Tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on a bacterial reverse mutation assay according to OECD Guideline 471 the test substance was considered to be non-mutagenic with and without metabolic activation (reference 7.6.1-1).

Based on an in vitro mammalian cell gene mutation assay (V79/HPRT) according to OECD Guideline 476 the test substance did not show a mutagenic potential (reference 7.6.1-2).

Based on chromosomal aberration test in human lymphocytes according to OECD guideline 473 the test item is considered to be non-clastogenic (reference 7.6.1-3)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 July 2017 - 28 August 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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

cf. Sofuni, 1993

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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:

ß-Naphthoflavone/Phenobarbital induced rat liver S9

Test concentrations with justification for top dose:

5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate
top dose: maximum recommended concentration according to OECD guideline 471

Vehicle / solvent:

- Vehicle/solvent used: ultrapure water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

other: daunomycin, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertant colonies

Evaluation criteria:

A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid and
- a biologically relevant increase in the mean number of revertants above a threshold of 2¬fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment - a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration

A test material is defined as negative or non-mutagenic in this assay if
- the assay is considered valid and
- none of the above mentioned criteria are met

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:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

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:

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

Vehicle controls validity:

valid

Untreated 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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: at 5000 µg/plate, only at the beginning of the experiment

Table 1: Summary 1st Series

Metabolic Activation	Test Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	H2O		25 ± 6	125 ± 9	17 ± 3	8 ± 2	29 ± 6
	Test item	5.00	26 ± 5	128 ± 9	22 ± 5	7 ± 4	34 ± 5
		15.8	36 ± 9	139 ± 19	18 ± 1	10 ± 5	37 ± 6
		50.0	22 ± 3	137 ± 14	15 ± 3	9 ± 3	36 ± 5
		158	20 ± 6	134 ± 21	18 ± 6	8 ± 1	41 ± 5
		500	25 ± 4	131 ± 14	20 ± 2	9 ± 4	39 ± 6
		1580	27 ± 4	145 ± 9	19 ± 6	12 ± 4	33 ± 1
		5000	22 ± 7^B	171 ± 13^B	22 ± 4^B	6 ± 1^B	40 ± 8^B
	DAUN	1.00	226 ± 27
	NaN3	2.00		1725 ± 52	906 ± 14
	9-AA	50.0				1235 ± 401
	NQO	2.00					1975 ± 85
With Activation	H2O		41 ± 5	138 ± 6	19 ± 3	10 ± 4	35 ± 6
	Test item	5.00	28 ± 1^C	143 ± 21	19 ± 3	11 ± 3	43 ± 3
		15.8	29 ± 10	149 ± 9	18 ± 15	10 ± 0	33 ± 3
		50.0	35 ± 5	136 ± 14	14 ± 7	9 ± 6	49 ± 4
		158	31 ± 4	152 ± 6	18 ± 4	9 ± 3	36 ± 6
		500	35 ± 5	192 ± 19	16 ± 4	8 ± 3	37 ± 1
		1580	31 ± 8	170 ± 22	20 ± 1	4 ± 2	47 ± 4
		5000	25 ± 4^B	146 ± 19^B	15 ± 1^B	4 ± 3^B	33 ± 2^B
	2-AA	2.00	969 ± 226	1720 ± 57
	2-AA	5.00			153 ± 28	351 ± 18
	2-AA	10.0					377 ± 34

 Key to Positive Controls

NaN3    Sodium azide

2-AA     2-Aminoanthracene

9-AA     9-Aminoacridine

DAUN   Daunomycin

NQO     4-Nitroquinoline-N-oxide

Key to Plate Postfix Codes

B          Precipitation at beginning of experiment

C          Contaminated

 

