Hexamethylene diisocyanate, trimers, reaction products with 2-hydroxyethyl acrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation (OECD 471): negative

Micronucleus test in mammalian cells (OECD 487): negative

Gene mutation in mammalian cells (OECD 490): negative

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:

04 May - 23 Nov 2016

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 in 2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ATCC, CCL-93
- Suitability of cells: The cells were chosen because of their stable karyotype and their low spontaneous induction rate of micronucleus formation under standardized culture conditions.
- Methods for maintenance in cell culture: Thawed cultures were set up in 75 cm² cell culture plastic flasks at 37°C in 5% CO2 atmosphere. 5x10E5 cells per flask were seeded in 15 mL of minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and subcultures were made every 3 - 4 days.

MEDIA USED
- Type and identity of media including CO2 concentration: MEM supplemented with 10% FBS, 100 U/100 µg/mL penicillin/streptomycin solution, 2 mM L-glutamine, 2.5 µg/mL amphotericin and 25 mM HEPES; 5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/ beta-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment: with and without metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000, 1500 and 2000 µg/mL
Experiment I without metabolic activation: 0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 2.5, 5.0, 7.5 and 10 µg/mL
Experiment I with metabolic activation: 25, 50, 100, 120, 140, 160, 180, 200, 225 and 250 µg/mL
Experiment II: without metabolic activation: 0.5, 1.0, 2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 and 7.5 µg/mL

A concentration of 2000 µg/mL was used in the pre-experiment since this is the highest recommended dose according to the guideline used. The concentrations used in the main experiments were based on cytotoxicity of the test item observed in the pre-experiment.

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

other: colchicin

Remarks:

- S9: ethylmethanesulphonate (1200 µg/mL in experiment I, 900 µg/mL in experiment II), colchicin (1.5 µg/mL in experiment I, 0.16 µg/mL in experiment II) + S9: cyclophosphamide (2.5 µg/mL in experiment I)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: approximately 50000 cells per 25 cm² cell culture flask

DURATION
- Preincubation period: approximately 48 h
- Exposure duration: 4 and 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR (cytogenetic assays): cytocalasin B (final concentration: 1.5 µg/mL)

STAIN (for cytogenetic assays): acridine orange solution

NUMBER OF REPLICATIONS: Duplicate cultures for every concentration/ control (except for the pre-experiment).

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: At the end of the cultivation, the complete culture medium was removed. Subsequently, cells were trypsinated and resuspended in medium. The cultures were transferred into tubes and incubated with hypotonic solution (0.4% KCl) for some minutes at room temperature. Cells were then fixed with methanol + glacial acetic acid (3+1) and resuspended gently. The suspension was dropped onto clean glass slides, heat dried and stained with acridine orange.

NUMBER OF CELLS EVALUATED: at least 2000 binucleated cells per concentration (1000 binucleated cells per slide)

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: clearly surrounded by a nuclear membrane, having an area of less than one-third that of the main nucleus, being located within the cytoplasm of the cell, not linked to the main nucleus via nucleoplasmic bridges; mononucleated and multinucleated cells and cells with more than six micronuclei were not considered

DETERMINATION OF CYTOTOXICITY
- Method: cytokinesis block proliferation index from 500 cells

Evaluation criteria:

A test substance was considered positive in the micronucleus test if:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
b) the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
c) any of the results are outside the distribution of the historical negative/solvent control data.
When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system. A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.

Statistics:

non-parametric chi²-test, p < 0.05

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 2.5 µg/mL and above (experiment I, -S9); at 200 µg/mL and above (experiment I, +S9); at 4.0 µg/mL and above (experiment II, -S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item was noted at concentrations of 125 µg/mL and above without metabolic activation and at concentrations of 250 µg/mL and above with metabolic activation.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: see tables 1 and 2

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see tables 1 and 2

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see table 3
- Negative historical control data: see table 3

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Table 1 Results of experiment I (4 h treatment with and without metabolic activation, 24 h fixation period)

