Reaction mass of calcium 2,6-bis(3-carboxylatopropanamido)hexanoate and isomers of calcium amino-(3-carboxylatopropanamido)hexanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA. Therefore, the substance is considered non-mutagenic in a bacterial reverse mutation assay.

The test item tested up to the maximum recommended concentration with and without mammalian metabolic activation system did not induce structural chromosome aberrations in Chinese Hamster lung cells. Thus, the test item is considered as not clastogenic in this system.

Furthermore, the substance tested both without and with metabolic activation, did not induce increases in mutant frequency in mammalian Chinese hamster ovary cells.

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:

February 10, 2017 - April 24, 2017

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 July, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5100 (Escherichia coli WP2 and WP2 UVRA Reverse Mutation Test)

Version / remarks:

August 1998

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:

June 2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations.
The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: S.ty.mur. TA98,100,1537,1535 rfa (cell wall), uvrB (DNA-repair) mutation

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:

Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test (Informatory Toxicity Test).
±S9 Mix: 5000; 1600; 500; 160; 50 and 16 μg/plate (Experiment I - plate incorporation method)
±S9 Mix: 5000; 1600; 500; 160; 50 and 16 μg/plate (Experiment II - pre-incubation method)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ultrapure water was applied as vehicle of the test item and the positive control substances SAZ and MMS; and DMSO was applied as vehicle for positive control substances 2AA, 9AA and NPD.

- Justification for choice of solvent/vehicle: In the study two vehicle control groups were used depending on the solubility of the test item and the solubility of positive control chemicals.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

(9AA), without metabolic activation, TA1537, 50 μg

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

(SAZ), without metabolic activation, TA100 and TA1535, 2 µg

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

(MMS), without metabolic activation, E.coli WP2 uvrA, 2 µL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

(2AA), with metabolic activation in all of Salmonella strains (2 µg) and in E.coli strain (50 µg)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylene-diamine

Remarks:

(NPD), without metabolic activation in TA98, 4 µg

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each.

Evaluation criteria:

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 TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain 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.
Conditions for the Validity of the Test
The tests (initial and confirmatory mutation experiments) 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 TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The E. coli WP2 uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titer is in the 10E9 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.

Statistics:

none

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: Test item was completely dissolved in ultrapure water.
- Precipitation: In the Initial Mutation Test no precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix); however in the Confirmatory Mutation Test (Pre-Incubation Test) slight precipitate was noticed on the plates at the highest examined concentration of 5000 μg/plate in the absence of exogenous metabolic activation (-S9 Mix). The obtained precipitate did not disturb the scoring of colonies and background lawn development in any case.

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each.
The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate. In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.
In the Informatory Toxicity Test inhibitory effect of the test item was not observed. The colony and background lawn development was not affected in any case. All of the obtained slight revertant colony number decreases or increases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system.
Slightly lower revertant colony numbers (within the biological variability range of the applied test system) were obtained in S. typhimurium TA98 at 16 and 5 μg/plate in the absence of exogenous metabolic activation (-S9 Mix).
The revertant colony numbers were above the vehicle control data range (within the historical control data and biological variability range) in S. typhimurium TA98 in the whole examined concentration range of 5000-5 μg/plate in the presence of exogenous metabolic activation (+S9 Mix).

HISTORICAL CONTROL DATA (Please refer to "Any other information on results incl.tables")
The spontaneous revertant colony numbers of ultrapure water vehicle control plates showed the characteristic mean numbers agreed with the actual historical control data ranges in the main experiments.In the Initial Mutation Test all of the obtained higher revertant colony numbers (higher than the revertant colony numbers of the vehicle control) remained within the corresponding historical control data ranges. In the Confirmatory Mutation Test all of the noticed increased revertant colony numbers remained in the corresponding historical control data ranges of the ultrapure water vehicle control, and were without any biological significance.

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

Range Finding Test (Informatory Toxicity Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester 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	21.7	1.03	31.0	1.63	114.7	1.06	124.3	1.06
DMSO Control	20.7	1.00	20.7	1.00	–	–	101.7	1.00
Ultrapure Water Control	21.0	1.00	19.0	1.00	108.7	1.00	117.0	1.00
5000	23.7	1.13	34.0	1.79	116.7	1.07	119.7	1.02
1600	20.0	0.95	32.7	1.72	91.0	0.84	125.7	1.07
500	21.3	1.02	26.3	1.39	100.0	0.92	123.3	1.05
160	22.0	1.05	33.7	1.77	97.0	0.89	127.7	1.09
50	22.0	1.05	31.0	1.63	100.3	0.92	124.0	1.06
16	15.3	0.73	34.3	1.81	100.7	0.93	129.3	1.11
5	15.3	0.73	34.7	1.82	96.7	0.89	118.0	1.01
NPD (4mg)	302.0	14.61	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1861.3	17.13	–	–
2AA (2mg)	–	–	1754.7	84.90	–	–	1608.0	15.82

MR: Mutation Rate

Ultrapure water was applied as vehicle of the test item and the positive control substance: SAZ and the DMSO was applied as vehicle for positive control substances: NPD and 2AA. The mutation rate of the test item, SAZ and untreated control is given referring to the ultrapure water; the mutation rate of NPD and 2AA is given referring to DMSO.

