	1.23	54.0	0.99
500	27.7	1.54	24.0	1.16	89.0	1.02	103.7	1.14	10.3	0.91	11.0	0.79	8.0	0.89	9.0	1.13	40.7	1.13	51.0	0.93
160	25.0	1.39	28.3	1.37	92.3	1.06	102.0	1.13	8.7	0.76	11.0	0.79	8.3	0.93	9.0	1.13	46.3	1.29	49.3	0.90
50	28.7	1.59	24.0	1.16	86.7	0.99	104.0	1.15	10.7	0.94	14.3	1.02	9.3	1.04	11.7	1.46	32.7	0.91	44.7	0.82
16	23.0	1.28	31.0	1.50	93.0	1.06	98.0	1.08	9.3	0.82	13.3	0.95	9.3	1.04	8.7	1.08	32.3	0.90	50.0	0.91
NPD (4 μg)	357.3	22.33	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 μg)	–	–	–	–	933.3	10.69	–	–	533.3	47.06	–	–	–	–	–	–	–	–	–	–
9AA (50 μg)	–	–	–	–	–	–	–	–	–	–	–	–	758.0	126.33	–	–	–	–	–	–
MMS (2 μL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	821.3	22.81	–	–
2AA (2 μg)	–	–	1828.0	85.69	–	–	1493.3	15.83	–	–	237.3	16.18	–	–	153.3	20.91	–	–	–	–
2AA (50 μg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	193.7	4.44

Table 2: Summary Table of the Results of the Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (μg/plate)	Salmonella typhimuriumtester strains																Escherichia coli WP2 uvrA
	-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	27.3	1.08	29.0	1.19	92.7	1.06	92.0	0.88	11.0	1.00	11.0	0.92	10.3	1.19	9.0	1.13	17.0	0.46	39.3	0.84
DMSO Control	23.0	1.00	22.7	1.00	–	–	94.3	1.00	–	–	13.0	1.00	7.3	1.00	6.3	1.00	–	–	38.7	1.00
Ultrapure Water Control	25.3	1.00	24.3	1.00	87.3	1.00	104.0	1.00	11.0	1.00	12.0	1.00	8.7	1.00	8.0	1.00	36.7	1.00	46.7	1.00
5000	5.3	0.21	19.7	0.81	24.3	0.28	52.3	0.50	0.0	0.00	4.3	0.36	1.3	0.15	6.3	0.79	14.7	0.40	26.7	0.57
1600	31.7	1.25	26.7	1.10	92.0	1.05	100.3	0.96	8.0	0.73	9.0	0.75	9.0	1.04	12.7	1.58	35.3	0.96	44.3	0.95
500	32.7	1.29	24.3	1.00	78.3	0.90	98.3	0.95	12.7	1.15	11.3	0.94	12.3	1.42	8.7	1.08	33.7	0.92	43.3	0.93
160	26.0	1.03	34.3	1.41	75.3	0.86	93.7	0.90	10.0	0.91	10.3	0.86	8.0	0.92	7.3	0.92	35.0	0.95	39.7	0.85
50	21.3	0.84	30.3	1.25	77.0	0.88	97.7	0.94	11.0	1.00	9.3	0.78	9.7	1.12	8.3	1.04	27.7	0.75	39.3	0.84
16	24.0	0.95	28.3	1.16	85.3	0.98	99.3	0.96	12.0	1.09	13.0	1.08	7.3	0.85	9.7	1.21	28.0	0.76	39.7	0.85
NPD (4 μg)	253.3	11.01	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2 μg)	–	–	–	–	797.3	9.13	–	–	509.3	46.30	–	–	–	–	–	–	–	–	–	–
9AA (50 μg)	–	–	–	–	–	–	–	–	–	–	–	–	528.0	72.00	–	–	–	–	–	–
MMS (2 μL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	741.3	20.22	–	–
2AA (2 μg)	–	–	1532.0	67.59	–	–	2826.7	29.96	–	–	164.0	12.62	–	–	121.7	19.21	–	–	–	–
2AA (50 μg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	243.7	6.30

Conclusions:

In an bacterial reverese mutation assay (AMES) according to OECD Guideline 471, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used.

Executive summary:

The mutagenic potential of the test item was determined in an in vitro bacterial reverse mutation assay (AMES) according to OECD Guideline 471. Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations (16, 50, 160, 500, 1600, 5000 µg/plate), including the controls, were tested in triplicate (positive and negative controls were run concurrently). nagative, vehicle and positive controls were valid. No cytotoxicity was observed up to the max. concentration. No precipitation was observed throughout the study. No substantial increases were observed in revertant colony numbers of any of the five tester 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 shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-11-23 to 2018-02-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Batch No.: D1608206

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

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures), Lot No 10H016
- Suitability of cells: Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations.
- proliferation rate: doubling time 12 - 14 h
- Methods for maintenance in cell culture: The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 ± 0.5 °C in a humidified atmosphere in an incubator, set at 5% CO2. The V79 cells for this study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2 mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10%). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5%.
- Modal number of chromosomes: diploid number, 2n = 22

MEDIA USED
- Type and identity of media including CO2 concentration: DME (Dulbecco’s Modified Eagle’s) medium, 5 % CO2
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver.

