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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

According to 1907/2006 (REACh) regulation, three in vitro genetic toxicity tests were performed:

- Bacterial Reverse Mutation Assay (OECD guidelines 471, EU method B.13/14) with Salmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 and Escherichia colistrain WP2uvrA (with and without metabolic activation); no toxicity and no mutagenicity was observed at the highest concentration of 5 mg/plate.

- In Vitro Mammalian Cell Micronucleus Test (OECD guidelines 487, EU method B.49) with mouse lymphoma L5178Y TK^+/-cells (with and without metabolic activation); no toxicity and no mutagenicity was observed at the highest concentration of 100 mg/mL.

- In Vitro Mammalian Cell Gene Mutation Test (OECD guidelines 476, EU method B.17) with mouse lymphoma L5178Y TK^+/-cells (with and without metabolic activation)up to 75μg/mL (3-h treatment) or 18.8μg/mL (24 -h treatment) without S9 mix and up 150μg/mL (3 -h treatment) or 200 µg/mL (24 -h treatment) with metabolic activation, showed mutagenic activity in the presence of a rat metabolizing system at concentration ≥ 75μg/mL in both experiments. In the absence of a rat metabolizing system, the results were inconclusive.

Link to relevant study records

Reference

Reference 1

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)

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

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.

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'.

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: other: chromosomal damage

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-23 to 2013-09-05

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: OECD guideline 487

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test concentrations with justification for top dose:

Experiments without S9 mix :
1.56, 3.13, 6.25, 9.38, 12.5, 18.8, 25, 37.5 and 50 µg/mL for the first experiment,
0.391, 0.781, 1.56, 3.13, 6.25, 9.38, 12.5 and 18.8 µg/mL for the second experiment,
3.13, 6.25, 9.38, 12.5, 18.8, 25, 37.5, 50 and 75 µg/mL for the third experiment.
Experiments with S9 mix: 12.5, 25, 37.5, 50, 62.5, 75, 87.5 and 100 µg/mL for both experiments

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: according to available solubility data since it allowed obtaining homogeneous suspensions of test item (to the naked eye)

Details on test system and experimental conditions:

TEST SYSTEM: L5178Y TK+/- cells which are an established cell line recommended by international regulations for in vitro mammalian cell gene mutation test were used in this study since they are suitable to reveal chemically induced micronuclei. The average cell cycle time is approximately 10-12 hours.

METHOD OF APPLICATION: in medium (RPMI 1640 medium containing 10% inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL))

DOSE FORMULATION: The test item was suspended in the vehicle at concentrations of:
-250 mg/mL for the first preliminary toxicity test,
-10 mg/mL for the second preliminary test and for each experiment of the main test.
The stock suspensions and further dilutions were prepared within the 4 hours before use, and then kept at room temperature and protected from light until use.

DURATION:
Without S9 mix: 3 h treatment + 24 h recovery (first et third experiment), 24 h treatment + 20 h recovery (second experiment, since the results of the first experiment were equivocal)
With S9 mix: 3 h treatment + 24 h recovery (both experiments)

SELECTION AGENT (mutation assays): no
SPINDLE INHIBITOR (cytogenetic assays): no
STAIN (for cytogenetic assays): 15 min 5% giemsa

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells per dose and 1000 monocucleated cells for the vehicle and positive control

DETERMINATION OF CYTOTOXICITY
- Method: population doubling (PD) is the log of the ratio of the final count at the time of harvesting (N) to the starting count (N0), divided by the log of 2.

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Other: no

OTHER:

Evaluation criteria:

Acceptance criteria
This study was considered valid if the following criteria are met:
-the Population Doubling (PD) of each vehicle control culture had to be ≥ 1 (indicating that cells have undergone mitotis),
-the mean background frequency of micronucleated cells in the vehicle control cultures should be within the historic negative control range for the Laboratory (Appendix 2),
-a statistically significant increase in the frequency of micronucleated cells had to be obtained in the positive controls over the background frequency of the vehicle control cultures.

