Cobalt disodium ethylenediaminetetraacetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A study according to OECD 471 (Ames-test) was conducted on Co(NH4)EDTA. A read across approach to CoNa2EDTA is reasonable (see the justification provided in section 13.2 of this dossier). No adverse effects occured. Thus the substance is not classified as genetic toxic.

Link to relevant study records

Reference

Reference 1

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 2

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.