Table 2: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-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	25.0	1.15	32.3	1.08	117.0	1.20	141.0	1.23	10.7	0.82	14.7	1.47	9.0	0.90	10.0	1.03	26.7	1.04	33.7	0.98
DMSO Control	25.0	1.00	22.3	1.00	–	–	124.7	1.00	–	–	11.0	1.00	10.7	1.00	15.3	1.00	–	–	33.3	1.00
Ultrapure Water Control	21.7	1.00	30.0	1.00	97.7	1.00	115.0	1.00	13.0	1.00	10.0	1.00	10.0	1.00	9.7	1.00	25.7	1.00	34.3	1.00
5000	20.3	0.94	38.0	1.27	97.3	1.00	115.3	1.00	15.3	1.18	10.0	1.00	7.3	0.73	10.3	1.07	28.0	1.09	41.3	1.20
1600	19.7	0.91	31.0	1.03	103.0	1.05	113.3	0.99	10.7	0.82	11.7	1.17	10.7	1.07	8.7	0.90	30.0	1.17	26.7	0.78
500	22.0	1.02	34.0	1.13	85.3	0.87	110.7	0.96	11.3	0.87	9.7	0.97	10.7	1.07	10.7	1.10	23.3	0.91	33.7	0.98
160	22.7	1.05	22.3	0.74	102.3	1.05	125.0	1.09	13.0	1.00	11.0	1.10	11.0	1.10	8.7	0.90	23.3	0.91	39.3	1.15
50	25.3	1.17	30.3	1.01	104.7	1.07	126.3	1.10	13.0	1.00	8.7	0.87	10.3	1.03	9.0	0.93	33.0	1.29	26.7	0.78
16	23.0	1.06	27.0	0.90	112.3	1.15	130.0	1.13	12.3	0.95	9.3	0.93	9.3	0.93	7.7	0.79	28.7	1.12	33.7	0.98
NPD (4mg)	184.0	7.36	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1077.3	11.03	–	–	890.7	68.51	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	944.0	88.50	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	941.3	36.68	–	–
2AA (2mg)	–	–	1616.0	72.36	–	–	3034.7	24.34	–	–	190.7	17.33	–	–	172.0	11.22	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	190.7	5.72

MR: Mutation Rate

Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Table 3: Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimuriumtester strains																Escherichia coli
	TA 98				TA 100				TA 1535				TA 1537				WP2uvrA
	-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	20.0	1.09	26.7	1.00	111.7	1.11	127.3	1.10	8.3	0.76	11.3	0.74	4.7	0.74	8.3	1.04	26.3	1.04	24.3	0.74
DMSO Control	17.3	1.00	24.0	1.00	–	–	96.0	1.00	–	–	13.3	1.00	5.3	1.00	9.7	1.00	–	–	30.7	1.00
Ultrapure Water Control	18.3	1.00	26.7	1.00	100.7	1.00	115.7	1.00	11.0	1.00	15.3	1.00	6.3	1.00	8.0	1.00	25.3	1.00	32.7	1.00
5000	24.3	1.33	39.3	1.48	90.0	0.89	123.0	1.06	12.3	1.12	14.7	0.96	5.3	0.84	9.7	1.21	26.3	1.04	39.0	1.19
1600	21.7	1.18	28.0	1.05	97.0	0.96	120.0	1.04	10.3	0.94	14.7	0.96	5.7	0.89	10.0	1.25	39.7	1.57	33.3	1.02
500	24.7	1.35	37.3	1.40	98.0	0.97	107.3	0.93	11.7	1.06	13.3	0.87	5.0	0.79	9.7	1.21	32.0	1.26	37.3	1.14
160	25.7	1.40	33.7	1.26	95.3	0.95	114.7	0.99	9.3	0.85	16.3	1.07	7.0	1.11	9.0	1.13	27.0	1.07	29.0	0.89
50	20.3	1.11	35.7	1.34	103.3	1.03	122.7	1.06	10.0	0.91	12.3	0.80	7.0	1.11	12.3	1.54	32.0	1.26	28.3	0.87
16	29.0	1.58	28.3	1.06	112.7	1.12	132.7	1.15	14.0	1.27	17.3	1.13	7.0	1.11	9.7	1.21	22.0	0.87	31.7	0.97
NPD (4mg)	328.7	18.96	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg)	–	–	–	–	1232.0	12.24	–	–	1021.3	92.85	–	–	–	–	–	–	–	–	–	–
9AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	492.0	92.25	–	–	–	–	–	–
MMS (2mL)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1093.3	43.16	–	–
2AA (2mg)	–	–	1514.7	63.11	–	–	1805.3	18.81	–	–	183.3	13.75	–	–	113.3	11.72	–	–	–	–
2AA (50mg)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	214.7	7.00

MR: Mutation Rate

Ultrapure water was applied as vehicle of the test item and the positive control substances: SAZ and MMS; and the DMSO was applied as vehicle for positive control substances: NPD, 9AA and 2AA. The mutation rate of the test item, SAZ, MMS and untreated control is given referring to the ultrapure water; the mutation rate of NPD, 9AA and 2AA is given referring to DMSO.

