Reaction product of 2,2'-oxydiethanol and 2-hydroxyethyl acrylate and 2-hydroxyethyl methacrylate and hexan-6-olide and trimethylhexa-1,6-diyl diisocyanate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD 471, Ames test): negative in S. typhimurium strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2uvrA with and without metabolic activation

Cytogenicity in mammalian cells (OECD 487, Micronucleus test): negative in mouse lymphoma L5178 cells with and without metabolic activation

Gene mutation in mammalian cells (OECD 490, Mouse lymphoma assay): negative in mouse lymphoma L5178 cells with and without metabolic activation

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Jan - 26 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministère de l'Économie et des Finances, vry-sur-Seine Cedex, France

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Japan Health Science Foundation
- Suitability of cells: recommended test system in international guidelines
- Number of passages if applicable: 24 and 39
- Methods for maintenance in cell culture: Cell cultures were maintained in suspension cell culture flasks at 37 °C and 5% CO2.

MEDIA USED
- Type and identity of media including CO2 concentration: The culture medium used was the R10 medium: Roswell Park Memorial Institute medium (RPMI 1640) + 10% horse serum, heat inactivated (v/v),
50 IU/mL penicillin, 50 µg/mL streptomycin + 1 mM sodium pyruvate + 0.05% pluronic acid.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no

Metabolic activation:

with and without

Metabolic activation system:

Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I
4 h treatment (with and without metabolic activation): 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250 and 500 µg/mL

Experiment I Repetition
4 h treatment (without metabolic activation): 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5 and 10 µg/mL

Experiment II
24 h treatment (without metabolic activation): 0.078, 0.156, 0.312, 0.625, 1.25, 2.5, 5 and 10 µg/mL

Test item concentrations were initially selected based on the solubility in the solvent ethanol. In cases where the test item is cytotoxic, the highest concentration should aim to reach a maximum cytotoxicity (cytostasis) of 55 ± 5%. In the first experiment (4 h) without metabolic activation, only two of the eight tested concentrations met the acceptability criteria, therefore the test was not valid. Cytotoxicity (cytostasis) of more than 71% was observed at concentrations of ≥ 15.6 µg/mL, but only when tested without metabolic activation. Therefore the first experiment (without metabolic activation) was repeated at lower concentrations.

Vehicle / solvent:

- Vehicle used: ethanol (1.0% (v/v))

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine (-S9), 0.1 µg/mL in ethanol (4 h exposure) and 0.01 µg/mL in ethanol (24 h exposure)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 2 x 10E6 cells/tube in 8 mL cultivation medium

DURATION
- Exposure duration: 4 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 4 h treatment: 20-24 h; 24 h treatment: 44-48 h

STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates in all experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the incubation period, the cell cultures were homogenised, centrifuged and underwent hypotonic treatment. Afterwards, the cells were fixed with methanol-acetic acid (3:1 ratio) and spread on glas slides for microscopic analysis.

NUMBER OF CELLS EVALUATED: at least 2000 binucleated cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: cytostasis
- Any supplementary information relevant to cytotoxicity: please refer to “Any other informations on materials and methods”

Evaluation criteria:

A test substance was considered positive (clastogenic or aneugenic) in the micronucleolus test if:
- At least one of the test concentrations showed a statistically significant increase in the frequency of micronucleated cells when compared to the concurrent negative control
- A dose effect was observed in at least one experimental condition when evaluated with an appropriate trend test
- The results were outside the data distribution range of historic negative controls

A test substance was considered negative (not clastogenic or aneugenic) in the micronucleus test if:
- None of the test concentrations showed a statistically significant increase in the frequency of micronucleated cells when compared with the concurrent negative control
- There was no concentration-related increase in the frequency of micronucleated cells when evaluated with an appropriate trend test
- All results were inside the distribution of the historical negative control data

Statistics:

Chi square test (p < 0.05)

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

HISTORICAL CONTROL DATA: please refer to Table 2 under “Any other informations on results incl. tables”

