Copper(2+), bis[N-[amino(imino-.kappa.N)methyl]urea-.kappa.O]-, nitrate (1:2)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-06-04 to 2014-01-09

Reliability:

2 (reliable with restrictions)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

copper(2+) bis(carbamimidoylurea) dinitrate

EC Number:

800-038-5

Cas Number:

1071838-81-7

Molecular formula:

Cu(C2H6N4O)2 (NO3)2

IUPAC Name:

copper(2+) bis(carbamimidoylurea) dinitrate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Target gene:

Thymidine Kinase (TK) locus in L5178Y TK +/- mouse lymphoma cells

Test concentrations with justification for top dose:

Experiments without S9 mix
Using a treatment volume of 1% in culture medium, the selected dose-levels were as follows:
2.34, 4.69, 9.38, 18.8, 37.5 and 75 μg/mL for the first experiment (3-hour treatment)
1.18, 2.35, 4.70, 7.05, 9.40 and 18.8 μg/mL for the second experiment (24-hour treatment).

Experiments with S9 mix
Using a treatment volume of 1% in culture medium, the selected dose-levels were as follows:
4.69, 9.38, 18.8, 37.5, 75 and 150 μg/mL for the first experiment
18.8, 37.5, 75, 100, 150 and 200 μg/mL for the second experiment.

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

L5178Y TK+/- cells are an established cell line recommended by international regulations for the in vitro
mammalian cell gene mutation test. These cells have demonstrated sensitivity to chemical mutagens, a
high cloning efficiency and a stable spontaneous mutant frequency. The average cell cycle time is
10-12 hours and the TK phenotypic expression time is 2 days.
L5178Y TK+/- cells, were obtained from ATCC (American Type Culture Collection, Manassas, USA),
through Biovalley (Marne-La-Vallée, France).
The cells were stored in a cryoprotective medium [10% horse serum and 10% dimethylsulfoxide (DMSO)]
in liquid nitrogen. Each batch of frozen cells was purged of spontaneous TK-/- mutants and checked for the
absence of mycoplasma. The cells were maintained in flasks as suspension culture in RPMI 1640 culture
medium supplemented by heat inactivated horse serum at 10%, v/v, in a 37°C, 5% CO2 humidified
incubator.

Evaluation criteria:

IWGT recommendations (d, e, f) were followed for the determination of a positive result, which should fulfill
the following criteria:
. at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control
(IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10-6,
. a dose-response relationship is demonstrated by a statistically significant trend test.
Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be
confirmed.
Noteworthy increases in the mutation frequency observed only at high-levels of cytotoxicity (Adj. RTG
lower than 10%), but with no evidence of mutagenicity at dose-levels with Adj. RTG between 10 and 20%,
are not considered as positive results.
A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value
between 10 and 20% if (g):
. there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of
mutagenicity in a series of data points between 100 and 20% Adj. RTG,
. there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also
a negative data point between 10 and 1% Adj. RTG.

Statistics:

A trend test was performed to assess the linear trend between the mutation frequency and the dose. This
statistical analysis was performed using SAS Enterprise Guide software (SAS version 9.2).
Only individual mutation frequencies obtained from cultures showing an Adj. RTG ≥ 10% were used in this
analysis.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

in the presence of a rat metabolizing system

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation

Under the experimental conditions of this study, the test item, Copper Guanylurea Nitrate (CuGUN) showed
positive result in the mouse lymphoma assay, in the presence of a rat metabolizing system. In the absence of a rat metabolizing system, the results were inconclusive.

Executive summary:

The objective of this study was to evaluate the potential of the test item, Copper Guanylurea Nitrate (CuGUN), to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells.

Methods: After a preliminary toxicity test, the test item Copper Guanylurea Nitrate (CuGUN), was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Cultures of 20 mL at 5 x 105 cells/mL (3-hour treatment) or cultures of 50 mL at 2 x 105 cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO2 humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once. Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype. The test item was dissolved in dimethylsulfoxide (DMSO).

Results: The cloning efficiencies, the suspension growths and the mutation frequencies of the vehicle controls were

as specified in the acceptance criteria. Moreover, the induced mutation frequencies obtained for the

positive controls met the acceptance criteria specified in the study plan. The study was therefore

considered as valid.

Since the test item was found severely cytotoxic in the preliminary test, the selection of the highest

dose-level for the main experiments was based on the level of toxicity, according to the criteria specified in

the international guidelines (decrease in Adj. RTG).