Table 4: Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

			Bacterial strains
Historical control data of untreated control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.0	105.0	10.5	8.1	25.4
		SD	3.7	25.7	1.4	2.3	5.2
		Minimum	9	66	3	2	11
		Maximum	39	155	23	19	45
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.5	117.1	11.8	9.0	33.9
		SD	4.3	18.1	1.4	1.9	5.2
		Minimum	12	75	4	2	17
		Maximum	46	166	23	20	56
			Bacterial strains
Historical control data of DMSO control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	20.4	100.1	10.3	7.9	24.7
		SD	3.6	24.8	1.3	2.4	4.6
		Minimum	10	64	3	2	11
		Maximum	38	147	23	20	45
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	26.5	113.8	11.8	8.8	33.7
		SD	4.1	18.3	1.5	1.9	5.0
		Minimum	15	71	3	3	16
		Maximum	47	162	25	20	57
			Bacterial strains
Historical control data of Water control	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	21.9	104.7	10.5	7.6	26.1
		SD	3.7	25.9	1.5	2.2	5.5
		Minimum	12	68	3	2	12
		Maximum	35	154	24	16	48
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	27.4	117.3	11.4	8.7	34.9
		SD	4.0	18.5	1.3	2.2	4.9
		Minimum	15	83	4	3	18
		Maximum	43	167	22	16	57
			Bacterial strains
Historical control data of positive controls	‑S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	260.1	977.2	847.3	478.6	724.5
		SD	31.8	150.6	126.3	104.5	65.0
		Minimum	123	521	359	110	320
		Maximum	664	1970	1855	1601	1313
	+S9		TA98	TA100	TA1535	TA1537	E. coli
		Average	1222.7	1436.4	164.1	147.0	257.7
		SD	274.9	318.3	33.1	20.1	72.5
		Minimum	386	583	85	69	140
		Maximum	2676	2988	498	399	477

SD: Standard deviation

 

Conclusions:

The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the substance is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

A bacterial reverse mutation assay according OECD TG 471, EU method B.13/14 and EPA OTS 789.5100 was performed to investigate the mutagenic potential in two independent experiments, in a plate incorporation test (Initial Mutation Test) and in a pre-incubation test (Confirmatory Mutation Test).

In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated.

The test item was dissolved in ultrapure water. In the Initial and Confirmatory Mutation Tests the following concentrations were examined: 5000, 1600, 500, 160, 50 and 16 μg/plate. Each assay was conducted with and without metabolic activation (±S9 Mix).

The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently.

No substantial 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 S9 Mix in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

The positive controls showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

In the performed experiments the revertant colony numbers of the untreated and DMSO control plates in the different experimental phases were slightly higher or lower than the ultrapure water vehicle control plates. The higher or lower revertant counts of these controls remained in the historical control data ranges.

In the performed experiments inhibitory effect of the test item was not observed in any case. Signs of cytotoxicity were not observed in either tested strains with and/or without metabolic activation.

In the Confirmatory Mutation Test slight precipitate was noticed on the plates in the examined bacterial strains at the highest examined concentration level of 5000 μg/plate in the absence of exogenous metabolic activation (-S9 Mix).

The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the substance 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:

November 28, 2016 - April 5, 2017

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:

29 July, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

30 May 2008

Deviations:

yes

Remarks:

There is a deviation from the guidelines regarding the maximum concentration. The maximum concentration was 2000 μg/ml, according to current Guideline (OECD 473, 29 July 2016) instead of 5000 μg/ml.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

yes

Remarks:

There is a deviation from the guidelines regarding the maximum concentration. The maximum concentration was 2000 μg/ml, according to current Guideline (OECD 473, 29 July 2016) instead of 5000 μg/ml.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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)
- Suitability of cells: Stability of karyotype and morphology makes them suitable for gene toxicity assays with low background aberrations.
- Cell cycle length, doubling time or proliferation index: doubling time 12-14 h
- Modal number of chromosomes: 2n=22
MEDIA USED
- Type and identity of media including CO2 concentration: DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine and 1 % of Antibiotic-antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated fetal bovine serum (final concentration 10 %), cultivated at 37°C in a humidified atmosphere containing 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Cytokinesis block (if used):

Colchicine

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:

The concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for soluble, lower-cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 473 (2016)).
Experiment A with 3/20 h treatment/sampling time
without and with S9 mix: 500, 1000 and 2000 μg/mL
Experiment B with 20/20 h treatment/sampling time
without S9 mix: 250, 500, 1000 and 2000 μg/mL
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 250, 500, 1000 and 2000 μg/mL
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 500, 1000 and 2000 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: medium (DME)
- Justification for choice of solvent/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:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DME (Dulbecco’s Modified Eagle’s) medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

(EMS), without metabolic activation, 0.4 and 1.0 μL/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DME (Dulbecco’s Modified Eagle’s) medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation, 5.0 μg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 5x10E5

DURATION
- Exposure duration: 3 hours (Experiment A and Experiment B with S9 Mix) and 20 hours (Experiment B without S9 Mix)
- Expression time (cells in growth medium): 20 hours (Experiment A) and 28 hours (Experiment B)

SPINDLE INHIBITOR: colchicine (0.2 μg/mL)

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 became plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies. For control of bias, all slides were coded and scored blind.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE
300 metaphase cells containing 2 N ± 2 centromeres were evaluated for structural aberrations from each experimental group.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was determined by cell counting, based on the cell counts Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative because:
– 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 laboratory historical negative control data.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity assay a 3-hour treatment in the absence of S9 Mix was performed with the concentration levels of: 500, 1000 and 2000 μg/mL. The obtained toxicity range was 3.33-22.82 %. At the 3-hour treatment (20 h sampling time) in the presence of S9 Mix the observed range of toxicity was -1.00- 14.54 % at concentration levels of 250, 500, 1000 and 2000 μg/mL. In case of 20/20, 28 h treatment/sampling times examined concentrations were: 125, 250, 500, 1000 and 2000 μg/mL. The observed toxicity range was -0.51-57.42 %. At the 3-hour treatment (28 h sampling time) in the presence of S9 Mix the obtained range of toxicity was 0.21-15.91% at concentration levels of 250, 500, 1000 and 2000 μg/mL.