Table 1: Experimental results of the micronucleus test

	Mononuclear cells number	Micronucleated cells number	P-value	Interpretation
Experiment I: 4 h + S9 mix
Vehicle control	994	6
	995	5
CP 1.5 µg/mL	979	21	1.84E-08	significant
	964	36
250 µg/mL	996	4	4.90E-01	non significant
	996	4
125 µg/mL	995	5	8.27E-01	non significant
	995	5
62.5 µg/mL	997	3	4.90E-01	non significant
	995	5
31.25 µg/mL	993	7	8.27E-01	non significant
	997	3
Negative control	999	1
	995	5
Experiment I (Repetition): 4 h - S9 mix
Vehicle control	995	5
	997	3
MMC 0.2 µg/mL	814	186	1.50E-94	significant
	781	219
Col 0.1 µg/mL	879	121	1.61E-43	non significant
	917	83
250 µg/mL	996	4	1.00E+00	non significant
	996	4
125 µg/mL	998	2	7.96E-01	non significant
	995	5
62.5 µg/mL	996	4	1.00E+00	non significant
	996	4
31.25 µg/mL	998	2	7.96E-01	non significant
	995	5
Negative control	997	3
	995	5
Experiment II: 24 h - S9 mix
Vehicle control	994	6
	996	4
MMC 0.02 µg/mL	916	84	3.34E-28	significant
	940	60
Col 0.01 µg/mL	961	39	1.23E-11	non significant
	969	31
250 µg/mL	997	3	4.66E-01	non significant
	996	4
125 µg/mL	998	2	4.66E-01	non significant
	995	5
62.5 µg/mL	997	3	3.16E-01	non significant
	997	3
31.25 µg/mL	998	2	6.37E-01	non significant
	994	6
Negative control	995	5
	997	3
CP: Cyclophosphamide; MMC: Mitomycin C; Col: Colchicine

Table 2: Historical control data

	Without S9						With S9
	4 h treatment (n=30)			24 h treatment (n=29)			4 h treatment (n=30)
	Vehicle*	Mitomycin	Colchicine	Vehicle*	Mitomycin	Colchicine	Vehicle*	Cyclophosphamide
Concentration (µg/mL)		0.2	0.1		0.02	0.01		1.5
Minimum	5	261	91	5	93	43	5	58
Maximum	13	489	326	14	228	231	15	233
Mean	8	368	180	8	135	76	9	128
Standard deviation	2	70	55	2	28	42	3	40
Vehicle*: culture medium or ethanol; data were generated in 2015-2018

Conclusions:

Under the experimental conditions of the in vitro micronucleus test the test substance did not induce micronuclei in mouse lymphoma L5178 cells with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 - 13 Dec 2018

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:

adopted 21 Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYEI National Institute of Pharmacy and Nutrition, Budapest, Hungary

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium strains) and trp operon (E. coli strain)

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Co-factor supplemented S9 mix, prepared from the livers of rats treated with phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

16, 50, 160, 500, 1600 and 5000 µg/plate with and without metabolic activation, 5000 µg/plate was selected as the highest test concentration based on the results of a preliminary study.

Vehicle / solvent:

- Vehicle: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-Nitro-1,2-phenylenediamine (NPD) in DMSO, 2-aminoanthracene (2AA) in DMSO

Remarks:

-S9: 9AA (50 µg/plate) for TA1537; SAZ (2 µg/plate) for TA100 and TA1535, MMS (2 µg/plate) for WP2uvrA; NPD (4 µg/plate) for TA98 / + S9: NPD (2µg/plate) for S. typhimurium strains and (50 µg/plate) for E. coli

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Initial mutation test: Plate incorporation
Confirmatory mutation test: Pre-incubation

DURATION
- Preincubation period: 20 min at 37 °C
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: triplicates in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: observation of bacterial growth inhibition

Evaluation criteria:

A material was considered mutagenic if:
- a dose-related increase in the number of revertant colonies occured and/or
- a reproducible biologically relevant positive response for at least one of the dose groups occured in at least one strain with or without metabolic activation
An increase was considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions was at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535 or TA1537 and Escherichia coli WP2uvrA the number of reversions was at least three times higher than the reversion rate of the vehicle control

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

Criteria for a Negative Response:
A test item was considered non-mutagenic if it produced 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.

Statistics:

Mean values and standard deviations were calculated.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation of the test item was observed at the concentration of 5000 µg/plate in the absence of metabolic activation (S9).

RANGE-FINDING/SCREENING STUDIES
Dose range finding tests were performed in S. typhimurium strains TA98 and TA100, based on the information about the solubility properties of the test item. A stock solution with a nominal concentration of 50 mg/mL was prepared in DMSO and diluted in 6 steps by factor √10. The revertant colony numbers and the inhibition of the background lawn was determined in a concentration range from 5 to 5000 µg/plate. Revertant colonies of the negative and the positive control confirmed the validity of the test system.