Test item	Concentration [µg/mL]	Cytostasis [%]	Relative cell growth [%]	Micronucleated cells frequency [%]
	without metabolic activation
Negative control	0	6	94	0.95
Solvent control	0	0	100	1.35
Test substance	0.5	8	92	0.75
	1.0	16	84	1.25
	2.5	47	53	1.25
EMS	1200	27	73	4.90
Colchicine	1.5	22	78	2.60
	with metabolic activation
Negative control	0	13	87	1.10
Solvent control	0	0	100	1.30
Test substance	25	1	99	0.65
	50	9	91	0.80
	100	12	88	0.70
	200	57	43	0.55
CPA	2.5	49	51	4.25

Negative control = cell culture medium, solvent control = DMSO or ethanol 1% v/v in cell culture medium, positive controls = ethylmethanesulfonate (EMS; without metabolic activation), colchicine (without metabolic activation), cyclophosphamide (CPA; with metabolic activation)

Cytostasis [%] = 100 – Relative Cell Growth [%]

Relative Cell Growth [%] = 100 x ((CBPI(test conc) – 1) / (CBPI(control – 1))

CBPI = Cytokinesis Block Proliferation Indix = ((mononucleate cells x 1) + (binucleate cells x 2) + (multinucleate cells x 3) / total number of cells

 

Table 2 Results of experiment II (24 h treatment without metabolic activation)

Test item	Concentration [µg/mL]	Cytostasis [%]	Relative cell growth [%]	Micronucleated cells frequency [%]
	without metabolic activation
Negative control	0	0	122	1.05
Solvent control	0	0	100	1.10
Test substance	2.0	10	90	0.83
	4.0	40	60	1.30
	6.0	60	40	1.25
EMS	900	54	46	4.55
Colchicine	0.16	15	85	4.60

Negative control = cell culture medium, solvent control = DMSO or ethanol 1% v/v in cell culture medium, positive controls = ethylmethanesulfonate (EMS; without metabolic activation), colchicine (without metabolic activation)

Cytostasis [%] = 100 – Relative Cell Growth [%]

Relative Cell Growth [%] = 100 x ((CBPI(test conc) – 1) / (CBPI(control – 1))

CBPI = Cytokinesis Block Proliferation Indix = ((mononucleate cells x 1) + (binucleate cells x 2) + (multinucleate cells x 3) / total number of cells

 

Table 3 Historical control data

	Negative control		Solvent Control		Positive controls
	Metabolic activation
	without	with	without	with	without (EMS)	without (Colchicine)	with (CPA)
Mean	0.91	1.07	0.88	1.03	4.19	4.21	4.00
SD	0.29	0.38	0.22	0.46	1.42	2.63	1.59
RSD	31.67	35.27	25.30	44.51	33.91	62.33	39.62
Min	0.45	0.50	0.55	0.55	2.25	1.85	2.30
Max	1.50	1.75	1.40	1.83	7.40	14.20	9.85
LCL	0.34	0.31	0.43	0.11	1.35	0.00	0.00
UCL	1.49	1.82	1.32	1.95	7.03	9.47	7.18
n	39	22	11	6	39	39	22

Negative control = cell culture medium, solvent control = DMSO or ethanol 1% v/v in cell culture medium, positive controls = ethylmethanesulfonate (EMS; without metabolic activation), colchicine (without metabolic activation), cyclophosphamide (CPA; with metabolic activation)

Mean: mean number of micronucleated cells (%), SD: standard deviation, RSD: relative standard deviation (%), Min: minimum number of micronucleated cells, Max: maximum number of micronucleated cells, LCL: lower control limit (95%, mean-2SD), UCL: upper control limit (95%, mean+2SD), n: number of assays

Conclusions:

Based on the results of the conducted study the test substance did not exhibit mutagenic properties in vitro.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 - 25 Apr 2016

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 in 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 1998

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and beta-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment/ Experiment I: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Experiment II: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate with and without metabolic activation

5000 µg/plate was chosen as maximal concentration since no toxic effects were observed in the pre-test/experiment I.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

yes

Remarks:

purified water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)

Remarks:

+ S9: 2-AA (2.5 µg/plate in TA 1535, TA 1537, TA 98, TA 100; 10 µg/plate in TA 102) - S9: sodium azide (10 µg/plate in TA 1535, TA 100), 4-NOPD (10 µg/plate in TA 98; 40 µg/plate in TA 1537), methylmethanesulfonate (1 µL/plate in TA 102)

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: number of revertants, clearing or rather diminution of the background lawn

Evaluation criteria:

A test item is considered as mutagenic if a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation. An increase is biologically relevant if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high or in tester strains TA 1535 and TA 1537 at least three times higher than the reversion rate of the solvent control.
A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Statistics:

Mean values and standard deviation were calculated.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II with and without metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
A pre-experiment (plate incorporation method) was performed with the tester strains TA 98 and TA 100 to evaluate the toxicity of the test item and to choose a concentration range for the main experiment I. The test substance was tested with and without metabolic activation at eight concentrations ranging from 3.16 - 5000 µg/plate. Since no toxicity or mutagenicity occured up to the highest concentration the range from 31.6 to 5000 µg/plate was selected for the main experiment. The results of the pre-experiment were reported as a part of the main experiment I.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The historical control data represent 688 - 1285 experiments for TA 1535, TA 1537, TA 98, TA 100 and TA 102 (in 2012 - 2014; see tables 1 and 2).
- Negative (solvent/vehicle) historical control data: The historical control data represent 684 - 1281 experiments for TA 1535, TA 1537, TA 98, TA 100 and TA 102 (in 2012 - 2014; see tables 1 and 2).

Table 1 Historical control data without metabolic activation

Strain		Mean	SD	Min	Max	RSD [%]	n
TA 98	NC	22.3	4.8	13	48	21.6	1159
	4-NOPD	443.7	183.1	192	2213	41.3	1172
TA 100	NC	95.5	18.1	61	182	18.9	1281
	NaN3	704.8	272.7	132	1498	38.7	1285
TA 1535	NC	10.9	5.1	4	35	46.8	1043
	NaN3	858.3	320.2	34	1472	37.3	1042
TA 1537	NC	7.5	2.4	2	27	31.4	1043
	4-NOPD	93.2	27.3	30	273	29.3	1054
TA 102	NC	230.5	47.8	136	415	20.8	684
	MMS	1733.5	408.3	162	3181	23.6	688

NC = negative control, 4-NOPD = 4-nitro-o-phenylene-diamine, NaN₃= sodium azide, MMS = methylmethanesulfonate

Table 2 Historical control data without metabolic activation

Strain		Mean	SD	Min	Max	RSD [%]	n
TA 98	NC	29.9	6.2	13	61	20.9	1157
	2-AA	2318.0	573.6	128	63606	24.7	1156
TA 100	NC	103.4	17.4	68	194	16.8	1286
	2-AA	1839.6	455.1	69	3132	24.7	1284
TA 1535	NC	9.1	3.3	4	34	36.1	1042
	2-AA	100.0	60.6	19	1011	60.6	1041
TA 1537	NC	7.6	2.7	3	31	35.3	1040
	2-AA	218.6	85.2	28	489	39.0	1039
TA 102	NC	290.9	65.3	91	495	22.4	683
	2-AA	663.9	176.6	137	2001	26.6	688

NC = negative control, 2-AA = 2-aminoanthracene

Table 3 Test results of experiment I (plate incorporation)

