Copper, diamminebis(nitrato-kO)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Copper, diamminebis(nitrato-kO)

EC Number:

604-942-9

Cas Number:

15414-04-7

Molecular formula:

CuH6N4O6

IUPAC Name:

Copper, diamminebis(nitrato-kO)

Method

Target gene:

HIS locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

hepatic microsomes from rat livers induced by Aroclor 1254

Test concentrations with justification for top dose:

0, 50, 150, 500, 1500, 5000 µg/plate in the first assay (with and without metabolic activation) and in the second assay (without metabolic activation)
0, 15, 50, 150, 500, 1500, 5000 µg/plate in the second assay (with metabolic activation with pre-incubation)

Justification for top dose: The test item MNP-363 induced no toxicity on the background growth whatever the dose and the strain tested, both with and without metabolic activation, except a slight toxicity in strain TA100 in presence of metabolic activation at the highest dose of 5000 µg/plate. Moreover, marked decreases in the number of revertants were noted when compared to the solvent control in strains TA100 and TA102, both with and without S9-mix. Therefore, the maximum dose retained for the first mutagenicity assay was maintained at 5000 µg/plate in all strains both with and without metabolic activation.

Vehicle / solvent:

sterile water

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-anthramine

Details on test system and experimental conditions:

2 independent assays performed with and without metabolic activation, the second assay with S9-mix being performed according to the pre-incubation protocol due to negative results in the first assay.

Rationale for test conditions:

Test conditions according to OECD guideline 471

Evaluation criteria:

A test item is considered to be clearly positive if, in any of the experimental conditions examined all the following criteria are fulfilled:
- at least one of the test doses exhibits a biologically significant increase (2 or 3 fold increase in the mean number of revertants depending on the strain) compared with the concurrent negative control and,
- the increase is dose-related,
- and the mean numbers of revertants of the test doses are outside the distribution of negative control data

Statistics:

Dunnett's method (Mahon et al, 1989) was used to allow comparison of the mean value for each dose to the mean value for the corresponding solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the 2nd assay with metabolic activation, slight toxicity at 1500 µg/plate and important toxicity at 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the 2nd assay with metabolic activation, slight toxicity at 1500 µg/plate and important toxicity at 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the 2nd assay with metabolic activation, slight toxicity at 1500 µg/plate and important toxicity at 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the 1st assay, slight toxicity at 5000 µg/plate (- S9 mix) and moderate toxicity at 5000 µg/plate (+ S9 mix). In the 2nd assay (+ S9 mix), slight toxicity at 1500 µg/plate and important toxicity at 5000 µg/plate.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the 2nd assay with metabolic activation, slight toxicity at 1500 µg/plate and important toxicity at 5000 µg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the first assay, some statistically significant decreases in the number of revertants were noted in all strains (except in strain TA1537) with and/or without metabolic activation.
In the second assay, in strains TA1537, TA98 and TA102, a very important decrease in the number of revertants was noted at 1500 µg/plate. This dose was thus not exploitable for the assessment of mutagenicity.
To end, some statistically significant decreases in the number of revertants were noted in strains TA98, TA100 and TA102 with and/or without metabolic activation.

Applicant's summary and conclusion

Conclusions:

The mutagenic activity of the test item MNP-363 was assessed by means of the Ames’ test in the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102 tested both in presence and in absence of metabolic activation, in two independent assays according to OECD guideline (OECD 471, 1997), using either the maximum recommended dose, or the highest dose compatible with the toxic activity of the test item (i.e. 1500 or 500 µg/plate).
The validity criteria for the assay were considered as fulfilled. The study is thus valid. Under these experimental conditions, no mutagenic activity was revealed.