Dipotassium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cobaltate(2-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Several studies on Cobalt salts and organic Cobalt complexes were conducted. None indicated a genetic toxic effect of EDTA-CoK2.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Principles of method if other than guideline:

NTP-Standard Protocol

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague Dawley rat liver S9 enzymes and cofactor mix

Test concentrations with justification for top dose:

50; 75; 100 µg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S 9: Mitomycin C; + S9: Cyclophosphamide

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: Chinese hamster Ovary (CHO)

Table 1: Results of the Chromosome Aberrations Test for Na3EDTA.
Study Result: Negative
Activation	Trial	Trial Call
No Activation	1	Negative
Induced Rat Liver S9	2	Negative
Trial #:1   Activation: No Activation   Date: 10/17/1984   Harvest Time: 13.5 hrs   Trial Call: Negative
		Dose	Total Cells Examined	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Abs.
		µg/mL		No. of	Abs	% Cells	No. of	Abs	% Cells	No. of	Abs	% Cells	No. of	Abs	% Cells
				Abs.	Per	With	Abs.	Per	With	Abs.	Per	With	Abs.	Per	With
					Cell	Abs.		Cell	Abs.		Cell	Abs.		Cell	Abs.
Abs: Aberrations
Vehicle Control:	Dimethylsulfoxide	10	100	1	0.01	1	0	0	0	1	0.01	1	0	0	0
Positive Control:	Mitomycin C	0.25	100	29	0.29	26	24	0.24	22	5	0.05	5	0	0	0
		1	50	31	0.62	46	24	0.48	44	7	0.14	14	0	0	0
Test Chemical:	Ethylenediamine tetraacetate, trisodium salt (EDTA)	25	100	1	0.01	1	0	0	0	1	0.01	1	0	0	0
		50	100	2	0.02	2	1	0.01	1	1	0.01	1	0	0	0
		75	100	5	0.05	5	3	0.03	3	2	0.02	2	0	0	0
		100	100	1	0.01	1	0	0	0	1	0.01	1	0	0	0
	Trend:			1.106			1.331			0.333
	Probability:			0.134			0.092			0.37
Trial #:2   Activation: Induced Rat Liver S9   Date: 10/31/1984   Harvest Time: 14.0 hrs   Trial Call: Negative
		Dose	Total Cells Examined	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Abs.
		µg/mL		No. of	Abs	% Cells	No. of	Abs	% Cells	No. of	Abs	% Cells	No. of	Abs	% Cells
				Abs.	Per	With	Abs.	Per	With	Abs.	Per	With	Abs.	Per	With
					Cell	Abs.		Cell	Abs.		Cell	Abs.		Cell	Abs.
Abs: Aberrations
Positive Control:	Cyclophosphamide	15	100	55	0.55	40	29	0.29	22	26	0.26	22	0	0	0
Vehicle Control:	Dimethylsulfoxide	10	100	3	0.03	3	2	0.02	2	1	0.01	1	0	0	0
Test Chemical:	Ethylenediamine tetraacetate, trisodium salt (EDTA)	25	100	1	0.01	1	1	0.01	1	0	0	0	0	0	0
		50	100	4	0.04	4	2	0.02	2	1	0.01	1	1	0.01	1
		75	100	4	0.04	4	2	0.02	2	2	0.02	2	0	0	0
		100	100	3	0.03	3	1	0.01	1	2	0.02	2	0	0	0
	Trend:			0.686			-0.156			1.164
	Probability:			0.247			0.562			0.122

Conclusions:

Interpretation of results:
negative

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

The test material represents the EDTA moiety of the registered substance.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

NTP-Standard Protocol

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced male Sprague Dawley rat liver S9 enzymes and cofactor mix

Test concentrations with justification for top dose:

50; 75; 100 µg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S 9: Mitomycin C; + S9: Cyclophosphamide

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: Chinese hamster Ovary (CHO)