HISTORICAL CONTROL DATA: Please refer to "Any other information on results incl. tables"

Table 1: Mean Percentage of cells with structural chromosome aberrations (Experiment A)

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/ 150 cells
				incl. gaps	excl. gaps
Solvent control (DME)	-	3 h	20 h	5	3
Retardan 200 P
500µg/mL	-	3 h	20 h	5	2
1000µg/mL	-	3 h	20 h	7	4
2000µg/mL	-	3 h	20 h	6	3
Pos. Control (EMS)	-	3 h	20 h	48**	35**
Solvent control (DME)	+	3 h	20 h	6	3
Retardan 200 P
500µg/mL	+	3 h	20 h	8	3
1000µg/mL	+	3 h	20 h	9	4
2000µg/mL	+	3 h	20 h	7	3
Pos. Control (Cycl.)	+	3 h	20 h	47**	40**

Positive control (-S9): Ethyl methanesulfonate (1.0 µL/mL),Positive control (+S9): Cyclophosphamide (5.0 µg/mL);** = p < 0.01 to the concurrent negative control and to the historical control

Table 2: Mean Percentage of cells with structural chromosome aberrations (Experiment B)

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/ 150cells
				incl. gaps	excl. gaps
Solvent control (DME)	-	20 h	20 h	5	3
Retardan 200 P
250 µg/mL	-	20 h	20 h	6	3
500µg/mL	-	20 h	20 h	7	3
1000µg/mL	-	20 h	20 h	8	4
2000µg/mL	-	20 h	20 h	7	3
Pos. Control (EMS)	-	20 h	20 h	50**	43**
Solvent control (DME)	-	20 h	28 h	5	3
Retardan 200 P
250 µg/mL	-	20 h	28 h	7	4
500µg/mL	-	20 h	28 h	7	3
1000µg/mL	-	20 h	28 h	7	4
2000µg/mL	-	20 h	28 h	7	4
Pos. Control (EMS)	-	20 h	28 h	45**	39**

Positive control (-S9): Ethyl methanesulfonate (0.4 µL/mL);** = p < 0.01 to the concurrent negative control and to the historical control

Table 3: Mean Percentage of cells with structural chromosome aberrations (Experiment B)- continued

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/ 150 cells
				incl. gaps	excl. gaps
Solvent control (DME)	+	3 h	28 h	5	2
Retardan 200 P
500µg/mL	+	3 h	28 h	6	4
1000µg/mL	+	3 h	28 h	6	3
2000µg/mL	+	3 h	28 h	7	3
Pos. Control (Cycl.)	+	3 h	28 h	48**	39**

Positive control (+S9): Cyclophosphamide (5.0 µg/mL); ** = p < 0.01 to the concurrent negative control and to the historical control

Table 4: Number of Polyploid cells and Endoreduplicated cells (Experiment A)

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Solvent control (DME)	-	3 h	20 h	0.0	0.0
Retardan 200 P
500µg/mL	-	3 h	20 h	0.0	0.0
1000µg/mL	-	3 h	20 h	0.0	0.0
2000µg/mL	-	3 h	20 h	0.0	0.0
Pos. Control (EMS)*	-	3 h	20 h	0.0	0.0
Solvent control (DME)	+	3 h	20 h	0.0	0.0
Retardan 200 P
500µg/mL	+	3 h	20 h	0.0	0.0
1000µg/mL	+	3 h	20 h	0.0	0.0
2000µg/mL	+	3 h	20 h	0.0	0.0
Pos. Control (Cycl.)**	+	3 h	20 h	0.0	0.0

* Ethyl methanesulfonate (1.0 µL/mL); ** Cyclophosphamide (5.0 µg/mL); The number of polyploid and endoreduplicated cells was determined in 150 cells of each test group.

Table 5: Number of Polyploid cells and Endoreduplicated cells (Experiment B)

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Solvent control (DME)	-	20 h	20 h	0.0	0.0
Retardan 200 P
250 µg/mL	-	20 h	20 h	0.0	0.0
500µg/mL	-	20 h	20 h	0.0	0.0
1000µg/mL	-	20 h	20 h	0.0	0.0
2000µg/mL	-	20 h	20 h	0.0	0.0
Pos. Control (EMS)	-	20 h	20 h	0.0	0.0
Solvent control (DME)	-	20 h	28 h	0.0	0.0
Retardan 200 P
250 µg/mL	-	20 h	28 h	0.0	0.0
500µg/mL	-	20 h	28 h	0.0	0.0
1000µg/mL	-	20 h	28 h	0.0	0.0
2000µg/mL	-	20 h	28 h	0.0	0.0
Pos. Control (EMS)	-	20 h	28 h	0.0	0.0

Ethyl methanesulfonate (0.4 µL/mL)

Table 6: Number of Polyploid cells and Endoreduplicated cells (Experiment B)- continued

Concentration (µg/mL)	S9 mix	Treatment time	Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Solvent control (DME)	+	3 h	28 h	0.0	0.0
Retardan 200 P
500µg/mL	+	3 h	28 h	0.0	0.0
1000µg/mL	+	3 h	28 h	0.0	0.0
2000µg/mL	+	3 h	28 h	0.0	0.0
Pos. Control (Cycl.)**	+	3 h	28 h	0.0	0.0

**Cyclophosphamide (5.0 µg/mL); The number of polyploid and endoreduplicated cells was determined in 150 cells of each test group.

