copper sulfate;N'-[2-(2-aminoethylamino)ethyl]ethane-1,2-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A positive Ames test is available for a category member.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

REPORTING FORMAT FOR THE CATEGORY APPROACH

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
See the documentation for the category approach.

2. CATEGORY APPROACH JUSTIFICATION (ENDPOINT LEVEL
See the documentation for the category approach.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his C, his D, his G and tryp E

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction, microsome fraction prepared from Sprague Dawley rat liver homogenate, is provided by MOLTOXTM (POB Box 1189 - 157 Industrial Park Dr - Boone, NC 28607 - USA) (S9 Moltox-11101-5-3805 validated on 07.2017 – expiry date: 25.05.2019).

Test concentrations with justification for top dose:

5000, 3000, 1500, 500, 150 and 50 μg.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
for test item: water
for controls: DMSO (Sigma-D5879- SZBG1310V), Acetone (CARLOERBA- P0051010- D7A063237A) and NaCl 0.15M (DUTSCHER- 06981713-3012587)
- Justification for choice of solvent/vehicle:
highly soluble in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine and cis-Platinum (II) Diammine Dichloride

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): 1-9 x10^8 bacteria

DURATION
Plates are incubated at 37° C over a 48-72 hour period.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: number of colonies/plate

Rationale for test conditions:

Protocol test conditions.

Evaluation criteria:

Ensure that the criteria of validity of the study are well respected namely:
 the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
 the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
 the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
 the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
 Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).

Statistics:

no statistics were performed

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at 3000 and 5000 microgram/L

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity compatible with the maximum tolerated 75% in the presence of the higher dose 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity compatible with the maximum tolerated 75% in the presence of the higher dose 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

At 3000 and 5000 microgram/L

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity compatible with the maximum tolerated 75% in the presence of the higher dose 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity compatible with the maximum tolerated 75% in the presence of the higher dose 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity compatible with the maximum tolerated 75% in the presence of the higher dose 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity compatible with the maximum tolerated 75% in the presence of the higher dose 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxicity compatible with the maximum tolerated 75% in the presence of the higher dose 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Sterility controls
1. Test items: Results show the absence of any bacterial growth in the presence of the various concentrations of the test item.
2. "S9-mix": Results show the absence of any bacterial growth in the presence of "S9-mix".

Bacteriostatic activity control
Results show a cytotoxicity compatible with the maximum tolerated 75% in the presence of the higher dose 5000 μg/plate. The test item is tested at these doses (5 000, 1 500, 500, 150 and 50 μg/plate) for the first assay. Due to the result of the first assay (increase in the number of revertant colonies in Salmonella typhimurium TA 1535, TA 98 and TA 100), one supplementary dose of 3000 μg/plate is studied for the second assay in Salmonella typhimurium TA 1535, TA 98 and TA 100.

Mutation assay interpretation
* There is no significant difference between the number of spontaneous reversions, the number of reversions obtained for the positive controls (without and with metabolic activation), and the mean of corresponding experimental historical values obtained in the laboratory.
* There is an increase in the number of revertant colonies in the presence of the higher dose of the test item (5 000 μg/plate) without metabolic activation in Salmonella typhimurium TA 1535, and TA 100.
* Results are confirmed in an independent experiment with the dose of 3 000 μg /plate.

Conclusions:

Solutions of the test item Reaction product of copper sulfate and tetraethylenepentamine BATCH: 170432 (LEMI code: GQQ021117), provided by BMS MICRO-NUTRIENTS NV, induce a mutagenic change in Salmonella typhimurium TA 1535 and TA 100 without metabolic activation, according to the OECD Guidelines n°471.

Executive summary:

Solutions obtained from GQQ021117, have been tested for their capacity to induce reverse mutation in four Salmonella typhimurium strains and one Escherichia coli WP2 (uvr Ā) (pKM101) strain. This study was performed in the absence and presence of metabolic activation. Two independent assays were carried out:

For assay n° 1, various concentrations of were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).

For assay n° 2, various concentrations of were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)).

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental“historical”values obtained in the laboratory.

These results validate the two tests.
There is an increase in the number of revertant colonies in the presence of the higher dose of the test item (5 000 μg/plate) without metabolic activation in Salmonella typhimurium TA 1535 and TA 100. Results are confirmed in an independent experiment with the dose of 3 000 μg /plate.