Test concentrations with justification for top dose:

3-hour treatment, harvest after 20 hours: 62.5, 125, 250, 500 µg/mL (with and without S9 mix)
20-hour treatment, harvest after 20 hours: 3.9, 7.8, 15.6, 31.3 µg/mL (without S9 mix)
20-hour (without S9 mix) treatment, harvest after 28 hours: 3.9, 7.8, 15.6, 31.3 µg/mL
3-hour (with S9 mix) treatment, harvest after 28 hours: 62.5, 125, 250, 500 µg/mL

Top dose was determined in an experimental pre-test on cytotoxicity.

Vehicle / solvent:

- Vehicle used:DME medium
- Justification for choice of vehicle: This vehicle is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test, and its suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DME 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
- Cell density at seeding: 5 x 10E5

DURATION
- Exposure duration: 3 h or 20 h
- Expression time:
3 h treatment: 17 or 25 h
20 h treatment: 8 h
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): 20 or 28 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.2 μg/mL)

STAIN (for cytogenetic assays): 5 % Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes free of cytoplasm) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration, negative and positive controls and were equally divided among the duplicates (150 metaphases/slide).

DETERMINATION OF CYTOTOXICITY
- Method: the Relative Increase in Cell Counts (RICC) was calculated as cytotoxicity parameter.

OTHER EXAMINATIONS:
Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally, the number of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.

Evaluation criteria:

– The percentage of cells with structural chromosome aberration(s) was evaluated.
– Different types of structural chromosome aberrations were listed with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment(s) were recorded.
– Individual culture data were summarised in tabular form.

Statistics:

For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

clear cytotoxicity of about 50% was observed after test item treatment in the absence and presence of metabolic activation.

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 relevant changes
- Effects of osmolality: No relevant changes
- Precipitation: No precipitation of the test item was observed at any of the applied concentrations.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: see "Any other informationon results"

Table 1: Results of V79 Chromosome Aberration Assay, Experiment A; 3 h treatment without S9 mix / 20 h sampling time

Non Activation Test Conditions	RICC	Cytotoxicity	Scored Metaphases	No of Aberrant Cells		% of Aberrant Cells	No of Aberrations		Aberrations
									Chromosome			Chromatid			others
				Gap+	Gap-	Gap-	Gap+	Gap-	gap	del	exchange	gap	del	exchange
Solvent Control a	100	0	150	7	2	1.333	7	2	1	0	0	4	1	1	-
Solvent Control b			150	8	3	2.000	8	3	3	0	1	2	2	0	-
Mean				8	3	1.667	8	3
Pos. Control a	48	52	150	44	34	22.667	70	45	15	10	7	10	11	17	-
Pos. Control b			150	38	34	22.667	57	40	8	7	8	9	8	17	-
Mean				41**	34**	22.667	64**	43**
62.5 µg/mL a	97	3	150	9	4	2.667	9	4	2	0	1	3	2	1	-
62.5 µg/mL b			150	7	3	2.000	7	3	1	0	1	3	2	0	-
Mean				8	4	2.333	8	4
125 µg/mL a	85	15	150	8	2	1.333	8	2	1	0	1	5	1	0	-
125 µg/mL b			150	8	3	2.000	8	3	2	1	1	3	1	0	-
Mean				8	3	1.667	8	3
250 µg/mL a	63	37	150	7	3	2.000	77	3	2	1	1	2	1	0	-
250 µg/mL b			150	8	4	2.667	8	4	1	1	2	3	1	0	-
Mean				8	4	2.333	8	4
500 µg/mL a	48	52	150	8	3	2.000	8	3	2	0	0	3	2	1	-
500 µg/mL b			150	9	4	2.667	10	5	0	1	0	5	4	0	-
Mean				9	4	2.333	9	4

Table 2: Results of V79 Chromosome Aberration Assay, Experiment A; 3 h treatment with S9 mix / 20 h sampling time