Table 1: Summary 2nd Series

Metabolic Activation	Test Material	Concentr. [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	WP2 uvrA
Without Activation	H2O		31 ± 11	125 ± 6	20 ± 5	8 ± 3	44 ± 6
	Test item	158	22 ± 5	127 ± 3	29 ± 5	8 ± 1	49 ± 4
		500	37 ± 4	152 ± 7	21 ± 4	7 ± 2	33 ± 4
		1580	24 ± 7	125 ± 14	27 ± 5	8 ± 3	47 ± 9
		2810	24 ± 2	135 ± 17	21 ± 6	8 ± 1	50 ± 6
		5000	20 ± 3^B	138 ± 11^B	26 ± 11^B	7 ± 4^B	51 ± 11^B
	DAUN	1.00	265 ± 4
	NaN3	2.00		1721 ± 43	989 ± 2
	9-AA	50.0				804 ± 190
	NQO	2.00					1988 ± 166
With Activation	H2O		40 ± 3	122 ± 12	17 ± 4	11 ± 5	47 ± 11
	Test item	158	30 ± 5	140 ± 17	17 ± 5	9 ± 3	51 ± 9
		500	39 ± 9	157 ± 15	19 ± 5	11 ± 3	47 ± 10
		1580	35 ± 6	154 ± 12	15 ± 8	11 ± 5	44 ± 5
		2810	25 ± 3	142 ± 12	11 ± 6	4 ± 2	41 ± 4
		5000	29 ± 3^B	128 ± 16^B	11 ± 8^B	8 ± 2^B	43 ± 4^B
	2-AA	2.00	349 ± 55	781 ± 57
	2-AA	5.00			162 ± 21	349 ± 59
	2-AA	10.0					309 ± 7

 Key to Positive Controls

NaN3    Sodium azide

2-AA     2-Aminoanthracene

9-AA     9-Aminoacridine

DAUN   Daunomycin

NQO     4-Nitroquinoline-N-oxide

Key to Plate Postfix Codes

B           Precipitation at beginning of experiment

 

 

Table 3: Historical Data

Negative Controls
Strain	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
S9 Mix	Without	With	Without	With	Without	With	Without	With	Without	With
Compound	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent	Solvent
Total Plates	234	234	229	234	170	170	190	190	190	189
Number of Values	45	45	44	45	29	29	34	34	34	34
Minimum	19	20	94	90	15	6	4	6	22	28
Maximum	52	58	152	151	38	28	11	15	39	45
Mean	38	44	120	125	25	21	8	9	30	37
Standard Deviation	7.3	7.6	12.4	12.5	5.8	4.2	1.6	1.9	5.2	4.4
Positive Controls
Strain	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
S9 Mix	Without	With	Without	With	Without	With	Without	With	Without	With
Compound	DAUN	2-AA	NaN3	2-AA	NaN3	2-AA	9-AA	2-AA	NQO	2-AA
Total Plates	117	117	115	117	85	85	95	95	95	95
Number of Values	45	45	44	45	29	29	34	34	34	34
Minimum	84	112	821	437	351	43	247	88	872	187
Maximum	779	3015	2376	3429	2149	758	1485	705	2275	696
Mean	285	816	1524	1411	869	186	673	299	1704	375
Standard Deviation	155.6	532.0	238.7	736.6	279.1	126.1	291.1	174.9	337.2	140.5

 

Conclusions:

The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.

Executive summary:

The present study was conducted to investigate the test material for its mutagenic potential in a bacterial reverse gene mutation assay in the absence and presence of a rat liver metabolizing system (S9 mix).

The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with (3-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively.

Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.

Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. It was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 June 2017 - 23 August 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:

2016-07-29

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing; Techni-cal University, 64287 Darmstadt, Germany)
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line.
- Doubling time: 12 - 16 h
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37 °C in 75 cm² plastic flasks. About 2-3×10E6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %). The cells were sub-cultured once or twice weekly. All incubations were done at 37 °C with 1.5 % carbon dioxide (CO2) in humidified air.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: For seeding of the cell cultures the complete culture medium was MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10 % FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
- 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:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

The maximum concentration was limited by phase separation visible at the end of the treatment time (4 hours), in accordance with the OECD 476.
Experiment I
4 hours treatment without S9 mix: 0.13; 0.24; 0.31; 0.40 (PS) µL/mL
4 hours treatment with S9 mix: 0.13; 0.24; 0.31; 0.40 (PS) µL/mL

Experiment IA:
without S9 mix: 0.13; 0.24; 0.31; 0.40 (PS); 0.52 (PS) µL/mL

PS= Phase Separation visible to the unaided eye at the end of treatment

Vehicle / solvent:

Solvent/vehicle: deionized water, final concentration in culture medium 10 %
Justification for choice of solvent/vehicle: Solubility and relatively low cytotoxicity in accordance with OECD 476

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

300 µg/mL, without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

2.3 µg/mL, with metabolic activation

Details on test system and experimental conditions:

Two independent experiments were performed. In experiment I the treatment duration was 4 hours with and without metabolic activation. Due to equivocal results in experiment I in the absence of S9 mix, this part of the experiment was repeated under identical experimental conditions (reported as Experiment IA). Two parallel cultures were used throughout the assay.