Table 1: Results of the Chromosome Aberrations Test for Na3EDTA.
Study Result: Negative
Activation	Trial	Trial Call
No Activation	1	Negative
Induced Rat Liver S9	2	Negative
Trial #:1   Activation: No Activation   Date: 10/17/1984   Harvest Time: 13.5 hrs   Trial Call: Negative
		Dose	Total Cells Examined	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Abs.
		µg/mL		No. of	Abs	% Cells	No. of	Abs	% Cells	No. of	Abs	% Cells	No. of	Abs	% Cells
				Abs.	Per	With	Abs.	Per	With	Abs.	Per	With	Abs.	Per	With
					Cell	Abs.		Cell	Abs.		Cell	Abs.		Cell	Abs.
Abs: Aberrations
Vehicle Control:	Dimethylsulfoxide	10	100	1	0.01	1	0	0	0	1	0.01	1	0	0	0
Positive Control:	Mitomycin C	0.25	100	29	0.29	26	24	0.24	22	5	0.05	5	0	0	0
		1	50	31	0.62	46	24	0.48	44	7	0.14	14	0	0	0
Test Chemical:	Ethylenediamine tetraacetate, trisodium salt (EDTA)	25	100	1	0.01	1	0	0	0	1	0.01	1	0	0	0
		50	100	2	0.02	2	1	0.01	1	1	0.01	1	0	0	0
		75	100	5	0.05	5	3	0.03	3	2	0.02	2	0	0	0
		100	100	1	0.01	1	0	0	0	1	0.01	1	0	0	0
	Trend:			1.106			1.331			0.333
	Probability:			0.134			0.092			0.37
Trial #:2   Activation: Induced Rat Liver S9   Date: 10/31/1984   Harvest Time: 14.0 hrs   Trial Call: Negative
		Dose	Total Cells Examined	Total Aberrations			Complex Aberrations			Simple Aberrations			Other Abs.
		µg/mL		No. of	Abs	% Cells	No. of	Abs	% Cells	No. of	Abs	% Cells	No. of	Abs	% Cells
				Abs.	Per	With	Abs.	Per	With	Abs.	Per	With	Abs.	Per	With
					Cell	Abs.		Cell	Abs.		Cell	Abs.		Cell	Abs.
Abs: Aberrations
Positive Control:	Cyclophosphamide	15	100	55	0.55	40	29	0.29	22	26	0.26	22	0	0	0
Vehicle Control:	Dimethylsulfoxide	10	100	3	0.03	3	2	0.02	2	1	0.01	1	0	0	0
Test Chemical:	Ethylenediamine tetraacetate, trisodium salt (EDTA)	25	100	1	0.01	1	1	0.01	1	0	0	0	0	0	0
		50	100	4	0.04	4	2	0.02	2	1	0.01	1	1	0.01	1
		75	100	4	0.04	4	2	0.02	2	2	0.02	2	0	0	0
		100	100	3	0.03	3	1	0.01	1	2	0.02	2	0	0	0
	Trend:			0.686			-0.156			1.164
	Probability:			0.247			0.562			0.122

Conclusions:

Interpretation of results:
negative

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

The study is well-documented by the publication and thus acceptable for assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

A total of 63 chemicals were tested for mutagenicity in a four-laboratory study (Inveresk Research International; Litton Bionetics, Inc.; New York Medical College and SRI International).

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Remarks:

and TA1538

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

uninduced and Aroclor 1254-induced liver S9 mix of male Fischer 344 rats, B6C3F1 mice, and Syrian hamsters

Test concentrations with justification for top dose:

0.3; 1; 3; 10; 33; 100; 333; 1000; 3333; 10000 µg/plate

Vehicle / solvent:

dest. water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine; 2-2(furyl)-3-(5-nitro-2-furyl)acrylamide

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

A total of 63 chemicals were tested for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, and Escherichia coli WP2 uvrA. The results for the test material trisodium hydrogen ethylenediaminetetraacetate was negative and so the test material was judged nonmutagenic.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

The complex EDTA-CoK2 consist of the organic moiety EDTA, the central atom Co2+ and the sodium ions. As the K+ ions are known to be non-toxic only the EDTA-Co complex must be regarded as toxic. For both, free EDTA and Co2+ compounds well-documented studies are available, which shows their toxicity. In general, the complex with the chelated Co2+ ion should be less toxic than the free Co2+ ion. Based on that and that the complex EDTA-Co has an average stability, a worst-case assessment where the EDTA-CoK2 complex dissociate in its components can be done and so a read-across is possible to free EDTA and other Co2+ compounds.

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

A total of 63 chemicals were tested for mutagenicity in a four-laboratory study (Inveresk Research International; Litton Bionetics, Inc.; New York Medical College and SRI International).

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Remarks:

and TA1538

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

uninduced and Aroclor 1254-induced liver S9 mix of male Fischer 344 rats, B6C3F1 mice, and Syrian hamsters

Test concentrations with justification for top dose:

0.3; 1; 3; 10; 33; 100; 333; 1000; 3333; 10000 µg/plate

Vehicle / solvent:

dest. water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-aminoanthracene; N-methyl-N'-nitro-N-nitrosoguanidine; 2-2(furyl)-3-(5-nitro-2-furyl)acrylamide

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

A total of 63 chemicals were tested for mutagenicity in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, and TA1538, and Escherichia coli WP2 uvrA. The results for the test material trisodium hydrogen ethylenediaminetetraacetate was negative and so the test material was judged nonmutagenic.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The study is well-documented by the publication and thus acceptable for assessment.