HISTORICAL CONTROL DATA
- Positive historical control data:
-S9: TA 98 average 285 ± 25.5 (range 152-598); TA100 average 1214.0 ± 80.0 (range 609-2272); TA1535 average 1024.3 ± 120.3 (range 407-2597); TA1537 average 594.4 ± 36.4 (range 136-2048); WP2uvrA average 858.4 ± 164.7 (range 330-1760)
+S9: TA 98 average 1395.7 ± 261.4 (range 286-3211); TA100 average 1727.4 ± 244.1 (range 712-3435); TA1535 average 160.7 ± 30.0 (range 91-328); TA1537 average 145.8 ± 18.9 (range 70-315); WP2uvrA average 204.9 ± 3.9 (range 133-367)

- Negative (DMSO) historical control data:
-S9: TA 98 average 18.1 ± 1.1 (range 9-36); TA100 average 87 ± 3.6 (range 58-131); TA1535 average 10.8 ± 0.6 (range 4-23); TA1537 average 8.5 ± 1.3 (range 3-20); WP2uvrA average 24.6 ± 3.3 (range 10-54)
+S9: TA 98 average 23 ± 0.8 (range 11-42); TA100 average 102.4 ± 7.7 (range 69-148); TA1535 average 11 ± 0.4 (range 3-23); TA1537 average 9.2 ± 1.3 (range 3-21); WP2uvrA average 31.4 ± 5.8 (range 12-59)

- Untreated historical control data:
-S9: TA 98 average 19.7 ± 1.7 (range 8-40); TA100 average 94.6 ± 2.4 (range 67-133); TA1535 average 10.9 ± 0.4 (range 4-21); TA1537 average 8.9 ± 1.3 (range 3-20); WP2uvrA average 25.2 ± 5.2 (range 11-52)
+S9: TA 98 average 24.5 ± 1.4 (range 11-43); TA100 average 112.9 ± 6.1 (range 74-159); TA1535 average 11 ± 0.5 (range 3-20); TA1537 average 9.2 ± 1.2 (range 3-20); WP2uvrA average 31.7 ± 6.4 (range 13-60)

Table 1: Results of the initial mutation test

Initial Mutation Test (Plate Incorporation Test)
Concentrations (mg/plate)	Salmonella typhimurium tester 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	20.7	1.32	31.0	1.63	71.7	1.04	100.7	1.13	9.7	1.32	10.0	1.00	5.7	1.13	7.0	0.88	31.7	0.95	30.7	0.77
DMSO Control	15.7	1.00	19.0	1.00	68.7	1.00	89.0	1.00	7.3	1.00	10.0	1.00	5.0	1.00	8.0	1.00	33.3	1.00	39.7	1.00
Ultrapure Water Control	–	–	–	–	75.0	1.00	–	–	13.3	1.00	–	–	–	–	–	–	31.3	1.00	–	–
5000	12.0	0.77	21.3	1.12	78.7	1.15	88.0	0.99	5.0	0.68	12.0	1.20	4.3	0.87	7.3	0.92	29.0	0.87	42.0	1.06
1600	17.0	1.09	25.0	1.32	79.3	1.16	85.3	0.96	9.0	1.23	9.3	0.93	6.0	1.20	9.0	1.13	30.0	0.90	45.7	1.15
500	20.7	1.32	19.0	1.00	79.7	1.16	82.0	0.92	8.7	1.18	12.3	1.23	4.7	0.93	7.3	0.92	33.0	0.99	34.3	0.87
160	17.0	1.09	20.7	1.09	69.0	1.00	83.7	0.94	7.0	0.95	10.0	1.00	5.7	1.13	8.3	1.04	29.7	0.89	42.3	1.07
50	16.3	1.04	20.0	1.05	70.3	1.02	85.7	0.96	9.0	1.23	8.3	0.83	6.7	1.33	8.3	1.04	35.7	1.07	42.0	1.06
16	17.0	1.09	25.3	1.33	83.0	1.21	90.3	1.01	9.7	1.32	11.7	1.17	8.3	1.67	8.0	1.00	34.7	1.04	31.7	0.80
NPD (4mg/plate)	326.7	20.85	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg/plate)	–	–	–	–	944.0	12.59	–	–	717.3	53.80	–	–	–	–	–	–	–	–	–	–
9AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	442.0	88.40	–	–	–	–	–	–
MMS (2mL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	1222.7	39.02	–	–
2AA (2mg/plate)	–	–	1005.3	52.91	–	–	1160.0	13.03	–	–	126.0	12.60	–	–	161.3	20.17	–	–	–	–
2AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	207.7	5.24