Table 7: Historical Control Data

A. 3h/20h treatment/sampling time without S9-mix

	number of aberrant cells/ 150 cells
	negative control		positive control (Ethyl methanesulfonate)
	incl. Gaps	excl. Gaps	incl. Gaps	excl. Gaps
Mean	5.70	2.65	40.20	30.85
SD	0.63	0.59	3.83	3.41
Lower confidence interval	4.27	1.31	31.53	23.13
Upper confidence interval	7.13	3.99	48.87	38.57
n	10	10	10	10

B. 3h/20h treatment/sampling time with S9-mix

	number of aberrant cells/150cells
	negative control		positive control (Cyclophosphamide)
	incl. Gaps	excl. Gaps	incl. Gaps	excl. Gaps
Mean	5.75	2.80	46.40	39.65
SD	0.74	0.55	2.10	1.83
Lower confidence interval	4.07	1.56	41.66	35.51
Upper confidence interval	7.43	4.04	51.14	43.79
n	10	10	10	10

C. 20h/20h treatment/sampling time without S9-mix

	number of aberrant cells/150cells
	negative control		positive control (Ethyl methanesulfonate)
	incl. Gaps	excl. Gaps	incl. Gaps	excl. Gaps
Mean	5.70	2.85	45.20	37.85
SD	1.00	0.59	2.32	2.13
Lower confidence interval	3.44	1.51	39.94	33.02
Upper confidence interval	7.96	4.19	50.46	42.68
n	10	10	10	10

D. 20h/28h treatment/sampling time without S9-mix

	number of aberrant cells/ 150cells
	negative control		positive control (Ethyl methanesulfonate)
	incl. Gaps	excl. Gaps	incl. Gaps	excl. Gaps
Mean	5.45	2.75	45.95	36.85
SD	0.67	0.59	2.29	2.20
Lower confidence interval	3.93	1.41	40.77	31.87
Upper confidence interval	6.97	4.09	51.13	41.83
n	10	10	10	10

E. 3h/28h treatment/sampling time with S9-mix

	number of aberrant cells/ 150 cells
	negative control		positive control (Cyclophosphamide)
	incl. Gaps	excl. Gaps	incl. Gaps	excl. Gaps
Mean	5.75	2.75	45.90	38.90
SD	0.50	0.59	2.07	3.29
Lower confidence interval	4.62	1.41	41.21	31.47
Upper confidence interval	6.88	4.09	50.59	46.33
n	10	10	10	10

n           = number of experiments

SD        = standard deviation

Conclusions:

The substance tested up to the maximum recommended concentration with and without mammalian metabolic activation system (three hours treatment) did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system (twenty hours treatment) did not induced structural chromosome aberrations in Chinese Hamster lung cells. Thus, the test item is considered as not clastogenic in this system.

Executive summary:

A study according OECD TG 473 was conducted to detect chromosome aberrations in Chinese hamster lung cells (V79).

The test item was suspended in cell medium (DME, Dulbecco’s Modified Eagle’s) and the concentrations tested were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 300 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix: 500, 1000 and 2000 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 250, 500, 1000 and 2000 μg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 250, 500, 1000 and 2000 μg/mL

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 500, 1000 and 2000 μg/mL                 

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to the maximum recommended concentration. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to the maximum recommended concentration in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberrations were inside the distribution of the laboratory historical negative control data.

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.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The number of aberrations found in the solvent controls was compatible with the laboratory 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.

The test item tested up to the maximum recommended concentration with and without mammalian metabolic activation system (three hours treatment) did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system (twenty hours treatment) did not induced structural chromosome aberrations in Chinese Hamster lung cells. Thus, the test item is considered as not 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:

November 28, 2016 - July 6, 2017

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

yes

Remarks:

Negative results were not confirmed as the confirmation of negative results is not required by the most current OECD Guideline; maximum recommended concentration was 2000 μg/mL instead of 5000 μg/mL.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

yes

Remarks:

Negative results were not confirmed as the confirmation of negative results is not required by the most current OECD Guideline; maximum recommended concentration was 2000 μg/mL instead of 5000 μg/mL.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial forward mutation assay

Target gene:

The method is based on the detection of mutations (either induced or spontaneously generated) in the hprt locus located on the X chromosome. HPRT (hypoxanthine-guanine phosphoribosyl transferase) is a cellular enzyme that allows cells to salvage hypoxanthine and guanine from surrounding medium for use in DNA synthesis.

