Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

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.

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

Constituent 1
Chemical structure
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 / strainopen allclose all
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
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 resultsopen allclose all
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.

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 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 experimentalhistorical”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.