Table 2: Results of the confirmatory mutation test

Confirmatory Mutation Test (Pre-Incubation Test)
Concentrations (mg/plate)	Salmonella typhimurium tester 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	19.7	1.04	23.0	0.96	67.3	1.09	82.0	1.14	9.3	0.93	11.3	0.97	5.0	1.00	7.7	1.10	33.7	1.20	26.7	0.90
DMSO Control	19.0	1.00	24.0	1.00	62.0	1.00	71.7	1.00	10.0	1.00	11.7	1.00	5.0	1.00	7.0	1.00	28.0	1.00	29.7	1.00
Ultrapure Water Control	–	–	–	–	80.7	1.00	–	–	10.7	1.00	–	–	–	–	–	–	32.7	1.00	–	–
5000	16.3	0.86	18.3	0.76	72.0	1.16	89.0	1.24	9.3	0.93	12.0	1.03	5.7	1.13	7.3	1.05	18.0	0.64	33.3	1.12
1600	18.7	0.98	21.7	0.90	78.0	1.26	83.0	1.16	8.3	0.83	11.0	0.94	5.0	1.00	8.3	1.19	26.3	0.94	35.7	1.20
500	14.3	0.75	18.3	0.76	71.7	1.16	92.7	1.29	7.0	0.70	12.7	1.09	3.0	0.60	8.3	1.19	23.0	0.82	41.0	1.38
160	18.0	0.95	24.7	1.03	72.3	1.17	96.3	1.34	8.7	0.87	9.7	0.83	6.3	1.27	7.3	1.05	21.7	0.77	45.0	1.52
50	14.0	0.74	28.7	1.19	71.0	1.15	94.3	1.32	7.7	0.77	11.3	0.97	7.0	1.40	7.0	1.00	27.3	0.98	36.7	1.24
16	15.7	0.82	22.7	0.94	53.3	0.86	88.0	1.23	7.0	0.70	9.3	0.80	6.3	1.27	7.0	1.00	22.7	0.81	38.3	1.29
NPD (4mg/plate)	588.0	30.95	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–
SAZ (2mg/plate)	–	–	–	–	875.3	10.85	–	–	1034.0	96.94	–	–	–	–	–	–	–	–	–	–
9AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	388.0	77.60	–	–	–	–	–	–
MMS (2mL/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	917.7	28.09	–	–
2AA (2mg/plate)	–	–	1272.0	53.00	–	–	1373.7	19.17	–	–	166.7	14.29	–	–	98.0	14.00	–	–	–	–
2AA (50mg/plate)	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	–	233.0	7.85

MR: Mutation Rate        

NPD: 4-Nitro-1,2-phenylenediamine

SAZ: Sodiumazide

9AA: 9-Aminoacridine

MMS: Methylmethanesulfonate

2AA: 2-aminoanthracene

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

Conclusions:

Under the tested conditions, the test compound was not mutagenic in any of the four tested Salmonella typhimurium strains (TA 98, TA 100, TA 1535 and TA 1537), nor in the E. coli strain WP2uvrA with and without metabolic activation up to 5000 µg/plate.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 May - 17 Jun 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted Jul 2019

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 30 May 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

adopted Aug 1998

Qualifier:

according to guideline

Guideline:

other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use

Version / remarks:

adopted Nov 2011

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYEI National Institute of Pharmacy and Nutrition, Budapest, Hungary

Type of assay:

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

Target gene:

tk locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA)
- Suitability of cells: Recommended test system in international guidelines
- Doubling time: 10.05 h (8 - 11 h)
- Methods for maintenance in cell culture: Cells were incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in the air.
- Modal number of chromosomes: 40

MEDIA USED
- Type and identity of media: Roswell Park Memorial Institute (RPMI) medium containing penicillin/streptomycin (10000 U/mL and 10 mg/mL, respectively), 0.3 g/L L-glutamine and 0.2 mg/mL pyruvic acid. The medium was supplemented with 5, 10 or 20% heat-inactivated horse serum and 0.5, 0.5 or 0 mg/mL Kolliphor P188, respectively, and used for exposure (RPMI 5), growth and expression (RPMI 10) and selection period (RPMI 20).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with a suspension of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose-range finding study
With and without S9 mix: 5, 10, 20, 50, 100, 300, 625, 1250 and 2500 µg/mL (3 h)
Without S9 mix: 5, 10, 20, 50, 100, 300, 625, 1250 and 2500 µg/mL (24 h)