METHOD OF APPLICATION: in medium
- Cell density at seeding: Two to four days after sub-cultivation stock cultures were trypsinized at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion was stopped by adding complete culture medium with 10 % FBS and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.2 % in saline. Prior to the trypsin treatment the cells were rinsed with PBS. Approximately 0.7 to 1.2×10E7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment IA: 4 hours without metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time: 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1.5x10E6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95 % confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95 % confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Pre-experiment: Not effected (pH 7.45 in the solvent control versus pH 7.01 at 2.0 µL/mL)
- Effects of osmolality: Pre-experiment: No relevant increase (284 mOsm in the solvent control versus 318 mOsm at 2.0 µL/mL)
- Evaporation from medium: Not examined
- Phase separation: determined at 0.40 µL/mL with and without metabolic activation
- Other confounding effects: None


RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 2 mg/mL, 2 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20 % relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation or phase separation should be evaluated at the beginning and at the end of treatment by the unaided eye.
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 0.04 µL/mL and 2 µL/mL were used. The highest concentration was chosen with respect to the current OECD Guideline 476.
In the pre-experiment toxic effects were observed after 4 hours treatment at 0.65 µL/mL and above in the absence of S9 mix and at 2 µL/mL in the presence of S9 mix.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Phase separation occurred at 1.14 µL/mL and above in the presence and absence of metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
The concentrations used in the main experiment were selected based on the data of the pre-experiment. The maximum concentration was 1.14 µL/mL. The individual concentrations were spaced by a factor of 1.3.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.


COMPARISON WITH HISTORICAL CONTROL DATA: Complies


ADDITIONAL INFORMATION ON CYTOTOXICITY:
In experiment I no relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % in both cultures occurred up to the maximum concentration with and without metabolic activation.
In experiment IA no relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % in both cultures occurred up to the maximum concentration without metabolic activation.

Summary Table

Culture I

				relative	relative	rel. adjusted	mutant	95 %
	conc.		S9	cloning	cell	cloning	colonies/	confidence
	µL/mL	PS	mix	efficiency I	density	efficiency I	10E6	interval
				%	%	%	cells
Experiment I without S9 mix
Solvent control with water			-	100.0	100.0	100.0	9.7	1.7 - 30.2
Positive control (EMS)	300.0 µg/mL		-	92.7	81.7	75.7	325.5	1.7 - 30.2
Test item	0.13		-	73.0	125.0	91.3	25.4	1.7 - 30.2
Test item	0.24		-	80.7	87.3	70.5	30.9	1.7 - 30.2
Test item	0.31		-	91.1	83.3	75.9	34.5	1.7 - 30.2
Test item	0.40	PS	-	95.5	80.0	76.4	32.5	1.7 - 30.2
Test item	0.52	PS	-	#	#	#	#	#
Test item	0.67	PS	-	#	#	#	#	#
Test item	0.88	PS	-	#	#	#	#	#
Test item	1.14	PS	-	#	#	#	#	#
Experiment I with S9 mix
Solvent control with water			+	100.0	100.0	100.0	11.2	2.0 - 29.4
Positive control (DMBA)	2.3 µg/mL		+	84.2	93.9	79.0	145.6	2.0 - 29.4
Test item	0.13		+	93.6	78.2	73.2	33.6	2.0 - 29.4
Test item	0.24		+	97.9	75.0	73.4	14.9	2.0 - 29.4
Test item	0.31		+	95.2	89.8	85.5	30.3	2.0 - 29.4
Test item	0.40	PS	+	108.8	83.6	90.9	16.8	2.0 - 29.4
Test item	0.52	PS	+	#	#	#	#	#
Test item	0.67	PS	+	#	#	#	#	#
Test item	0.88	PS	+	#	#	#	#	#
Test item	1.14	PS	+	#	#	#	#	#
Experiment IA without S9 mix
Solvent control with water			-	100.0	100.0	100.0	32.1	1.7 - 30.2
Positive control (EMS)	300.0 µg/mL		-	99.3	104.8	104.0	247.9	1.7 - 30.2
Test item	0.13		-	100.3	72.5	72.8	9.6	1.7 - 30.2
Test item	0.24		-	101.1	93.6	94.6	17.9	1.7 - 30.2
Test item	0.31		-	100.4	88.5	88.9	16.9	1.7 - 30.2
Test item	0.40	PS	-	101.2	68.4	69.2	16.6	1.7 - 30.2
Test item	0.52	PS	-	96.4	77.6	74.8	31.5	1.7 - 30.2
Test item	0.67	PS	-	#	#	#	#	#
Test item	0.88	PS	-	#	#	#	#	#
Test item	1.14	PS	-	#	#	#	#	#

