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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

239-147-2

EC Name:

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

Cas Number:

15096-41-0

Molecular formula:

C10H23Cl4Cr2O5

IUPAC Name:

complexation reaction product of Tetrachloro-μ-hydroxy(μ-methacrylato-O:O')dichromium, isopropylalcohol and water

Test material form:

liquid

Method

Species / 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:

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

Controls

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Any other information on results incl. tables

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

 

Applicant's summary and conclusion

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.