Non Activation Test Conditions	RICC	Cytotoxicity	Scored Metaphases	No of Aberrant Cells		% of Aberrant Cells	No of Aberrations		Aberrations
									Chromosome			Chromatid			others
				Gap+	Gap-	Gap-	Gap+	Gap-	gap	del	exchange	gap	del	exchange
Solvent Control a	100	0	150	8	4	2.667	8	4	2	1	1	2	2	0	-
Solvent Control b			150	7	4	2.667	7	4	2	1	1	1	2	0	-
Mean				8	4	2.667	8	4
Pos. Control a	47	53	150	50	42	28.000	83	59	9	6	6	21	21	26	-
Pos. Control b			150	46	38	25.333	71	46	9	6	4	16	16	20	-
Mean				48**	40**	26.667	77**	53**
62.5 µg/mL a	98	2	150	7	3	2.000	7	3	2	0	0	3	3	0	-
62.5 µg/mL b			150	9	3	2.000	11	5	2	0	1	4	4	0	-
Mean				8	3	2.000	9	4
125 µg/mL a	79	21	150	7	3	2.000	7	3	1	0	0	3	3	0	-
125 µg/mL b			150	10	5	3.333	10	5	3	1	0	2	2	2	-
Mean				9	4	2.667	9	4
250 µg/mL a	59	41	150	7	3	2.000	8	3	3	1	1	1	1	0	-
250 µg/mL b			150	9	2	1.333	9	2	3	0	1	1	1	0	-
Mean				8	3	1.667	9	3
500 µg/mL a	44	56	150	10	5	3.333	10	5	2	0	2	3	3	0	-
500 µg/mL b			150	8	4	2.667	8	4	2	0	0	4	4	0	-
Mean				9	5	3.000	9	5

Table 3: Results of V79 Chromosome Aberration Assay, Experiment B; 20 h treatment without S9 mix / 20 h sampling time

Non Activation Test Conditions	RICC	Cytotoxicity	Scored Metaphases	No of Aberrant Cells		% of Aberrant Cells	No of Aberrations		Aberrations
									Chromosome			Chromatid			others
				Gap+	Gap-	Gap-	Gap+	Gap-	gap	del	exchange	gap	del	exchange
Solvent Control a	100	0	150	6	3	2.00	6	3	1	1	0	2	2	0	-
Solvent Control b			150	7	4	2.667	7	4	0	0	3	3	1	0	-
Mean				7	4	2.333	7	4
Pos. Control a	48	52	150	43	37	24.667	76	51	1	9	7	15	13	22	-
Pos. Control b			150	45	39	26.000	60	40	5	6	5	15	13	16	-
Mean				44**	38**	25.333	68**	46**
62.5 µg/mL a	97	3	150	6	3	2.000	6	3	1	0	2	2	1	0	-
62.5 µg/mL b			150	8	4	2.667	9	4	1	1	1	4	2	0	-
Mean				7	4	2.333	8	4
125 µg/mL a	90	10	150	7	3	2.000	7	3	2	0	1	2	2	0	-
125 µg/mL b			150	9	3	2.000	9	3	2	1	1	4	1	0	-
Mean				8	3	2.000	8	3
250 µg/mL a	64	36	150	8	3	2.000	8	3	3	0	2	2	1	0	-
250 µg/mL b			150	5	2	1.333	5	2	1	0	0	2	2	0	-
Mean				7	3	1.667	7	3
500 µg/mL a	44	56	150	7	3	2.000	7	3	3	0	0	1	3	0	-
500 µg/mL b			150	8	4	2.667	9	4	1	0	1	4	2	1	-
Mean				8	4	2.333	8	4

Table 4: Results of V79 Chromosome Aberration Assay, Experiment B; 20 h treatment without S9 mix / 28 h sampling time

Non Activation Test Conditions	RICC	Cytotoxicity	Scored Metaphases	No of Aberrant Cells		% of Aberrant Cells	No of Aberrations		Aberrations
									Chromosome			Chromatid			others
				Gap+	Gap-	Gap-	Gap+	Gap-	gap	del	exchange	gap	del	exchange
Solvent Control a	100	0	150	6	3	2.00	6	3	1	0	2	2	1	0	-
Solvent Control b			150	8	4	2.667	8	4	2	0	2	2	2	0	-
Mean				7	4	2.333	7	4
Pos. Control a	44	56	150	44	39	26.000	64	42	6	6	6	16	14	16	-
Pos. Control b			150	48	40	26.667	68	45	8	6	5	15	15	19	-
Mean				46**	40**	26.333	66**	44**
62.5 µg/mL a	97	3	150	7	4	2.667	8	4	1	0	2	3	2	0	-
62.5 µg/mL b			150	6	3	2.000	6	3	1	1	0	2	2	0	-
Mean				7	4	2.333	7	4
125 µg/mL a	90	10	150	6	3	2.000	7	3	1	1	1	3	1	0	-
125 µg/mL b			150	8	3	2.000	8	3	3	1	0	2	1	1	-
Mean				7	3	2.000	8	3
250 µg/mL a	65	35	150	7	3	2.000	7	3	2	0	0	2	3	0	-
250 µg/mL b			150	7	3	2.000	7	3	0	0	1	4	2	0	-
Mean				7	3	2.000	7	3
500 µg/mL a	46	54	150	7	3	2.000	8	3	4	0	3	1	0	0	-
500 µg/mL b			150	8	4	2.667	9	5	2	0	1	2	2	2	-
Mean				8	4	2.333	9	4