Experiment I
With S9 mix: 20, 50, 100, 200, 240 and 320 µg/mL (3 h)
Without S9 mix: 4, 8, 16, 24, 32 and 40 µg/mL (3 h)

Experiment II
Without S9 mix: 2.5, 5, 10, 12, 14, 16, 18, 20 and 22 µg/mL (24 h)


The selection of the concentrations used in the main experiments was based on data from the range-finding experiment. Toxicity was noted at ≥ 20 µg/mL after 3 h exposure without S9 mix, at ≥ 100 µg/mL after 3 h exposure with S9 mix and at ≥ 20 µg/mL after 24 h exposure without S9 mix.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: The solubility of the test item in the vehicle was shown in a foregoing range-finding study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 1 x 10E7 cells/tube for 3 h exposure and 6 x 10E6 cells/tube for 24 h exposure in sterile flask (culturing surface 75 cm2)

DURATION
- Exposure duration: 3 and 24 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10 – 11 days

SELECTION AGENT (mutation assays): 300 µg/mL trifluorothymidine

NUMBER OF REPLICATIONS: Duplicate cultures in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency and relative total growth

OTHER: Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations.

Evaluation criteria:

A test item is considered to be clearly positive (considered to be able to induce mutation in this test system), if the following criteria are met:
- The assay is valid (please refer to “acceptability criteria” under “Any other information on materials and methods incl. tables”).
- The increase in mutant frequencies above the concurrent background exceeds the Global Evaluation Factor (GEF) and the increase is concentration-related.

A test item is considered to be negative (considered to be unable to induce mutations in this test system), if the following criteria are met:
- The assay is valid (please refer to “acceptability criteria” under “Any other information on materials and methods incl. tables”).
- There is no concentration-related response or, if there is an increase in mutant frequencies, it does not exceed the GEF.
- When there is no culture showing an relative total growth (RTG) value between 10-20%, but there is no evidence of mutagenicity in the 20-100% RTG range, and there is at least one data point between 20-25% RTG or there is no evidence of mutagenicity in the 25-100% RTG range and there is also a negative data point slightly below 10% RTG.

Statistics:

For defining positive and negative responses, the biological relevance of the increased mutant frequency (MF) was considered first. Instead of statistical analysis the evaluation of the test relies on the use of a predefined induced mutant frequency (i.e. increase in MF above concurrent control), designated the Global Evaluation Factor (GEF), which is based on the analysis of the distribution of the negative control. For this assay the GEF is 126 x 10E6. However, as an additional analysis the heterogeneity of the obtained data was tested and the statistical analysis of mutant frequencies (total wells with clones) was carried out by analysis of variance (ANOVA) and Dunnett’s Test (2-sided, α = 0.05) by IBM® SPSS® Statistics, Version 25 (2017) statistical software program. The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: The pH and osmolality values (± S9) were within acceptable ranges.
- Precipitation: The cell growth caused slight opalescence in the cultures, however, precipitate was not observed in the cell suspensions after the 3 h and 24 h treatments in the cultures.

RANGE-FINDING/SCREENING STUDIES:
Solubility and cytotoxicity were determined in a preliminary range-finding study. L5178Y cells were treated with test item concentrations of 5, 10, 20, 50, 100, 300, 625, 1250 and 2500 µg/mL for 3 h in the presence and absence of S9 mix and for 24 h in the absence of S9 mix, respectively. Toxicity was observed at ≥ 20 µg/mL after 3 h exposure without S9 mix, at ≥ 100 µg/mL after 3 h exposure with S9 mix and at ≥ 20 µg/mL after 24 h exposure without S9 mix. Slightly opalescent suspension was noted at 100 µg/mL in the absence of S9 mix and opalescent or slightly opalescent suspension was noted at ≥ 300 µg/mL in the presence and absence of S9 mix.