 

Culture II

				relative	relative	rel. adjusted	mutant	95%
	conc.		S9	cloning	cell	cloning	colonies/	confidence
	µL/mL	PS	mix	efficiency I	density	efficiency I	10 exp. 6	interval
				%	%	%	cells
Experiment I without S9 mix
Solvent control with water			-	100.0	100.0	100.0	18.4	1.7 - 30.2
Positive control (EMS)	300.0 µg/mL		-	89.2	77.5	69.1	317.8	1.7 - 30.2
Test item	0.13		-	102.4	86.9	88.9	24.6	1.7 - 30.2
Test item	0.24		-	100.0	79.8	79.8	19.1	1.7 - 30.2
Test item	0.31		-	96.3	80.6	77.7	31.3	1.7 - 30.2
Test item	0.40	PS	-	92.8	63.7	59.1	21.7	1.7 - 30.2
Test item	0.52	PS	-	#	#	#	#	#
Test item	0.67	PS	-	#	#	#	#	#
Test item	0.88	PS	-	#	#	#	#	#
Test item	1.14	PS	-	#	#	#	#	#
Experiment I with S9 mix
Solvent control with water			+	100.0	100.0	100.0	18.4	2.0 - 29.4
Positive control (DMBA)	2.3 µg/mL		+	92.4	98.0	90.5	179.6	2.0 - 29.4
Test item	0.13		+	95.4	86.7	82.7	22.6	2.0 - 29.4
Test item	0.24		+	97.5	77.0	75.1	16.2	2.0 - 29.4
Test item	0.31		+	98.1	97.5	95.7	17.5	2.0 - 29.4
Test item	0.40	PS	+	84.5	87.4	73.8	26.6	2.0 - 29.4
Test item	0.52	PS	+	#	#	#	#	#
Test item	0.67	PS	+	#	#	#	#	#
Test item	0.88	PS	+	#	#	#	#	#
Test item	1.14	PS	+	#	#	#	#	#
Experiment IA without S9 mix
Solvent control with water			-	100.0	100.0	100.0	27.0	1.7 - 30.2
Positive control (EMS)	300.0 µg/mL		-	98.7	72.1	71.1	185.5	1.7 - 30.2
Test item	0.13		-	103.7	86.6	89.8	26.0	1.7 - 30.2
Test item	0.24		-	101.6	110.7	112.5	21.0	1.7 - 30.2
Test item	0.31		-	102.6	79.9	82.0	19.6	1.7 - 30.2
Test item	0.40	PS	-	102.1	78.5	80.2	21.0	1.7 - 30.2
Test item	0.52	PS	-	107.4	68.9	74.0	25.9	1.7 - 30.2
Test item	0.67	PS	-	#	#	#	#	#
Test item	0.88	PS	-	#	#	#	#	#
Test item	1.14	PS	-	#	#	#	#	#

 CE = Cloning efficiency

PS = phase separation visible to the unaided eye at the end of treatment

# culture was not continued to avoid analysis of too many concentrations with phase separation

Statistical analysis:

Linear Regression Analysis
experimental group	p-value
Experiment I, culture I without S9 mix	0.033^s
Experiment I, culture II without S9 mix	0.508
Experiment I, culture I with S9 mix	0.799
Experiment I, culture II with S9 mix	0.520
Experiment IA, culture I without S9 mix	0.895
Experiment IA, culture II without S9 mix	0.467

T-Test
experimental group	p-value
Experiment I without S9 mix, 0.2 µL/mL	0.010^s
Experiment I without S9 mix, 0.3 µL/mL	0.000^s
Experiment I without S9 mix, 0.4 µL/mL	0.008^s
Experiment I with S9 mix, 0.1 µL/mL	0.002^s
Experiment I, with S9 mix, 0.3 µL/mL	0.040^s
Experiment IA without S9 mix, 0.52 µL/mL	0.675

S = significant (p< 0.05)

Conclusions:

In an in vitro mammalian cell gene mutation assay (V79/HPRT) according to OECD Guideline 476, the test substance did not show a mutagenic potential.