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)

MEDIA USED
- Type and identity of media including CO2 concentration: Ham's F12 medium supplemented with 1 % of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %), cell were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. During treatment, serum content was reduced to 5 % (F12-5).
- 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:

Treatment concentrations for the mutation assay were selected on the basis of the result of a pre-test on toxicity.
5-hour treatment period without S9-mix:
125, 250, 500, 1000 and 2000 μg/mL
5-hour treatment period with S9-mix:
125, 250, 500, 1000 and 2000 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: medium
- Justification for choice of solvent/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:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ham's F12 medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation, 1.0 μL/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation, 20 μg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 5x10E6 cells/dish

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 19 hours
- Selection time: 8 Days

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

NUMBER OF REPLICATIONS: 2

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: cloning efficiency

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a 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.
Test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.
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 done 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 heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Solubility: The test item was suspended in Ham's F12 medium. A homogen suspension was obtained up to a concentration of 100 mg/mL.
- Precipitation: not observed

RANGE-FINDING/SCREENING STUDIES:
A Pre-test on Toxicity was performed to establish an appropriate concentration range for the main mutation assay, both in the absence and in the presence of metabolic activation (S9-mix). Toxicity was determined by comparing the colony forming ability of the treated groups to the negative (solvent) control. The Relative survival in percent of cells treated with the test item in presence and absence of metabolic activation was not significantly different from the solvent control.

HISTORICAL CONTROL DATA: Please refer to "any other information including tables"

Table 1: Summarized results of the concentration selection cytotoxicity assay

Test group	Dose µg/ml	S9-mix	Treatment/ time/ hour	Number of colonies/200 cells/dish			Mean	Relative^a survival in percent
				dish 1	dish 2	dish 3
Solvent Control (Ham's F12 medium)	–	–	5	204	205	202	203,7	100
Test item	125	–	5	203	204	205	204,0	100
	250	–	5	205	201	201	202,3	99
	500	–	5	199	203	204	202,0	99
	1000	–	5	204	204	200	202,7	100
	1500		5	201	203	199	201,0	99
	2000	–	5	202	200	204	202,0	99
Solvent Control (Ham's F12 medium)	–	+	5	202	206	200	202,7	100
Test item	125	+	5	204	201	201	202,0	100
	250	+	5	205	202	199	202,0	100
	500	+	5	201	200	197	199,3	98
	1000	+	5	197	204	201	200,7	99
	1500	+	5	202	203	200	201,7	100
	2000	+	5	200	198	203	200,3	99

a Relative to sovent control

Table 2 Main mutation assay summary of results without S9-Mix

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control a	204.7	±	1.53	100	100	1	0	1	1	3	6	101	5.94
Pos. control (EMS 1.0 µL/mL) a	51.7	±	1.53	25	60	198	194	198	203	206	999	61	1637.70**
TEST ITEM
125 µg/mL a	202,3	±	1,53	99	99	0	1	0	1	3	5	100	5.00
250 µg/mL a	203,3	±	2,89	99	99	3	1	3	0	0	7	100	7.00
500 µg/mL a	203,3	±	1,53	99	98	2	2	0	0	1	5	99	5.05
1000 µg/mL a	204,3	±	2,89	100	98	0	1	2	0	2	6	99	6.06
2000 µg/mL a	203,3	±	1,53	99	98	0	2	0	3	2	7	99	7.07

a = parallel of second culture; abs.C.E. = Absolute Cloning Efficiency; EMS=Ethyl methanesulfonate; ** = p < 0.01 to the concurrent negative control and to the historical control

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control b	203.0	±	0.00	100	100	1	0	3	0	2	6	100	6.00
Pos. control (EMS 1.0 µL/mL) b	52.3	±	1.53	26	60	191	195	200	203	197	986	60	1643.33**
TEST ITEM
125 µg/mL b	200,3	±	0,58	99	100	0	1	2	1	1	5	100	5.00
250 µg/mL b	201,3	±	1,53	99	99	1	0	1	3	3	8	99	8.08
500 µg/mL b	202,7	±	0,58	100	99	4	0	0	0	2	6	99	6.06
1000 µg/mL b	200,7	±	1,15	99	99	0	1	2	1	3	7	99	7.07
2000 µg/mL b	202,3	±	1,53	100	99	2	2	2	1	0	4	99	4.04

b = parallel of first culture;abs.C.E. = Absolute Cloning Efficiency;EMS=Ethyl methanesulfonate;** = p < 0.01 to the concurrent negative control and to the historical control

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control c	202.3	±	2.52	100	100	3	1	0	0	2	6	101	5.94
Pos. control (EMS 1.0µL/mL) c	53.0	±	2.65	26	61	204	210	199	203	196	1012	62	1632.25**
TEST ITEM
125 µg/mL c	202,0	±	2,65	100	99	1	0	4	0	1	6	101	5.94
250 µg/mL c	203,0	±	1,73	100	100	1	0	2	2	2	7	101	6.93
500 µg/mL c	201,0	±	3,46	99	98	0	0	3	3	2	8	99	8.08
1000 µg/mL c	202,3	±	2,08	100	99	1	1	1	1	1	5	100	5.00
2000 µg/mL c	200,0	±	2,65	99	98	3	1	2	0	1	7	99	7.07

c = parallel of second culture;abs.C.E. = Absolute Cloning Efficiency;EMS=Ethyl methanesulfonate;** = p < 0.01 to the concurrent negative control and to the historical control

