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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

701-400-4

Molecular formula:

C8H23N5CuSO4

IUPAC Name:

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

Method

Target gene:

his C, his D, his G and tryp E

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain 5

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

Controls

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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

Test results 6

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

Test results 7

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.

Applicant's summary and conclusion

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 Reaction product of copper sulfate and tetraethylenepentamine 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

Reaction product of copper sulfate and tetraethylenepentamine

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.