With or without S9-Mix	Test substance concentration [µg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± standard deviation)
		TA 1535	TA 1537	TA 98	TA 100	TA 102
-	0 (water)	17 ± 5.0	8 ± 3.1	25 ± 7.0	80 ± 6.1	245 ± 6.0
-	0 (DMSO)	22 ± 4.0	7 ± 5.5	16 ± 7.4	76 ± 9.0	209 ± 21.4
-	31.6	16 ± 1.5	8 ± 2.6	17 ± 8.1	54 ± 9.2	245 ± 22.4
-	100	15 ± 3.8	9 ± 0.6	23 ± 6.7	56 ± 3.5	217 ± 12.7
-	316	18 ± 6.0	9 ± 0.6	20 ± 4.0	40 ± 10.0	192 ± 11.2
-	1000	17 ± 1.7	11 ± 2.1	34 ± 4.2	55 ± 11.5	199 ± 11.8
-	2500	17 ± 10.8	9 ± 4.0	26 ± 5.0	79 ± 8.2	187 ± 28.3
-	5000	18 ± 7.2	9 ± 1.0	21 ± 5.1	65 ± 17.0	194 ± 21.0
Positive controls, - S9	NaN3 10 µg/plate	856 ± 24.5			579 ± 35.5
	4-NOPD 10 µg/plate			418 ± 40.4
	4-NOPD 40 µg/plate		115 ± 6.4
	MMS 1 µL					1284 ± 113.5
+	0 (water)	13 ± 5.0	7 ± 2.6	15 ± 5.3	73 ± 10.1	274 ± 33.5
+	0 (DMSO)	12 ± 0.6	9 ± 3.5	16 ± 4.0	72 ± 7.1	255 ± 35.8
+	31.6	10 ± 6.0	13 ± 3.8	15 ± 3.1	92 ± 15.2	286 ± 43.7
+	100	15 ± 4.6	14 ± 3.2	11 ± 1.5	90 ± 1.5	257 ± 14.5
+	316	13 ± 2.1	11 ± 2.0	14 ± 1.5	94 ± 7.2	260 ± 23.9
+	1000	13 ± 7.6	10 ± 2.0	18 ± 0.6	96 ± 6.7	238 ± 12.7
+	2500	11 ± 7.0	9 ± 3.2	17 ± 2.6	85 ± 18.6	238 ± 11.5
+	5000	15 ± 3.2	13 ± 4.2	15 ± 3.8	71 ± 14.0	239 ± 63.8
Positive controls, + S9	2-AA, 2.5 µg/plate	1348 ± 126.9	290 ± 28.1	694 ± 165.7	1348 ± 126.9
	2-AA, 10 µg/plate					691 ± 86.0

NaN₃= sodium azide; 4-NOPD = 4-nitro-o-phenylene diamine; MMS = methylmethanesulfonate; 2-AA =2-aminoanthracene

 

Table 4 Test results of experiment II (pre-incubation)

With or without S9-Mix	Test substance concentration [µg/plate]	Mean number of revertant colonies per plate (average of 3 plates ± standard deviation)
		TA 1535	TA 1537	TA 98	TA 100	TA 102
-	0 (water)	29 ± 7.0	8 ± 0.0	21 ± 2.6	80 ± 10.1	269 ± 6.2
-	0 (DMSO)	19 ± 3.2	8 ± 3.0	16 ± 3.6	67 ± 12.1	243 ± 30.6
-	31.6	20 ± 1.2	9 ± 3.6	26 ± 1.5	64 ± 9.0	291 ± 20.2
-	100	22 ± 11.0	9 ± 3.6	23 ± 4.7	56 ± 3.2	274 ± 20.7
-	316	26 ± 10.0	6 ± 2.3	19 ± 4.4	54 ± 6.2	260 ± 9.3
-	1000	25 ± 2.1	8 ± 0.6	20 ± 2.1	69 ± 9.1	262 ± 29.8
-	2500	30 ± 5.1	10 ± 1.2	19 ± 2.3	68 ± 2.0	247 ± 39.0
-	5000	27 ± 8.1	8 ± 4.9	18 ± 3.5	65 ± 1.5	225 ± 15.4
Positive controls, - S9	NaN3 10 µg/plate	1023 ± 14.4			536 ± 12.2
	4-NOPD 10 µg/plate			463 ± 31.4
	4-NOPD 40 µg/plate		112 ± 5.1
	MMS 1 µL					1862 ± 148.3
+	0 (water)	11 ± 2.6	11 ± 4.0	29 ± 3.6	66 ± 18.0	460 ± 30.0
+	0 (DMSO)	14 ± 2.3	6 ± 1.5	23 ± 1.0	75 ± 14.6	343 ± 19.0
+	31.6	13 ± 5.5	8 ± 2.5	31 ± 5.3	67 ± 7.5	343 ± 9.5
+	100	19 ± 4.6	12 ± 2.1	22 ± 4.6	66 ± 7.6	320 ± 19.1
+	316	16 ± 4.2	8 ± 1.0	23 ± 1.5	67 ± 6.0	335 ± 10.2
+	1000	14 ± 8.3	10 ± 3.1	28 ± 7.6	71 ± 12.3	324 ± 15.4
+	2500	10 ± 1.2	7 ± 3.2	30 ± 3.6	72 ± 5.0	315 ± 36.1
+	5000	12 ± 5.3	9 ± 6.2	25 ± 11.4	76 ± 5.8	327 ± 17.0
Positive controls, + S9	2-AA, 2.5 µg/plate	128 ± 14.8	139 ± 18.1	1185 ± 190.8	1318 ± 108.5
	2-AA, 10 µg/plate					803 ± 62.1