Table 5: Results of V79 Chromosome Aberration Assay, Experiment B; 3 h treatment with S9 mix / 28 h sampling time

Non Activation Test Conditions	RICC	Cytotoxicity	Scored Metaphases	No of Aberrant Cells		% of Aberrant Cells	No of Aberrations		Aberrations
									Chromosome			Chromatid			others
				Gap+	Gap-	Gap-	Gap+	Gap-	gap	del	exchange	gap	del	exchange
Solvent Control a	100	0	150	7	4	2.667	7	4	1	0	3	2	1	0	-
Solvent Control b			150	8	3	2.000	8	3	0	0	0	5	2	1	-
Mean				8	4	2.333	8	4
Pos. Control a	45	55	150	48	37	24.667	72	47	11	7	7	14	10	23	-
Pos. Control b			150	41	34	22.667	69	43	11	6	10	15	9	18	-
Mean				45**	36**	23.667	71**	45**
62.5 µg/mL a	98	2	150	7	4	2.667	7	4	3	0	2	0	2	0	-
62.5 µg/mL b			150	7	3	2.000	7	3	2	2	1	2	0	0	-
Mean				7	4	2.333	7	4
125 µg/mL a	81	19	150	6	2	1.333	6	2	4	1	0	0	1	0	-
125 µg/mL b			150	10	4	2.667	10	4	1	1	1	5	2	0	-
Mean				8	3	2.000	8	3
250 µg/mL a	61	39	150	10	4	2.667	10	4	3	0	0	3	4	0	-
250 µg/mL b			150	7	3	2.000	7	3	4	0	1	0	2	0	-
Mean				9	4	2.333	9	4
500 µg/mL a	42	58	150	9	3	2.000	9	3	3	0	1	3	2	0	-
500 µg/mL b			150	9	4	2.667	9	4	3	0	1	2	3	0	-
Mean				9	4	2.333	9	4

Table 6: Mean Percentage of Cells with Structural Chromosome Aberration(s); Experiment A

Concentration (μg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/ 150 cells
				incl. gaps	excl. gaps
Solvent control (DME medium)	-	3 h	20 h	8	3
62.5 μg/mL	-	3 h	20 h	8	4
125 μg/mL	-	3 h	20 h	8	3
250 μg/mL	-	3 h	20 h	8	4
500 μg/mL	-	3 h	20 h	9	4
Pos. Control (EMS)	-	3 h	20 h	41**	34**
Solvent control (DME medium)	+	3 h	20 h	8	4
62.5 μg/mL	+	3 h	20 h	8	3
125 μg/mL	+	3 h	20 h	9	4
250 μg/mL	+	3 h	20 h	8	3
500 μg/mL	+	3 h	20 h	9	5
Pos. Control (Cycl.)	+	3 h	20 h	48**	40**
Positive control (-S9): Ethyl methanesulfonate (1.0 μL/mL) Positive control (+S9): Cyclophosphamide (5.0 μg/mL) ∗∗: p<0.01

Table 7: Mean Percentage of Cells with Structural Chromosome Aberration(s); Experiment B

Concentration (μg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/ 150 cells
				incl. gaps	excl. gaps
Solvent control (DME medium)	-	20 h	20 h	7	4
3.9 μg/mL	-	20 h	20 h	7	4
7.8 μg/mL	-	20 h	20 h	8	3
15.6 μg/mL	-	20 h	20 h	7	3
31.3 μg/mL	-	20 h	20 h	8	4
Pos. Control (EMS)	-	20 h	20 h	44**	38**
Solvent control (DME medium)	-	20 h	28 h	7	4
3.9 μg/mL	-	20 h	28 h	7	4
7.8 μg/mL	-	20 h	28 h	7	3
15.6 μg/mL	-	20 h	28 h	7	3
31.3 μg/mL	-	20 h	28 h	8	4
Pos. Control (EMS)	-	20 h	28 h	46**	40**
Solvent control (DME medium)	+	3 h	28 h	8	4
62.5 μg/mL	+	3 h	28 h	7	4
125 μg/mL	+	3 h	28 h	8	3
250 μg/mL	+	3 h	28 h	9	4
500 μg/mL	+	3 h	28 h	9	4
Pos. Control (Cycl.)	+	3 h	28 h	45**	36**
Positive control (-S9): Ethyl methanesulfonate (0.4 μL/mL) Positive control (+S9): Cyclophosphamide (5.0 μg/mL) ∗∗: p<0.01