Executive summary:

The study was performed to investigate the potential of tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD Guideline 476. The treatment period was 4 hours at concentrations of 0.13, 0.24, 0.31, and 0.40 µL/mL with and without metabolic activation (S9 mix, Phenobarbital/beta-naphthoflavone induced rat liver). No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells.Therefore, it is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31 May 2017 - 05 September 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:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Chromosome Aberration Test in mammalian cells

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: healthy non-smoking donors
- Suitability of cells: Yes. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances.
- Sex, age and number of blood donors: Blood Experiment I: female donor (26 years old), Experiment II: female donor (31 years old)
- Whether whole blood or separated lymphocytes were used : whole blood was used

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Dulbecco's Modified Eagles Medium/Ham's F12; 5.5 % CO2
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 0.01, 0.02, 0.04, 0.07, 0.12, 0.21, 0.37, 0.65, 1.14, 2.00 µL/mL

Without metabolic activation:
Experiment I: 0.01, 0.02, 0.04, 0.07, 0.12, 0.21, 0.37, 0.65, 1.14, 2.00 µL/mL
Experiment II: 0.04, 0.07, 0.12, 0.21, 0.37, 0.65, 1.14, 2.00 µL/mL

Vehicle / solvent:

- Vehicle/solvent used: Deionized water
- Justification for choice of solvent/vehicle: solubility and low cytotoxicity in accordance with OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: about 1.5

NUMBER OF CELLS EVALUATED: 150 per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: in 250 metaphase cells per culture
- Determination of endoreplication: in 250 metaphase cells per culture

Evaluation criteria:

Evaluation of the slides was performed according to the standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" using microscopes with 100 x oil immersion objectives. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison with the controls by counting 1000 cells per culture in duplicate. 150 well-spread metaphases were evaluated per culture for structural aberrations. Only metaphases containing a number of centromeres equal to a number of 46 ± 2 were included in the analysis. Breaks, fragments, deletions, exchanges and chromosomal disintegrations are recorded as structural chromosomal aberrations. Gaps were recorded as well, but they are not included in the calculation of the aberration rates since gaps are achromatic lesions of unknown biological relevance for which a clear relationship to treatment cannot be established. In addition, the number of polyploid cells in 250 metaphase cells per culture (% polyploid metaphases) was evaluated.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Other confounding effects: Phase separation was observed in Experiment I at 0.65 µL/mL and above in the absence of S9 mix and at 0.37 µL/mL and above in the presence of S9 mix. In Experiment II, phase separation was observed at 1.14 µL/mL and above in the absence of S9 mix.

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the main experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index

Table 1: Summary of results

Exp.

Prepa- ration interval

Test item concen- tration in µL/mL

Poly- ploid cells in %

Endo- mitotic cells in %

Mitotic indices in % of control

Aberrant cells in % incl. gaps*

Aberrant cells in % excl. gaps*

Aberrant cells in %  carrying exchanges

Exposure period 4 hrs without S9 mix

I	22 hrs	Solvent control^1	0.0	0.0	100.0	0.3	0.3	0.0
		Positive control^2	n.d.	n.d.	71.8	9.3	9.3^S	1.7
		0.21	0.0	0.0	87.5	1.3	1.3	0.3
		0.37	0.2	0.0	88.7	0.0	0.0	0.0
		0.65^PS	0.2	0.0	94.2	0.7	0.7	0.0

Exposure period 22 hrs without S9 mix

II	22 hrs	Solvent control^1	0.2	0.0	100.0	1.3	1.0	0.0
		Positive control^3	n.d.	n.d.	44.3	17.3	17.0^S	3.7
		0.37	0.0	0.0	100.0	1.7	1.3	0.0
		0.65	0.0	0.0	87.3	1.0	1.0	0.0
		1.14^PS	0.0	0.0	94.5	0.3	0.0	0.0