Principles of method if other than guideline:

Induction of TK mutations in mouse lymphoma cells (L5178Y/TK+/- to TK-/-). Treatment was for 3 hrs in the absence of metabolic activation

GLP compliance:

not specified

Type of assay:

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

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

soft-agar media containing RPMI-1640, 10% horse serum, no antibiotics, 1mM sodium pyruvate, 0.02% (w/v) Pluronic F-68 and 0.37% Difco Bacto agar

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

5.69 - 57.11 µg/ml
6 cultures treated with metal compound and 3 solvent controls

Vehicle / solvent:

saline

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

not specified

Details on test system and experimental conditions:

3 hours at 37°C treatment, following 48 hours at 37°C expression period
incubation for 7 days in TFT containing medium (4µg/mL)

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

The test substance cobalt dichloride hexahydrate did not induce gene mutations in mouse lymphoma cells at the given concentrations and under the conditions applied in the test.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

The complex EDTA-CoK2 consist of the organic moiety EDTA, the central atom Co2+ and the sodium ions. As the K+ ions are known to be non-toxic only the EDTA-Co complex must be regarded as toxic. For both, free EDTA and Co2+ compounds well-documented studies are available, which shows their toxicity. In general, the complex with the chelated Co2+ ion should be less toxic than the free Co2+ ion. Based on that and that the complex EDTA-Co has an average stability, a worst-case assessment where the EDTA-CoK2 complex dissociate in its components can be done and so a read-across is possible to free EDTA and other Co2+ compounds.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

Induction of TK mutations in mouse lymphoma cells (L5178Y/TK+/- to TK-/-). Treatment was for 3 hrs in the absence of metabolic activation

GLP compliance:

not specified

Type of assay:

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

Target gene:

thymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

soft-agar media containing RPMI-1640, 10% horse serum, no antibiotics, 1mM sodium pyruvate, 0.02% (w/v) Pluronic F-68 and 0.37% Difco Bacto agar

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

5.69 - 57.11 µg/ml
6 cultures treated with metal compound and 3 solvent controls

Vehicle / solvent:

saline

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

not specified

Details on test system and experimental conditions:

3 hours at 37°C treatment, following 48 hours at 37°C expression period
incubation for 7 days in TFT containing medium (4µg/mL)

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

The test substance cobalt dichloride hexahydrate did not induce gene mutations in mouse lymphoma cells at the given concentrations and under the conditions applied in the test.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 18 to 04 June, 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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus

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:

Rat liver S9 tissue fraction

Test concentrations with justification for top dose:

5000, 2500, 1250, 625 and 313 µg/plate.
On the basis of the results obtained in the preliminary toxicity test, 5000 µg/plate was selected as the maximum dose level for the Main assays.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: The test item was found to be soluble at 50 mg/mL. This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.

Untreated negative controls:

yes

Remarks:

Solvent

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Absence of S9

Untreated negative controls:

yes

Remarks:

Solvent

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Presence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in the first main assay a plate-incorporation method was used. In the second a pre-incubation method was used.

DURATION
The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were held at 4°C for 24 hours or immediately scored by counting the number of revertant colonies on each plate.

NUMBER OF REPLICATIONS: three replicate plates were used at each test point.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Solubility test

As indicated by the Sponsor, solubility of the test item was evaluated in a preliminary trial using sterile water fro injection. This solvent is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at 50 mg/mL. This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.

Toxicity test

The test item Cobalt diammonium EDTA was assayed in the toxicity test at a maximum dose level of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate.

No precipitation of the test item was observed at the end of the incubation period at any concentration.

Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

Main Assays

Two Main Assays were performed. The maximum concentration of the test item to be used in the main experiments should be determined taking into consideration cytotoxicity and solubility in the final treatment mixture. On the basis of the results obtained in the preliminary toxicity test, 5000 µg/plate (the upper limit to testing indicated in the Study Protocol) was selected as the maximum dose level for the Main assays.

In Main Assay I, using the plate incorporation method, the test item was assayed at 5000, 2500, 1250, 625 and 313 µg/plate with all tester strains.

No toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation.

As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the same concentrations and including a pre-incubation step for all treatments. Neither toxicity, nor relevant increase in the number of revertant colonies was observed at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period, at any concentration, in any experiment.

The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Analysis of results

Acceptance criteria

The assay was considered valid if the following criteria were met:

1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.

2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.

3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

Criteria for outcome of the assays

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Evaluation

Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data.

The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid.

The test item did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Conclusions:

Not genotoxic

Executive summary:

Method

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy, according to the OECD guideline 471. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone. The test item was used as a solution in sterile water for injection.