NaN₃= sodium azide; 4-NOPD = 4-nitro-o-phenylene diamine; MMS = methylmethanesulfonate; 2-AA =2-aminoanthracene

Conclusions:

Based on the results of the conducted study the test substance did not exhibit mutagenic properties in bacterial cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Dec 2016 - 31 Jan 2017

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 in 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2008

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

clone TK+/- -3.7.2C

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins Munich cell bank
- Suitability of cells: The cells have been used successfully in in vitro experiments for many years and are characterised by their high proliferation rate and cloning efficiency.
- Cell cycle length, doubling time or proliferation index: 10 - 12 h doubling time
- Methods for maintenance in cell culture: Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.
- Modal number of chromosomes: 40 ± 2

MEDIA USED
- Type and identity of media including CO2 concentration: Complete Culture Medium: RPMI 1640 supplemented with 10% horse serum, 100U/100 µg/mL penicillin/ streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 225 mM HEPES, 2.5 µg/mL amphotericin B; Treatment Medium: same as Complete Culture Medium but with 5% horse serum; Selective Medium: same as Complete Culture Medium but with 20% horse serum and 5 µg/mL TFT; 5% CO2 for all media
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes by culturing cells in RPMI 1640 supplemented with 9.0 µg/mL hypoxanthine, 15.0 thymidine, 22.5 µg/mL glycine and 0.1 µg/mL methotrexate, 5% CO2

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Wistar rats treated with phenobarbital (80 mg/kg bw) and beta-naphthoflavone (100 mg/kg bw)

Test concentrations with justification for top dose:

Pre-experiment:
With and without S9 mix: 7.8, 15.6, 31.3, 62.5, 125, 250, 500, 1000 µg/mL (4 h)

Main experiment:
Without S9 mix: 2.5, 5, 10, 12, 15, 18, 20, 22 µg/mL (4 h)
With S9 mix: 75, 100, 125, 150, 200, 260 µg/mL (4 h)

The selection of the concentrations used in the main experiment was based on data from the pre-experiments.

Vehicle / solvent:

- Vehicle/solvent used: DMSO (1% (v/v))
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL. Based on the results of the solubility test, DMSO was used as solvent.

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO (1% (v/v)

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 3E5 cells/mL

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 48 h
- Selection time: 12 d
- Fixation time: 14 d

SELECTION AGENT (mutation assays): Trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 2 replications each in one experiment (cloning efficiency), 4 replications each in one experiment (mutagenicity)

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

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

Evaluation criteria:

The test substance was considered mutagenic if:
a) the induced mutant frequency meets or exceeds the Global Avaluation Factor (GEF) of 126 mutants per 10E6 cells and
b) a dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the muatant frequency, an increased occurence of small colonies (≥ 40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal abberrations.
A test substance is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Statistics:

non-parametric Mann-Whitney test

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with and without metabolic activation; see tables 1 and 2 under "Any informations on results incl. tables

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value detected with the test substance was within the physiological range.
- Precipitation: No precipitation of the test item was noted in the experiments.