HISTORICAL CONTROL DATA
- Positive historical control data:
NQO, 3h (-S9 mix): mean ± SD: 843.85 ± 143.85 (range 483.42-1070.89)
CP, 3h (+S9 mix): mean ± SD: 1227.13 ± 245.52 (range 901.06-1887.55)
CP, 24 h (-S9 mix): mean ± SD 848.19 ± 137.58 (range 566.84-1129.54)

- Negative (solvent/vehicle) historical control data:
DMSO, 3h (-S9 mix): mean ± SD: 87.51 ± 18.65 (range 48.71-126.30)
DMSO, 3h (+S9 mix): mean ± SD: 91.59 ± 19.46 (range 51.42-131.75)
DMSO, 24 h (-S9 mix): mean ± SD: 92.81 ± 21.80 (range 46.36-139.26)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was observed after 3 h of exposure in the presence of S9 mix at ≥ 8 µg/mL, after 3 h of exposure in the absence of S9 mix at 100 µg/mL and at ≥ 5 µg/mL after 24 h exposure without S9 mix.
After 3 h exposure without S9 mix relative total growth (RTG) was depressed by 32%, 66% and 86% at 8, 16 and 24 µg/mL, respectively. At 32 and 40 µg/mL the high cytotoxicity did not allow plating of these for doses for mutagenicity and cloning efficiency.
After 3 h exposure with S9 mix RTG was reduced to 51%, 78% and 87% at 100, 200 and 240 µg/mL, respectively. At 320 µg/mL high cytotoxicity was observed (results were excluded from final conclusion)
After 24 h exposure without S9 mix, RTG was depressed by 33%, 67%, 85%, 93%, 100% and 100% at 5, 10, 12, 14, 16 and 18 µg/mL, respectively. 12 µg/mL was considered as highest evaluable and valid concentration level for evaluation.

Table 1: Experimental results

Concentration [µg/mL]	SG	RTG	CEV	MF (x10E-6)	GEF
3 h treatment without S9 mix
DMSO	9.01	100.00	91.54	84.56	210.60
4	7.50	97.93	107.62	91.53
8	4.96	67.83	112.86	81.84
16	3.13	34.46	90.81	113.61
24	1.63	14.21	72.11	161.49
32^##	0.12	^#	^#	^#
40^##	0.05	^#	^#	^#
NQO (0.2 µg/mL)	5.85	47.98	67.59	736.4
3 h treatment with S9 mix
DMSO	11.73	100.00	98.17	101.69	227.70
20	10.39	100.00	111.98	107.41
50	10.24	86.94	97.82	103.33
100	6.04	49.42	94.21	131.84
200	2.25	21.69	111.02	148.41
240	1.66	12.93	89.62	162.21
320^##	0.58	5.75	114.36	193.54^##
CP (5 µg/mL)	4.14	11.47	31.92	1332.89
24 h treatment without S9 mix
DMSO	40.56	100.00	100.44	109.14	235.10
2.5	32.60	87.68	109.57	133.20
5	24.35	67.30	112.57	153.90
10	14.07	32.88	95.21	194.35
12	5.93	14.51	99.63	213.60
14^##	2.78	6.99	102.45	273.23^##
16^##	0.47	1.08	93.27	463.18^##
18^##	0.31	0.20	25.60	1099.06^##
20^##	^#	^#	^#	^#
22^##	^#	^#	^#	^#
NQO (0.1 µg/mL)	5.36	10.29	78.16	592.56
SG: suspension growth; RTG: relative total growth; CEV: cloning efficiency; MF: mutant frequency; GEF: global evaluation factor #: data could not be calculated; ##: excluded from the evaluation (cytotoxicity > 90%) NQO: 4 -nitroquinoline-n-oxide CP: cyclophosphamide

Conclusions:

Under the experimental conditions of the gene mutation assay the test item did not induce gene mutations at the TK locus in L5178Y mouse lymphoma cells with and without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in bacteria

A bacterial gene mutation assay with the test substance was performed in accordance with OECD guideline 471 and in compliance with GLP (Vertesi, 2019a). In two independent experiments, the four Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2uvrA were exposed to the test item, positive or vehicle control both in the presence and absence of metabolic activation. In the first experiment the plate incorporation method was applied, in the second run the pre-incubation method was used. Based on the results of a preliminary range finding study, test item concentrations of 16, 50, 160, 500, 1600 and 5000 µg/plate were chosen. No signs of toxicity were observed up to and including the limit concentration. The mean number of revertant colonies and the development of background lawn were used to evaluate the mutation rate of the test compound. Positive and vehicle controls were in the range of historical data and confirmed the validity of the test system. The mean number of revertant colonies observed for the test item was comparable to the number of revertant colonies of the vehicle controls, with and without metabolic activation. Under the condition of this experiment there is no mutagenic potential towards the selected S. typhimurium and E. coli strains.