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control d	201.3	±	1.15	100	100	2	1	2	1	1	7	101	6.93
Pos. control (EMS 1.0 µL/mL) d	52.7	±	.53	26	61	204	199	196	201	207	1007	61	1650.81**
TEST ITEM
125 µg/mL d	200,0	±	1,00	99	100	0	0	5	1	0	6	101	5.94
250 µg/mL d	201,3	±	1,15	100	100	3	2	1	1	1	8	101	7.92
500 µg/mL d	200,7	±	2,89	100	99	2	0	1	1	1	5	100	5.00
1000 µg/mL d	200,3	±	0,58	100	99	3	0	0	3	2	8	100	8.00
2000 µg/mL d	199,7	±	0,58	99	99	1	1	0	2	2	6	99	6.06

d= parallel of second culture;abs.C.E. = Absolute Cloning Efficiency;EMS=Ethyl methanesulfonate;** = p < 0.01 to the concurrent negative control and to the historical control

Table 3 Main mutation assay summary of results with S9-Mix

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control a	201.3	±	2.52	100	100	0	3	2	3	0	8	100	8.00
Pos. control (DMBA 20 µg/mL) a	113.0	±	2.65	56	75	103	111	114	101	117	546	75	728.00**
TEST ITEM
125 µg/mL a	202,3	±	1,53	100	100	0	1	4	1	0	6	99	6.06
250 µg/mL a	199,3	±	1,15	99	100	1	2	0	0	4	7	100	7.00
500 µg/mL a	202,0	±	1,00	100	98	0	3	0	1	3	7	98	7.14
1000 µg/mL a	201,7	±	3,51	100	98	2	3	0	3	0	8	98	8.16
2000 µg/mL a	199,7	±	0,58	99	100	1	2	2	0	2	7	100	7.00

a = parallel of first culture; abs.C.E. = Absolute Cloning Efficiency; DMBA=7,12-Dimethyl benzanthracene; ** = p < 0.01 to the concurrent negative control and to the historical control

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control b	202.0	±	1.00	100	100	0	1	1	1	2	5	100	5.00
Pos. control (DMBA 20 µg/mL) b	112.0	±	2.00	55	74	113	117	99	108	116	553	75	737.33**
TEST ITEM
125 µg/mL b	200,3	±	0,58	99	99	1	0	3	0	2	6	99	6.06
250 µg/mL b	200,0	±	0,00	99	99	1	1	1	3	1	7	99	7.07
500 µg/mL b	201,3	±	0,58	100	99	0	0	2	4	1	7	99	7.07
1000 µg/mL b	200,0	±	2,00	99	99	2	1	0	2	0	5	99	5.05
2000 µg/mL b	200,7	±	0,58	99	99	2	2	1	1	2	8	99	8.08

b = parallel of first culture;abs.C.E. = Absolute Cloning Efficiency;DMBA=7,12-Dimethyl benzanthracene;** = p < 0.01 to the concurrent negative control and to the historical control

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control c	202.3	±	2.89	100	100	0	0	3	3	1	7	101	6.93
Pos. control (DMBA20 µg/mL) c	114.3	±	1.53	57	70	120	114	110	118	112	574	71	808.45**
TEST ITEM
125 µg/mL c	204,3	±	1,15	101	100	0	1	1	2	2	6	101	5.94
250 µg/mL c	203,3	±	2,08	100	98	4	1	1	0	1	7	99	7.07
500 µg/mL c	200,3	±	2,52	99	99	4	0	0	1	2	7	100	7.00
1000 µg/mL c	199,7	±	3,06	99	100	2	0	2	1	0	5	101	4.95
2000 µg/mL c	200,3	±	2,08	99	99	0	3	1	1	2	7	100	7.00

c = parallel of second culture;abs.C.E. = Absolute Cloning Efficiency;DMBA=7,12-Dimethyl benzanthracene

NON ACTIVATION TEST CONDITION	SURVIVAL TO TREATMENT				REL. POPU- LATION GROWTH (%) OF CONTROL	MUTANT COLONIES DISH NUMBER					TOTAL MUTANT COLONIES	ABSOLUTE C.E. %	MUTANT FREQ. X 10^-6
	MEAN COLONY NUMBERS.D.			PERCENT VEH. CONTROL		1	2	3	4	5
Solvent control d	202.0	±	1.00	100	100	0	1	1	4	2	8	101	7.92
Pos. control (DMBA 20 µg/mL) d	112.0	±	2.00	55	70	119	110	107	115	112	563	70	804.28**
TEST ITEM
125 µg/mL d	201,0	±	1,73	100	99	3	3	0	0	1	7	100	7.00
250 µg/mL d	200,7	±	0,58	99	99	0	0	0	4	2	6	99	6.06
500 µg/mL d	200,7	±	2,08	99	99	1	0	1	3	3	8	100	8.00
1000 µg/mL d	200,7	±	2,08	99	99	0	1	5	2	0	8	100	8.00
2000 µg/mL d	200,7	±	1,15	99	99	0	1	0	4	0	5	100	5.00

d = parallel of second culture;abs.C.E. = Absolute Cloning Efficiency;DMBA=7,12-Dimethyl benzanthracene

Table 4 Historical control data (for solvent control mutant frequency 2016 -2017)

	Without S9 mix	With S9 mix
	5-hour treatment	5-hour treatment
Mean	6.37	6.66
SD	0.57	0.87
Range	4.90 – 8.82	4.12 – 11.76
Lower confidence interval	5.11	4.73
Upper confidence interval	7.63	8.59
n	11	11

Table 5 Historical control data (for positive control mutant frequency)

	Without S9 mix EMS	With S9 mix DMBA
	5-hour treatment	5-hour treatment
Mean	1516.10	750.93
SD	26.13	14.53
Range	1357.81 – 1636.92	690.00-810.29
Lower confidence interval	1457.87	718.55
Upper confidence interval	1574.32	783.30
n	11	11

EMS    =   Ethyl methanesulphonate; DMBA=   7,12-Dimethylbenzanthracene; SD =   Standard deviation; n =   number of experiments

Conclusions:

The test item tested both without and with metabolic activation, did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells. Thus, it was not mutagenic under the conditions of this study.