RANGE-FINDING/SCREENING STUDIES: The toxicity of the test substance was investigated in a pre-experiment up to a maximum concentration of 1 mg/mL. This concentration was determined based on a solubility test. Eight concentrations were tested without and with metabolic activation: 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL. The experimental conditions for the pre-experiment were the same as for the main experiment. After a 2 day growth period the relative suspension growth of the treated cell cultures was calculated.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
see table 3

OTHER
-Colony sizing showed no clastogenic effects induced by the test substance.
-The statistical analysis of the results displayed that some of the mutant frequencies were significantly increased over those of the solvent controls, but there was no evidence for a dose-response relationship. Therefore, this effect was considered as not biologically relevant (see table 2).

Table 1 Results of the main experiment without metabolic activation

Concentration [µg/mL]	RCE [%]	RTG [%]	MF [mutants/ 10E6 cells]	IMF [mutants/ 10E6 cells]	GEF exceeded
0 (NC)	101.0	93.0	61.3	/	/
0 (SC)	100.0	100.0	62.4	/	/
2.5	86.4	77.8	73.8	11.4	-
5	98.3	86.9	86.2	23.8	-
10	93.6	62.3	69.0	6.6	-
12	92.1	64.1	84.3	21.9	-
15	89.2	34.4	109.6*	47.2	-
18	81.3	18.4	102.1*	39.8	-
20	90.6	1.8	117.8*	55.5	-
22	87.8	10.3	148.2*	85.8	-
EMS 300 µg/mL	71.2	64.0	767.8*	705.4	+
MMS 10 µg/L	86.4	82.4	434.0*	371.6	+

NC = negative control, SC = solvent control, RCE = relative cloning efficiency, RTG = relative total growth, MF = mutant frequency, IMF = induced mutant frequency, GEF = global evaluation factor, EMS = ethylmethanesulfonate, MMS = methylmethanesulfonate

+ GEF exceeded

- GEF not exceeded

* statistical significant increase in mutant frequency compared to solvent controls (p < 0.05)

 

Table 2 Results of the main experiment with metabolic activation

Concentration [µg/mL]	RCE [%]	RTG [%]	MF [mutants/ 10E6 cells]	IMF [mutants/ 10E6 cells]	GEF exceeded
0 (NC)	97.3	96.4	60.1	/	/
0 (SC)	100.0	100.0	54.6	/	/
75	96.7	82.0	74.2	19.6	-
100	93.6	84.9	62.7	8.1	-
125	81.4	66.0	113.6*	59.0	-
150	87.9	73.6	62.0	7.4	-
200	89.3	46.0	89.5*	34.9	-
250	80.2	13.8	131.4*	76.8	-
260	107.1	10.8	149.5*	94.9	-
B[a]P (2.5 µg/L)	93.6	60.8	551.2*	496.6	+

NC = negative control, SC = solvent control, RCE = relative cloning efficiency, RTG = relative total growth, MF = mutant frequency, IMF = induced mutant frequency, GEF = global evaluation factor, B[a]P = benzo[a]pyrene

+ GEF exceeded

- GEF not exceeded

* statistical significant increase in mutant frequency compared to solvent controls (p < 0.05)

 

Table 3 Historical control data

	Negative control		Solvent Control		Positive controls
	Metabolic activation
	without	with	without	with	without (EMS; 300 µg/mL)	without (MMS; 10 µg/mL)	with (B[a]P; 2.5 µg/mL)
Mean	86.8	84.0	90.2	84.5	721.3	787.5	636.1
Min	50.1	50.1	50.9	50.6	318.7	376.4	303.6
Max	165.8	165.9	167.4	146.4	2919.0	2416.1	1267.2
SD	25.1	24.4	31.6	23.0	213.0	439.4	168.3
RSD [%]	28.9	29.0	35.0	27.3	29.5	55.8	26.5
n	437	643	68	68	206	249	253

Negative control = cell culture medium, solvent control = DMSO 1% v/v in cell culture medium, positive controls = ethylmethanesulfonate (EMS; without metabolic activation), methylmethanesulfonate (MMS; without metabolic activation), benzo[a]pyrene (with metabolic activation)

Mean: mean of mutant frequency [mutants/10E6 cells], Min: minimum of mutant frequency [mutants/10E6 cells], Max: maximum of mutant frequency [mutants/10E6 cells], SD: standard deviation, RSD: relative standard deviation [%], n: number of control values

Conclusions:

Based on the results of the conducted study the test substance did not exhibit mutagenic properties in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reliable in vitro studies regarding genetic toxicity are available for the test substance.