 

In vitro cytogenicity in mammalian cells

The potential of the test item to induce aneugenic and clastogenic effects in mammalian cells in vitro was analysed in a micronucleus test in cultured mouse lymphoma L5178 cells according to OECD guideline 487 and in compliance with GLP (Parmantier, 2019). The cells were exposed to the test item, positive and vehicle controls for 4 h (first experiment) in the presence and absence of metabolic activation (S9) and for 24 h (second experiment) in the absence of metabolic activation. Concentrations were chosen in a way that the highest concentration should reach a maximum cytotoxicity (cytostasis) of 55 ± 5%.

In the first experiment of 4 h treatment, selected concentrations were in the range of 3.9 to 500 µg/mL with and without metabolic activation. Without metabolic activation, cytotoxicity of > 71% was observed at concentrations ≥ 15.6 µg/mL and only two concentrations were valid for analysis. Thus, the 4 h exposure in the absence of metabolic activation was repeated at a lower concentration range of 0.078 to 10 µg/mL. In the second experiment of 24 h treatment in the absence of metabolic activation, the same concentration range was used. In each experiment, two parallel cultures were analysed and at least 2000 binucleated cells per concentration were evaluated for cytogenetic damage.

In both experiments at any concentration, treatment with the test item revealed no relevant increase in the frequency of micronucleated cells, both, in the presence and absence of metabolic activation.

Negative controls (culture medium) and solvent controls (1.0% (v/v) ethanol) showed reproducible frequencies of micronuclei which remained in the range of historic negative control data. Concurrent positive controls (cyclophosphamide (+S9), mitomycin C and colchicine (-S9)) induced a statistically significant increase in micronuclei frequencies when compared to negative/vehicle controls and the response was within the range of historic positive control data. Under the experimental conditions of the study, the test item has no clastogenic or aneugenic potential.

Gene mutation in mammalian cells in vitro

The test item was further tested for its mutagenic potential in mammalian cells in a mouse lymphoma assay in vitro according to OECD guideline 490 (Vertesi, 2019b) and compliant with GLP.Two independent experiments were performed in L5178Y mouse lymphoma cells (thymidine kinase locus). The cells were exposed with the test item, positive or vehicle control for 3 h (first experiment) with and without metabolic activation (S9 mix) and for 24 h (second experiment) in the absence of metabolic activation. Concentrations were selected based on the results of a foregoing solubility and cytotoxicity experiment and in the range of 20 to 320 µg/mL for 3 h exposure in the presence of S9 mix, in the range of 4 to 40 µg/mL for 3 h exposure in the absence of S9 mix and in the range of 2.5 to 22 µg/mL for 24 h exposure in the absence of S9 mix. 

Cytotoxicity was observed after treatment with the test item in all tests, both in the presence and absence of metabolic activation. In addition, the cell growth caused slight opalescence in the cultures, however, precipitate was not observed in the cell suspensions after the 3 h and 24 h treatments in the cultures.

Treatment with the test item did not induce a biologically relevant increase in mutation frequencies. Although a slight dose-related increase in mutation frequencies was noted at both, in the presence and absence of metabolic activation, the values remained within historical control data and were unequivocally below the Global Evaluation Factor (GEF).

The mutation frequency of the vehicle control (DMSO) was within the range of historical control data. The positive control substances cyclophosphamide (+S9 mix) and 4-nitroquinolone-n-oxide (–S9 mix) significantly increased the mutation frequency in L5178Y cells, thus demonstrating the sensitivity and validity of the test system.

Based on the experimental results, there was no evidence for a mutagenic potential of the test item in L5178Y cells, neither in the presence nor in the absence of metabolic activation. Thus, the test item was considered negative for mutagenicity in mammalian cells.

Conclusion

Three in vitro studies on genetic toxicity are available, one bacterial reverse mutation assay (Ames test), an in vitro cytogenicity assay (micronucleus assay) in mammalian cells and a gene mutation assay in mammalian cells (mouse lymphoma assay). All tests revealed negative results. Thus, the test item is considered to be negative for gene mutation in vitro.

Justification for classification or non-classification

The available data on genetic toxicity in vitro of the registered substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.