Table 8: Number of Cells and Endoreduplicated Cells; Experiment A

Concentration (μg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/ 150 cells
				incl. gaps	excl. gaps
Solvent control (DME medium)	-	3 h	20 h	0.0	0.0
62.5 μg/mL	-	3 h	20 h	0.0	0.0
125 μg/mL	-	3 h	20 h	0.0	0.0
250 μg/mL	-	3 h	20 h	0.0	0.0
500 μg/mL	-	3 h	20 h	0.0	0.0
Pos. Control (EMS)*	-	3 h	20 h	0.0	0.0
Solvent control (DME medium)	+	3 h	20 h	0.0	0.0
62.5 μg/mL	+	3 h	20 h	0.0	0.0
125 μg/mL	+	3 h	20 h	0.0	0.0
250 μg/mL	+	3 h	20 h	0.0	0.0
500 μg/mL	+	3 h	20 h	0.0	0.0
Pos. Control (Cycl.)**	+	3 h	20 h	0.0	0.0
* Ethyl methanesulfonate (1.0μL/mL) ** Cyclophosphamide (5.0μg/mL)

Table 9: Number of Cells and Endoreduplicated Cells; Experiment B

Concentration (μg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/ 150 cells
				incl. gaps	excl. gaps
Solvent control (DME medium)	-	20 h	20 h	0.0	0.0
3.9 μg/mL	-	20 h	20 h	0.0	0.0
7.8 μg/mL	-	20 h	20 h	0.0	0.0
15.6 μg/mL	-	20 h	20 h	0.0	0.0
31.3 μg/mL	-	20 h	20 h	0.0	0.0
Pos. Control (EMS)*	-	20 h	20 h	0.0	0.0
Solvent control (DME medium)	-	20 h	28 h	0.0	0.0
3.9 μg/mL	-	20 h	28 h	0.0	0.0
7.8 μg/mL	-	20 h	28 h	0.0	0.0
15.6 μg/mL	-	20 h	28 h	0.0	0.0
31.3 μg/mL	-	20 h	28 h	0.0	0.0
Pos. Control (EMS)*	-	20 h	28 h	0.0	0.0
Solvent control (DME medium)	+	3 h	28 h	0.0	0.0
62.5 μg/mL	+	3 h	28 h	0.0	0.0
125 μg/mL	+	3 h	28 h	0.0	0.0
250 μg/mL	+	3 h	28 h	0.0	0.0
500 μg/mL	+	3 h	28 h	0.0	0.0
Pos. Control (Cycl.)**	+	3 h	28 h	0.0	0.0
* Ethyl methanesulfonate (0.4 μL/mL) ** Cyclophosphamide (5.0 μg/mL) ∗∗: p<0.01

Conclusions:

In an in vitro mammalian cell chromosome aberration assay according to OECD guideline 473, the test item did not show clastogenic properties.

Executive summary:

The clastogenic potential of the test item was assessed in an n an in vitro mammalian cell chromosome aberration assay according to OECD guideline 473 in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations of the test item dissolved in DME medium were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix)

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix 62.5, 125, 250 and 500 μg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 3.9, 7.8, 15.6 and 31.3 μg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 3.9, 7.8, 15.6 and 31.3 μg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 62.5, 125, 250 and 500 μg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item. Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts. No relevant increase in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. In experiment A in the presence of metabolic activation, one value was slightly above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, CeTePox® 0214 did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2018-02-13 to 2018-003-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

29 July 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial forward mutation assay

Specific details on test material used for the study:

Batch No.: D1608206

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

Target gene:

hprt

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

sub-line KI

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:ECACC (European Collection of Cell Cultures)
- Suitability of cells: The system used in this study has been extensively validated.
- Methods for maintenance in cell culture: in Ham's F12 medium containing 10 % fetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air.

MEDIA USED
- Type and identity of media including CO2 concentration: Ham's F12 medium, 5 % CO2
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver (S9 mix)

Test concentrations with justification for top dose:

125, 250, 500, 1000, 1250, 1500, 1750 µg/mL
Test concentrations were determined in an pre-test on cytotxicity.

Vehicle / solvent:

- Vehicle used: Ham’s F12 medium
- Justification for choice of vehicle: This vehicle is compatible with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test, and its suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 5 x 10E6

DURATION
- Exposure duration: 5 h
- Expression time: 19 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SELECTION AGENT (mutation assays): hypoxanthine Ham's F12-SEL medium) containing 3.4 μg/mL of thioguanine (6-TG).

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After the selection period, the colonies were fixed, stained with Giemsa and counted for mutant selection and cloning efficiency determination.

DETERMINATION OF CYTOTOXICITY
- Method: Relative survival (%), based on Number of colonies/200cells/dish.

Rationale for test conditions:

As demanded by the OECD guideline 476.