Executive summary:

A study was conducted according OECD TG 476 to determine the mutagenic potential of the test item in mammalian Chinese hamster Ovary cells (CHO-K1). Concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver.

5-hour treatment period without S9-mix:

125, 250, 500, 1000 and 2000 μg/mL

5-hour treatment period with S9-mix:

125, 250, 500, 1000 and 2000 μg/mL

In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 476 (2016)).

Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was 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.

There was no precipitation of the test item suspension at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The validity of the test and the efficacy of the S9 mix were demonstrated by distinct 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 frequency found in the positive controls was within the range of historical laboratory control data.

The substance tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells, when tested up to maximum recommended concentration. Thus, it was not mutagenic under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames Test

A bacterial reverse mutation assay according OECD TG 471, EU method B.13/14 and EPA OTS 789.5100 was performed to investigate the mutagenic potential in two independent experiments, in a plate incorporation test (Initial Mutation Test) and in a pre-incubation test (Confirmatory Mutation Test).

In the Initial and Confirmatory Mutation Tests Salmonella typhimurium TA98, TA1537, TA1535 and TA100 strains and Escherichia coli WP2 uvrA were investigated.

The test item was dissolved in ultrapure water. In the Initial and Confirmatory Mutation Tests the following concentrations were examined: 5000, 1600, 500, 160, 50 and 16 μg/plate. Each assay was conducted with and without metabolic activation (±S9 Mix).

The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently.

No substantial 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 S9 Mix in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.

The positive controls showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains.

In the performed experiments the revertant colony numbers of the untreated and DMSO control plates in the different experimental phases were slightly higher or lower than the ultrapure water vehicle control plates. The higher or lower revertant counts of these controls remained in the historical control data ranges.

In the performed experiments inhibitory effect of the test item was not observed in any case. Signs of cytotoxicity were not observed in either tested strains with and/or without metabolic activation.

In the Confirmatory Mutation Test slight precipitate was noticed on the plates in the examined bacterial strains at the highest examined concentration level of 5000 μg/plate in the absence of exogenous metabolic activation (-S9 Mix).

The test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the substance is considered non-mutagenic in this bacterial reverse mutation assay.

 

Chromosome Aberration Assay

A study according OECD TG 473 was conducted to detect chromosome aberrations in Chinese hamster lung cells (V79).

The test item was suspended in cell medium (DME, Dulbecco’s Modified Eagle’s) and the concentrations tested were selected on the basis of cytotoxicity investigations made in a preliminary study (with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver). In the two independent experiments of the Chromosome Aberration Assay (Experiments A and B, both run in duplicate) at least 300 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below:

Experiment A with 3/20 h treatment/sampling time

without and with S9 mix: 500, 1000 and 2000 μg/mL

Experiment B with 20/20 h treatment/sampling time

without S9 mix: 250, 500, 1000 and 2000 μg/mL

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 250, 500, 1000 and 2000 μg/mL

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 500, 1000 and 2000 μg/mL                 

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, neither in the absence nor in the presence of metabolic activation, up to the maximum recommended concentration. There were no statistical differences between treatment and concurrent solvent and historical control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations did not show significant alterations compared to concurrent and historical controls, up to cytotoxic concentrations without S9 mix over a prolonged treatment period of 20 hours with harvest at 20 or 28 hours following treatment start. Further, a 3-hour treatment up to the maximum recommended concentration in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations.

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. The observed chromosome aberrations were inside the distribution of the laboratory historical negative control data.

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.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The number of aberrations found in the solvent controls was compatible with the laboratory 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.

The test item tested up to the maximum recommended concentration with and without mammalian metabolic activation system (three hours treatment) did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item tested up to cytotoxic concentrations, without mammalian metabolic activation system (twenty hours treatment) did not induced structural chromosome aberrations in Chinese Hamster lung cells. Thus, the test item is considered as not clastogenic in this system.

 

HPRT Assay

A study was conducted according OECD TG 476 to determine the mutagenic potential of the test item in mammalian Chinese hamster Ovary cells (CHO-K1). Concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver.

5-hour treatment period without S9-mix:

125, 250, 500, 1000 and 2000 μg/mL

5-hour treatment period with S9-mix:

125, 250, 500, 1000 and 2000 μg/mL

In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 476 (2016)).

Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was 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.

There was no precipitation of the test item suspension at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The validity of the test and the efficacy of the S9 mix were demonstrated by distinct 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 frequency found in the positive controls was within the range of historical laboratory control data.

The substance tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency in this in vitro test in Chinese hamster ovary cells, when tested up to maximum recommended concentration. Thus, it was not mutagenic under the conditions of this study.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item is not classified as mutagenic or clastogenic according to Regulation (EC) No 1272/2008 (CLP), as amended for the seventeenth time in Regulation (EU) 2021/849.