- Bacterial gene mutation:

The potential of the test substance to induce gene mutations in bacteria was assessed in a study performed according to OECD Guideline 471 and in compliance with GLP (Schreib, 2016). The test item was suspended in DMSO, which was also used as solvent control. Purified water served as negative control. Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were treated with the test material for 48 h using the plate incorporation (experiment I) and the pre-incubation (experiment II) method at concentrations ranging from 31.5 to 5000 µg/plate, in triplicate, both with and without liver microsomal activation. No precipitation and no toxic effects of the test item were observed in any tester strain used up to the highest dose group in experiment I and II with and without metabolic activation. The reduction in the number of revertants (below the indication factor of 0.5) observed in strain TA 100 at 316 µg/plate in the absence of metabolic activation was considered as not biologically relevant due to lack of a dose-response relationship. No biologically relevant increases in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Thus, under the conditions of this study, the test substance is considered to be non-mutagenic.

 

- Micronucleus test in mammalian cells:

The potential of the test substance to induce micronuclei in Chinese hamster V79 cells was assessed in a study performed according to OECD Guideline 487 and in compliance with GLP (Donath, 2016). The test item was suspended in DMSO, which was also used as vehicle control. Cell culture medium served as negative control. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h at concentrations of 0.5, 1.0 and 2.5 µg/mL without and 25, 50, 100 and 200 µg/mL with metabolic activation. Precipitation of the test item was observed at 100 µg/mL and above with metabolic activation. The second experiment was performed with a treatment time of 24 h without metabolic activation at 2.0, 4.0 and 6.0 µg/mL. No precipitation of the test item occurred in this experiment. The limit for discrimination between no cytotoxic and a cytotoxic effect is a CBPI value of 70% compared to the solvent control corresponding to 30% of cytostasis. According to this criterion based on laboratory experience no cytotoxicity was observed in experiment I with and without metabolic activation and in experiment II without metabolic activation. No biologically relevant increase of the micronucleus frequency was noted after treatment with the test item in experiment I with and without metabolic activation and in experiment II without metabolic activation. Appropriate reference mutagens, used as positive controls, induced distinct and statistically significant increases of the micronucleus frequency demonstrating the validity of the test system. Thus, under the conditions of this study, the test substance is considered to be non-mutagenic.

 

- Gene mutation in mammalian cells

The potential of the test substance to induce gene mutations in mouse lymphoma L5178Y cells was assessed in a study performed according to OECD Guideline 490 and in compliance with GLP (Schreib, 2017). The test item was suspended in DMSO, which was also used as solvent control. Cell culture medium served as negative control. The main experiment was performed with and without liver microsomal activation and a treatment period of 4 h at concentrations of 2.5, 5, 10, 12, 15, 18, 20 and 22 µg/mL without and 75, 100, 125, 150, 200, 250 and 260 µg/mL with metabolic activation. The selection of the concentrations was based on data from a pre-experiment to evaluate toxicity. Duplicate cultures were used for the determination of the cloning efficiency and four cultures were used for the determination of mutant frequencies. No precipitation of the test item occurred. Cytotoxicity was observed in the experiment without and with metabolic activation as evidenced by a relative total growth of 10.3% and 10.8% for the highest concentration, respectively. No biologically relevant increase of mutants was noted after treatment with the test item with and without metabolic activation. Additionally, colony sizing showed no clastogenic effect induced by the test item. Appropriate reference mutagens, used as positive controls, induced distinct and statistically significant increases of the mutation frequency demonstrating the validity of the test system. Thus, under the conditions of this study, the test substance is considered to be non-mutagenic.

 

Justification for classification or non-classification

The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.