Evaluation criteria:

Evaluation of Results
Providing that all acceptability criteria are fulfilled, the test item is considered to be clearly positive if, in any of the experimental conditions examined:
• at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
• the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
• none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
• there is no concentration-related increase when evaluated with an appropriate trend test,
• all results are inside the distribution of the historical negative control data (based 95% control limit).
The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

Statistical Analysis was performed with SPSS PC+ software for the following data:
• mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
• mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups
• The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

sub-line: KI

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1250 µg/mL and above

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 effect
- Effects of osmolality: no effect
- Precipitation: Not observed

Table 1: Summary of the results, 5 h treatment without S9 mix (parallel of the first culture a)

		SURVIVAL TO TREATMENT			REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10 E -6
		MEAN COLONY NUMBER	S.D.	PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control		202.0	2.65	100	100	0	2	1	1	2	6	102	5,,88
Pos. control EMS	1.0μL/mL	52.0	1.00	26	71	193	201	197	202	201	994	73	1361,64**
Test Item	125 μg/mL	201.7	1.53	100	98	1	2	0	0	2	5	100	5,00
	250 μg/mL	199.0	1.00	99	99	0	1	1	2	1	5	101	4,95
	500 μg/mL	188.7	0.58	93	100	1	0	2	1	1	5	102	4,90
	1000 μg/mL	181.7	1.15	90	99	0	0	1	3	2	6	101	5,94
	1250 μg/mL	161.3	1.53	80	98	0	1	2	1	2	6	100	6,00
	1500 μg/mL	39.7	0.58	20	98	2	0	0	1	3	6	100	6,00
	1750 μg/mL	0.0	0.00	0	0	-	-	-	-	-	-	-	-
abs.C.E. = Absolute Cloning Efficiency EMS = Ethyl methanesulfonate ** = p < 0.01 to the concurrent negative control and to the historical control

Table 2: Summary of the results, 5 h treatment without S9 mix (parallel of the first culture b)

		SURVIVAL TO TREATMENT			REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10 E -6
		MEAN COLONY NUMBER	S.D.	PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control		203.3	1.53	100	100	2	3	1	1	0	7	101	6,93
Pos. control EMS	1.0μL/mL	48.3	1.53	24	71	198	189	202	206	194	989	72	1373,61**
Test Item	125 μg/mL	202.7	1.53	100	99	0	0	3	2	1	6	100	6,00
	250 μg/mL	200.3	2.08	99	99	1	0	2	1	1	5	100	5,00
	500 μg/mL	189.7	0.58	93	99	1	0	0	1	3	5	100	5,00
	1000 μg/mL	184.0	1.00	90	99	1	1	0	1	2	5	100	5,00
	1250 μg/mL	163.3	4.93	80	99	1	2	0	2	1	6	100	6,00
	1500 μg/mL	42.7	2.52	21	99	0	0	2	2	1	5	100	5,00
	1750 µg/mL	0.0	0.00	0	0	-	-	-	-	-	-	-	-
abs.C.E. = Absolute Cloning Efficiency EMS = Ethyl methanesulfonate ** = p < 0.01 to the concurrent negative control and to the historical control

Table 3: Summary of the results, 5 h treatment without S9 mix (parallel of the first culture c)

		SURVIVAL TO TREATMENT			REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10 E -6
		MEAN COLONY NUMBER	S.D.	PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control		202.0	3.46	100	100	0	1	2	0	4	7	101	6,93
Pos. control EMS	1.0μL/mL	51.7	2.52	26	73	197	213	194	209	206	1019	74	1377,03**
Test Item	125 μg/mL	203.3	1.53	101	99	1	0	3	0	3	7	99	7,07
	250 μg/mL	202.7	1.53	100	100	1	0	0	3	1	5	100	5,00
	500 μg/mL	187.7	1.53	93	100	1	1	0	1	2	5	101	4,95
	1000 μg/mL	181.3	1.53	90	100	1	0	2	0	3	6	100	6,00
	1250 μg/mL	164.0	1.00	81	98	1	0	0	3	2	6	99	6,06
	1500 μg/mL	41.7	2.08	21	99	1	0	2	2	2	7	99	7,07
	1750 µg/mL	0.0	0.00	0	0	-	-	-	-	-	-	-	-
abs.C.E. = Absolute Cloning Efficiency EMS = Ethyl methanesulfonate ** = p < 0.01 to the concurrent negative control and to the historical control

Table 4: Summary of the results, 5 h treatment without S9 mix (parallel of the first culture d)

		SURVIVAL TO TREATMENT			REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10 E -6
		MEAN COLONY NUMBER	S.D.	PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control		202.0	2.00	100	100	1	0	0	3	2	6	101	5,94
Pos. control EMS	1.0μL/mL	53.0	2.00	26	71	207	213	196	191	204	1011	72	1404,17**
Test Item	125 μg/mL	200.7	1.15	99	99	0	2	0	2	1	5	100	5,00
	250 μg/mL	199.3	1.15	99	99	2	0	1	2	0	5	100	5,00
	500 μg/mL	187.7	1.53	93	100	0	2	1	1	1	5	100	5,00
	1000 μg/mL	180.7	1.15	89	99	2	1	0	1	2	6	100	6,00
	1250 μg/mL	162.0	1.00	80	99	1	1	1	1	1	5	99	5,05
	1500 μg/mL	39.0	1.00	19	99	0	0	2	2	2	6	100	6,00
	1750 µg/mL	0.0	0.00	0	0	-	-	-	-	-	-	-	-
abs.C.E. = Absolute Cloning Efficiency EMS = Ethyl methanesulfonate ** = p < 0.01 to the concurrent negative control and to the historical control