Exposure period 4 hrs with S9 mix

I	22 hrs	Solvent control^1	0.4	0.2	100.0	1.7	1.7	0.0
		Positive control^4	n.d.	n.d.	77.4	10.7	10.3^S	0.7
		0.12	0.2	0.0	87.9	1.0	1.0	0.0
		0.21	0.4	0.0	109.9	2.3	2.3	0.0
		0.37^PS	0.6	0.2	109.3	1.7	1.7	0.0

*       Including cells carrying exchanges

n.d.  Not determined

^PS    Phase separation occurred at the end of treatment

^S       Aberration frequency statistically significant higher than corresponding control values

¹       Deionized water 10.0 % (v/v)

²       EMS: 825µg/mL

³       EMS: 550 µg/mL

⁴       CPA: 7.5 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.
Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.

Executive summary:

The test item, dissolved in deionized water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation (S9 mix) according to OECD guideline 473.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 150 metaphases per culture were evaluated for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 2.00 μL/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests. 

Phase separation was observed in Experiment I at 0.65 μL/mL and above in the absence of S9 mix and at 0.37 μL/mL and above in the presence of S9 mix. In Experiment II, phase separation was observed at 1.14 μL/mL and above in the absence of S9 mix. No relevant influence on osmolarity or pH was observed. In both experiments, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In this study, neither with nor without metabolic activation, clastogenicity was observed at the concentrations evaluated. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.0 – 0.6 %) as compared to the rates of the solvent controls (0.0 – 0.4 %). In both experiments, either EMS (550 or 825 μg/mL) or CPA (7.5 μg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro.

Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Reverse Mutation Test (reference 7.6.1-1)

A study according to OECD guideline 471 was conducted to investigate the test material for its mutagenic potential in a bacterial reverse gene mutation assay in the absence and presence of a rat liver metabolizing system (S9 mix). The investigations for the mutagenic potential of the test item were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from rats pretreated with (3-Naphthoflavone/Phenobarbital was used. In this study, two experimental series were performed. The S9 mix used contained 10% S9 in the 1st and 20% S9 in the 2nd series, respectively. Solvent and positive control treatments were included for all strains. The mean numbers of revertant colonies were all within acceptable ranges for solvent control treatments, or were clearly elevated by positive control treatments, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. Following treatment of all bacteria tester strains with the test item in the absence and presence of S9 mix, no relevant increases in revertant numbers were observed. It was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic under the experimental conditions described.

HPRT (reference 7.6.1-2)

The study was performed to investigate the potential of tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD Guideline 476. The treatment period was 4 hours at concentrations of 0.13, 0.24, 0.31, and 0.40 µL/mL with and without metabolic activation (S9 mix, Phenobarbital/beta-naphthoflavone induced rat liver). No relevant and reproducible increase in mutant colony numbers/10E6 cells was observed in the main experiments up to the maximum concentration. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Under the experimental conditions reported the test substance did not induce gene mutations at the HPRT locus in V79 cells. Therefore, it is considered to be non-mutagenic in this HPRT assay.

Chromosomal aberration test (reference 7.6.1-3)

The test item, dissolved in deionized water, was assessed for its potential to induce chromosomal aberrations in human lymphocytes in vitro in the absence and presence of metabolic activation (S9 mix) according to OECD guideline 473. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 150 metaphases per culture were evaluated for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index. The highest treatment concentration in this study, 2.00 μL/mL was chosen with respect to the OECD Guideline for in vitro mammalian cytogenetic tests. Phase separation was observed in Experiment I at 0.65 μL/mL and above in the absence of S9 mix and at 0.37 μL/mL and above in the presence of S9 mix. In Experiment II, phase separation was observed at 1.14 μL/mL and above in the absence of S9 mix. No relevant influence on osmolarity or pH was observed. In both experiments, in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed phase separation. In this study, neither with nor without metabolic activation, clastogenicity was observed at the concentrations evaluated. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.0 – 0.6 %) as compared to the rates of the solvent controls (0.0 – 0.4 %).In both experiments, either EMS (550 or 825 μg/mL) or CPA (7.5 μg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosomal aberrations in human lymphocytes in vitro. Therefore, the test item is considered to be non-clastogenic in this chromosome aberration test.

Justification for classification or non-classification

Classification, Labeling, 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. The results indicate that the test substance is non-mutagenic. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EC) No 2017/776.