Experiments without S9 mix:

Using a treatment volume of 1% in culture medium, the selected dose-levels were as follows: . 2.34, 4.69, 9.38, 18.8, 37.5 and 75 μg/mL for the first experiment (3-hour treatment), . 1.18, 2.35, 4.70, 7.05, 9.40 and 18.8 μg/mL for the second experiment (24-hour treatment). At the end of the 3- and 24-hour treatment periods, no precipitate was observed in the culture medium.

Cytotoxicity: Following the 3-hour treatment, a moderate to severe toxicity was induced at dose-levels ≥ 37.5 μg/mL, as shown by a 36-99% decrease in Adj. RTG. Following the 24-hour treatment, a moderate to severe toxicity was noted at dose-levels ≥ 4.70 μg/mL, as shown by a 47-100% decrease in Adj. RTG.

Mutagenicity:

Following the 3-hour treatment, slight Increases in the Mutation Frequency (IMF values up to 47 x 10-6 mutants) were noted up to the dose-level of 37.5 μg/mL which showed a 36% decrease in Adj. RTG. These increases did not reach the GEF of 126 x 10-6 mutants but were dose-related (p = 0.0057). An increase in the MF exceeding the GEF was noted at 75 μg/mL (IMF of 611 x 10-6), however the toxicity induced at this dose-level was too severe (with a mean Adj. RTG at 1%, below the threshold of 10% for an acceptable toxicity level). Thus the corresponding increase in MF was considered not to be biologically relevant.

Following the 24-hour treatment, slight increases in the mutation frequency (IMF values up to 47 x 10-6 mutants) were noted up to the dose-level of 2.35 μg/mL which showed a 47% decrease in Adj. RTG. At the higher tested dose-levels, increases in the MF reaching or exceeding the GEF of 126 x 10-6 were noted. However the toxicity induced at these dose-levels was too severe (mean Adj. RTG below the threshold of 10% for an acceptable toxicity level) and therefore the mutation frequencies obtained cannot be taken into account in the evaluation, leading to only two dose-levels for the results interpretation.

Since none of the dose-levels tested in the first and second experiment induced a cytotoxicity close to the recommended level (i.e. 10 to 20% of Adj. RTG), it was suggested to undertake a third experiment using the longest treatment period of 24 hours and a narrower range of dose-levels. But it is important to notice that, for the test item, the relationship between concentration and toxicity is characterized by a steep slope such that small variance in concentration has a large impact on toxicity making it difficult to achieve the recommended level of toxicity. Therefore, it was decided by the Sponsor not to perform a third experiment since the probability to obtain dose-levels producing the recommended level of toxicity was low.

The overall results obtained in the absence of S9 mix were considered as inconclusive.

Experiments with S9 mix:

Using a treatment volume of 1% in culture medium, the selected dose-levels were as follows: 4.69, 9.38, 18.8, 37.5, 75 and 150 μg/mL for the first experiment,

18.8, 37.5, 75, 100, 150 and 200 μg/mL for the second experiment. No precipitate was observed in the culture medium at the end of the treatment periods.

Cytotoxicity

In the first and second experiments, a moderate to severe toxicity was induced at dose-levels ≥ 75 μg/mL, as shown by a 44-100% decrease in Adj. RTG.

Mutagenicity

In the first experiment, an increase in the mutation frequency was observed. This increase exceeded the GEF (IMF values up to 531 x 10-6 mutants) at dose-levels > 75 μg/mL, inducing acceptable levels of cytotoxicity (44 and 83% decrease in the Adj. RTG). Moreover a dose-response relationship was demonstrated (p<0.0001) at dose-levels inducing acceptable levels of cytotoxicity (i.e. < 90% decrease in the Adj. RTG). Consequently, these results met the criteria of a positive response.

In the second experiment, increases in the mutation frequency were observed up to the dose-level of 75 μg/mL which showed a 65% decrease in Adj. RTG. These increases did not reach the GEF of 126 x 10-6 mutants but were dose-related (p = 0.0056). Increases in the MF exceeding the GEF of 126 x 10-6 was noted at higher tested dose-levels (IMF up to 259 x 10-6), however the toxicity induced at these dose-levels was too severe (with a mean Adj. RTG below the threshold of 10% for an acceptable toxicity level). Thus the corresponding increase in MF was considered not to be biologically relevant.

Since dose-related increases in the MF were observed in both experiments and since the GEF was exceeded in the first experiment at dose-levels showing acceptable toxicity levels, the overall results were considered to meet the criteria of a positive response.

N.B.: No precipitate was observed in the culture medium at the end of the treatment periods and

positive genotoxic effects were observed at a maximum concentration 20-fold higher than the solubility limit but all results obtained at the solubility limit (10 μg/mL) were negative.