Observations

The test item was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period, at any concentration tested, in the absence or presence of S9 metabolism. Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

On the basis of the results obtained in the preliminary toxicity test, inMain Assay I, using the plate incorporation method, the test item was assayed at 5000, 2500, 1250, 625 and

313 µg/plate with all tester strains. No toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation.

As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the same concentrations and including a pre-incubation

step for all treatments. Neither toxicity, nor relevant increase in the number of revertant colonies was observed at any dose level, with any tester strain, in the absence or presence

of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period, at any concentration, in any experiment.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the

absence or presence of S9 metabolism.

Conclusion

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From May 18 to 04 June, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For the detailed read across justification see the document provided in section 13.2 of this dossier.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

assessment report

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine locus

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:

Rat liver S9 tissue fraction

Test concentrations with justification for top dose:

5000, 2500, 1250, 625 and 313 µg/plate.
On the basis of the results obtained in the preliminary toxicity test, 5000 µg/plate was selected as the maximum dose level for the Main assays.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: The test item was found to be soluble at 50 mg/mL. This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.

Untreated negative controls:

yes

Remarks:

Solvent

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Remarks:

Absence of S9

Untreated negative controls:

yes

Remarks:

Solvent

Negative solvent / vehicle controls:

yes

Remarks:

Water

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

Presence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in the first main assay a plate-incorporation method was used. In the second a pre-incubation method was used.

DURATION
The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were held at 4°C for 24 hours or immediately scored by counting the number of revertant colonies on each plate.

NUMBER OF REPLICATIONS: three replicate plates were used at each test point.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Solubility test

As indicated by the Sponsor, solubility of the test item was evaluated in a preliminary trial using sterile water fro injection. This solvent is compatible with the survival of the bacteria and the S9 metabolic activity. The test item was found to be soluble at 50 mg/mL. This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test.

Toxicity test

The test item Cobalt diammonium EDTA was assayed in the toxicity test at a maximum dose level of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate.

No precipitation of the test item was observed at the end of the incubation period at any concentration.

Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

Main Assays

Two Main Assays were performed. The maximum concentration of the test item to be used in the main experiments should be determined taking into consideration cytotoxicity and solubility in the final treatment mixture. On the basis of the results obtained in the preliminary toxicity test, 5000 µg/plate (the upper limit to testing indicated in the Study Protocol) was selected as the maximum dose level for the Main assays.

In Main Assay I, using the plate incorporation method, the test item was assayed at 5000, 2500, 1250, 625 and 313 µg/plate with all tester strains.

No toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation.

As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the same concentrations and including a pre-incubation step for all treatments. Neither toxicity, nor relevant increase in the number of revertant colonies was observed at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period, at any concentration, in any experiment.

The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.

Analysis of results

Acceptance criteria

The assay was considered valid if the following criteria were met:

1. Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.

2. The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.

3. No more than 5% of the plates should be lost through contamination or other unforeseen event.

Criteria for outcome of the assays

For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.

Evaluation

Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data.

The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each strain. No plates were lost through contamination or cracking. The study was accepted as valid.

The test item did not induce two-fold increases in the number of revertant colonies, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.

Conclusions:

Not genotoxic

Executive summary:

Method

The test item was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy, according to the OECD guideline 471. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbital and 5,6-benzoflavone. The test item was used as a solution in sterile water for injection.

Observations

The test item was assayed in the toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. No precipitation of the test item was observed at the end of the incubation period, at any concentration tested, in the absence or presence of S9 metabolism. Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

On the basis of the results obtained in the preliminary toxicity test, inMain Assay I, using the plate incorporation method, the test item was assayed at 5000, 2500, 1250, 625 and

313 µg/plate with all tester strains. No toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation.

As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the same concentrations and including a pre-incubation

step for all treatments. Neither toxicity, nor relevant increase in the number of revertant colonies was observed at any dose level, with any tester strain, in the absence or presence

of S9 metabolism. No precipitation of the test item was observed at the end of the incubation period, at any concentration, in any experiment.

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the

absence or presence of S9 metabolism.

Conclusion

It is concluded that the test item does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

Category 1: Substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans. Substances known to induce heritable mutations in the germ cells of humans.

Categoty 2: Substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests asIn vitromutagenicity tests such as these indicated in 3.5.2.3.8:

- in vitro mammalian chromosome aberration test;

- in vitro mammalian cell gene mutation test;

- bacterial reverse mutation tests

The substance did not create gene mutations in the strains of Salmonella typhimurium and E. Coli under the test conditions, therefore according to the 3.5. of the CLP Regulation EC n.1272/2008, it cannot be classified as mutagenic for germ cells.