Table 5: Summary of the results, 5 h treatment with S9 mix (parallel of the first culture a)

		SURVIVAL TO TREATMENT			REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10 E -6
		MEAN COLONY NUMBER	S.D.	PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control		201.3	2.08	100	100	1	1	2	2	1	7	102	6,86
Pos. control EMS	1.0μL/mL	110.3	2.08	55	76	104	115	119	98	101	537	78	688,46**
Test Item	125 μg/mL	200.7	4.16	100	100	2	0	0	2	2	6	101	5,94
	250 μg/mL	198.7	0.58	99	98	0	1	2	1	1	5	100	5,00
	500 μg/mL	173.0	2.65	86	98	0	1	2	1	1	5	100	5,00
	1000 μg/mL	159.0	1.00	79	99	2	2	0	1	1	6	101	5,94
	1250 μg/mL	118.3	2.08	59	99	1	2	1	1	0	5	101	4,95
	1500 μg/mL	92.3	2.08	46	97	0	0	3	2	0	5	99	5,05
	1750 µg/mL	35.7	0.58	18	98	2	0	0	3	0	5	100	5,00
abs.C.E. = Absolute Cloning Efficiency DMBA = 7,12-Dimethyl benzanthracene ** = p < 0.01 to the concurrent negative control and to the historical control

Table 6: Summary of the results, 5 h treatment with S9 mix (parallel of the first culture b)

		SURVIVAL TO TREATMENT			REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10 E -6
		MEAN COLONY NUMBER	S.D.	PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control		201.3	2.08	100	100	2	0	4	1	0	7	101	6,93
Pos. control EMS	1.0μL/mL	110.3	2.08	57	75	108	110	97	106	112	533	76	701,32**
Test Item	125 μg/mL	201.7	1.53	100	100	0	0	1	1	3	5	101	4,95
	250 μg/mL	197.7	1.15	98	98	0	2	0	1	3	6	99	6,06
	500 μg/mL	172.3	3.06	86	99	0	1	1	1	2	5	100	5,00
	1000 μg/mL	158.3	0.58	79	99	1	1	1	0	2	5	100	5,00
	1250 μg/mL	117.0	1.00	58	99	0	2	2	0	2	6	100	6,00
	1500 μg/mL	90.3	0.58	45	99	1	1	2	0	1	5	100	5,00
	1750 µg/mL	37.0	1.00	18	99	1	1	2	1	1	6	100	6,00
abs.C.E. = Absolute Cloning Efficiency DMBA = 7,12-Dimethyl benzanthracene ** = p < 0.01 to the concurrent negative control and to the historical control

Table 7: Summary of the results, 5 h treatment with S9 mix (parallel of the first culture c)

		SURVIVAL TO TREATMENT			REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10 E -6
		MEAN COLONY NUMBER	S.D.	PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control		202.0	3.0	100	100	1	1	0	2	2	6	100	6,00
Pos. control EMS	1.0μL/mL	117.0	1.00	58	73	118	111	106	108	114	557	73	763,01**
Test Item	125 μg/mL	198.7	3.06	98	99	3	1	0	2	0	6	99	6,06
	250 μg/mL	197.7	1.53	98	101	0	0	3	2	0	5	101	4,95
	500 μg/mL	175.7	1.53	87	99	1	0	1	2	1	5	99	5,05
	1000 μg/mL	158.7	1.53	79	101	2	0	0	2	1	5	101	4,95
	1250 μg/mL	115.3	1.53	57	100	1	1	0	2	1	5	101	4,95
	1500 μg/mL	88.7	1.15	44	99	0	1	2	2	1	6	99	6,06
	1750 µg/mL	42.7	2.31	21	100	2	2	0	0	2	6	101	5,94
abs.C.E. = Absolute Cloning Efficiency DMBA = 7,12-Dimethyl benzanthracene ** = p < 0.01 to the concurrent negative control and to the historical control

Table 8: Summary of the results, 5 h treatment with S9 mix (parallel of the first culture d)