Conclusion:

Solutions of the test item Reaction product of copper sulfate and tetraethylenepentamine BATCH: 170432 (LEMI code: GQQ021117), provided by BMS MICRO-NUTRIENTS NV, induce a mutagenic change inSalmonella typhimuriumTA 1535 and TA 100 without metabolic activation, according to the OECD Guidelines n°471.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo testing proposal has been submitted.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study planned

Justification for type of information:

TESTING PROPOSAL ON VERTEBRATE ANIMALS: genetic toxicity in vivo
[Please provide information for all of the points below. The information should be specific to the endpoint for which testing is proposed. Note that for testing proposals addressing testing on vertebrate animals under the REACH Regulation this document will be published on the ECHA website along with the third party consultation on the testing proposal(s).]

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out:
Reaction product of copper sulfate and tetraethylenepentamine

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies:
• Etude 2017-GQQ-1 (PH-17/0643): Study according to OECD 471 test, GLP-compliant. Positive without metabolic activation system, negative with metabolic activation system.
- Available non-GLP studies:
• Foureman 1994. Chemical Mutagenesis Testing in Drosophila. IX. Results of 50 Coded Compounds Tested for the National Toxicology Program. OECD 477 study with Triethylenetetramine (constituent of TEPA) for Drosophila. Non-GLP study. Negative.
- Historical human data:
No relevant historical human data was available for this substance.
- (Q)SAR:
• VEGA: Mutagenicity (Ames test) CONSENSUS model 1.0.2.: Mutagenic with a consensus score of 0.3 for 4 models. This was confirmed with the OECD 471 test listed under GLP studies.
• The applicability of QSARs for higher tier mutagenicity endpoints is limited and therefore not relevant.
- In vitro methods:
The available in vitro methods have been considered as part of the tiered approach to testing, as specified in REACH Guidance 7a. Additional in vitro mutagenicity tests are deemed unnecessary because (i) the Ames test was positive, which necessitates further in vivo tests irrespective of other in vitro test results, (ii) the Ames test indicates gene mutation effects and no chromosome effects and (iii) neither Cu nor TEPA are classified for mutagenicity, making chromosome effects unlikely and further in vitro tests for chromosome effects unnecessary.
- Weight of evidence:
“Reaction product of copper sulfate and tetraethylenepentamine” was tested in vitro with the OECD 471 study and returned indications of mutagenicity. QSAR predictions also suggested the genotoxic potential of the substance. As outlined in the Integrated Testing Strategy (ITS) for mutagenicity (ECHA, 2017), an appropriate in vivo somatic genotoxicity study should be performed after any positive in vitro genotoxicity test. No relevant in vivo genotoxicity studies were identified for this or related substances. Following the ECHA testing strategy, an in vivo test is now required to conclude on the mutagenicity of this substance.
- Grouping and read-across:
Copper sulfate is not expected to have genotoxic effects but the genotoxic effects of tetraethylenepentamine (TEPA) are unclear. No suitable in-vivo tests are available for TEPA or similar substances to use as read-across information. The in-vivo test used in the TEPA registration dossier is a read-across from an in-vivo micronucleus test for Triethylenetetramine (TETA). The OECD 471 test for “Reaction product of copper sulfate and tetraethylenepentamine” suggest mutagenic effects while the micronucleus test is used to detect clastogenecity. Therefore, no suitable in-vivo tests have been identified to read-across from.

- Substance-tailored exposure driven testing [if applicable]:
Not applicable.
- Approaches in addition to above [if applicable]:
Not applicable.
- Other reasons [if applicable]:
Not applicable.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
The substance is not classified for carcinogenicity or mutagenicity therefore genetic toxicity testing cannot be waived. As outlined in the Integrated Testing Strategy (ITS) for mutagenicity (ECHA, 2017), “if there is a positive result in any of the in vitro studies from Annex VII or VIII and there are no appropriate results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study should be proposed”. No in vivo genotoxicity data were identified for this substance. The observed mutagenicity effects in bacteria therefore necessitate the consideration of further in vivo testing “as a last resort” (ECHA, 2016).

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed:
To assess the potential to induce gene mutations in vivo, a transgenic rodent gene mutation assay (TGR; OECD Test Guideline 488) is proposed. As specified in the OECD 488 protocol, “an in vivo assay for gene mutations is useful for further investigation of a mutagenic effect detected by an in vitro system”. The principle of the TGR assay is to sample tissues of interest after an administration period, insert genomic DNA in bacterial hosts and subsequently detect mutations in these bacterial hosts. It is proposed to conduct the study in either mice or rats following oral gavage as this is the most likely route of exposure. The glandular stomach (site-of-contact) and the liver (systemic exposure) are proposed as focal tissues.

Qualifier:

according to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

Endpoint conclusion

Endpoint conclusion:

no study available