		SURVIVAL TO TREATMENT			REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10 E -6
		MEAN COLONY NUMBER	S.D.	PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control		201.0	1.0	100	100	1	1	1	3	1	7	100	7,00
Pos. control EMS	1.0μL/mL	117.0	1.00	58	74	97	105	113	102	105	522	74	705,41*002A
Test Item	125 μg/mL	198.7	1.53	99	99	0	0	2	1	2	5	99	5,05
	250 μg/mL	197.7	1.53	98	100	0	3	0	2	0	5	100	5,00
	500 μg/mL	175.7	1.53	87	99	1	1	1	2	0	6	99	6,06
	1000 μg/mL	158.7	1.53	79	99	3	0	1	1	2	7	99	7,07
	1250 μg/mL	115.3	1.53	57	100	1	2	0	1	2	6	100	6,00
	1500 μg/mL	88.7	1.15	44	99	0	1	2	0	2	5	99	5,05
	1750 µg/mL	42.7	2.31	21	99	1	1	2	1	1	6	100	6,00
abs.C.E. = Absolute Cloning Efficiency DMBA = 7,12-Dimethyl benzanthracene ** = p < 0.01 to the concurrent negative control and to the historical control

Conclusions:

In a mammalian cell forward mutation assay (HPRT) according to OECD guideline 476, the test item did not show genotoxic properties.

Executive summary:

The mutagenic properties of the test item were assessed in a mammalian cell forward mutation assay according to OECD guidelien476. The test item, dissolved in Ham’s F12 medium, was tested in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital andβ-naphthoflavone induced rat liver:

5-hour treatment period without S9-mix:

125, 250, 500, 1000, 1250, 1500 and 1750 μg/mL

5-hour treatment period with S9-mix:

125, 250, 500, 1000, 1250, 1500 and 1750 μg/mL

In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity. Phenotypic expression was evaluated up to 8 days following exposure.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in osmolality of the test system were noted at the different dose levels tested. The pH values of test item solutions showed a dose associated increase in the acceptable range compared to the concurrent control groups.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, neither in the absence nor in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups compared to the concurrent and historical control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical control data.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate (1.0 μL/mL) and 7,12-Dimethyl benz[a]anthracene (20 μg/mL). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data. Thus, the study is considered valid.

The test item tested up to cytotoxic concentrations with and without metabolic activation over a 5 hour treatment period did not induce statistically significant and biologically relevant increases in mutant frequency.

It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The mutagenic potential of the test item was determined in an in vitro bacterial reverse mutation assay (AMES) according to OECD Guideline 471. Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used in two independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test) and in a pre-incubation test (experiment II, Confirmatory Mutation Test). Each assay was conducted with and without metabolic activation (±S9 Mix). The concentrations (16, 50, 160, 500, 1600, 5000 µg/plate), including the controls, were tested in triplicate (positive and negative controls were run concurrently). nagative, vehicle and positive controls were valid. No cytotoxicity was observed up to the max. concentration. No precipitation was observed throughout the study. No substantial increases were observed in revertant colony numbers of any of the five tester 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 shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

The clastogenic potential of the test item was assessed in an n an in vitro mammalian cell chromosome aberration assay according to OECD guideline 473 in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations of the test item dissolved in DME medium were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix)

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix 62.5, 125, 250 and 500 μg/mL test item

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 3.9, 7.8, 15.6 and 31.3 μg/mL test item

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 3.9, 7.8, 15.6 and 31.3 μg/mL test item

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 62.5, 125, 250 and 500 μg/mL test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item. Clear cytotoxicity of about 50% was observed after test item treatment in all experimental parts.No relevant increase in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. In experiment A in the presence of metabolic activation, one value was slightly above the 95% control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

In conclusion, CeTePox® 0214 did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

The mutagenic properties of the test item were assessed in a mammalian cell forward mutation assay according to OECD guidelien476. The test item, dissolved in Ham’s F12 medium, was tested in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital andβ-naphthoflavone induced rat liver:

5-hour treatment period without S9-mix:

125, 250, 500, 1000, 1250, 1500 and 1750μg/mL

5-hour treatment period with S9-mix:

125, 250, 500, 1000, 1250, 1500 and 1750μg/mL

In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity. Phenotypic expression was evaluated up to 8 days following exposure.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in osmolality of the test system were noted at the different dose levels tested. The pH values of test item solutions showed a dose associated increase in the acceptable range compared to the concurrent control groups.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, neither in the absence nor in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups compared to the concurrent and historical control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical control data.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate (1.0μL/mL) and 7,12-Dimethyl benz[a]anthracene (20μg/mL). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data. Thus, the study is considered valid.

The test item tested up to cytotoxic concentrations with and without metabolic activation over a 5 hour treatment period did not induce statistically significant and biologically relevant increases in mutant frequency.

It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.

Conclusion: the test item did not show mutagenic properties in bacterial or mammalian cell mutation assays. Furthermore, the test item did not show clastogenic properties in a mammalian cell assay. Thus the test item is considered to be non-genotoxic.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

No genotoxic properties were documented. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.