Evaluation criteria
The biological relevance of the results should be considered first. Statistical methods are used as an aid in evaluating the test results but should not be the only determinant of a positive response. A result is considered as positive if at least a 2.5-fold increase in the number of micronucleated cells in comparison to the concurrent control is observed, with a statistically significant difference, at one or more concentrations. Concentration-related increases in the frequency of micronucleated cells and comparison to the vehicle control historical data should also be taken into account.

Statistics:

The statistical comparison was performed using the X2 test, in which p = 0.05 was used as the lowest level of significance.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: At 2500 µg/mL, the pH of the culture medium was approximately 7.4 (as for the vehicle control) .
- Effects of osmolality: at 2500 µg/ml, the osmolality was equal to 446 mOsm/kg H2O (466 for the vehicle control)
- Water solubility: According to available solubility data, a precipitate was expected in the culture medium at the dose-level of 2500 µg/mL. Thus, using a test item concentration of 250 mg/mL in the vehicle and a treatment volume of 1% in the culture medium, the highest selected dose-level was 2500 µg/mL.
- Precipitation: At the end of the treatment periods (3- and 24-hour treatments), a precipitate was observed in the culture medium at dose-levels ≥ 1250 µg/mL.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: strain/cell type: L5178Y TK+/- cells

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions of the study, the test item, CuGUN, did not induce any chromosome damage, or damage to the cell division apparatus, in cultures mammalian somatic cells, using L5178Y TK +/- mouse lymphoma cells, either in the presence or in absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item, Copper Guanylurea Nitrate (CuGUN), to induce an increase in the frequency of micronucleated cells, in L5178Y TK+/- mouse lymphoma cells.

After two preliminary toxicity tests, the test item Copper Guanylurea Nitrate (CuGUN), suspended in dimethylsulfoxide, was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

Without S9 mix: 3 h treatment + 24 h recovery ( First experiment)

Without S9 mix: 24 h treatment + 20 h recovery (Second experiment)

With S9 mix: 3 h treatment + 24 h recovery (First experiment)

With S9 mix: 3 h treatment + 24 h recovery (Second experiment)

Since the equivocal result observed in the first experiment without S9 mix was not confirmed during the second experiment (using a longer treatment duration), its reproducibility was checked in a third experiment (3-hour treatment without S9 mix). Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.

Results With two minor exceptions which were not considered to impact the validity or the integrity of the results, the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria, and positive controls showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid. Since the test item was found to be cytotoxic in the preliminary tests, the highest dose-level selected for the main experiment was based on the level of toxicity, according to the criteria specified in the international guidelines. Experiments without S9 mix The dose-levels selected for treatments were as follows: . 1.56, 3.13, 6.25, 9.38, 12.5, 18.8, 25, 37.5 and 50 μg/mL for the first experiment, . 0.391, 0.781, 1.56, 3.13, 6.25, 9.38, 12.5 and 18.8 μg/mL for the second experiment, . 3.13, 6.25, 9.38, 12.5, 18.8, 25, 37.5, 50 and 75 μg/mL for the third experiment.

Cytotoxicity Following the 3-hour treatment + 24-hour recovery period in the first experiment, a slight toxicity was noted at the highest tested dose-level of 50 μg/mL, as shown by a 31% decrease in the PD. Following the 24-hour treatment + 20-hour recovery period (second experiment), a severe toxicity was noted at dose-levels ≥ 6.25 μg/mL, as shown by a 100% decrease in the PD. Following the 3-hour treatment + 24-hour recovery period in the third experiment, a slight toxicity was noted at 3.13 μg/mL as shown by a 31% decrease in the PD and a moderate to severe toxicity was noted at dose-levels ≥ 25 μg/mL, as shown by a 42 to 100% decrease in the PD.

Micronucleus analysis The dose-levels selected for micronucleus analysis were as follows: . 18.8, 37.5 and 50 μg/mL for the first experiment, the latter being the highest tested dose-level, . 0.781, 1.56 and 3.13 μg/mL for the second experiment, the latter inducing a 15% decrease in the PD and higher dose-levels being too cytotoxic, . 12.5, 18.8 and 25 μg/mL for the third experiment, the latter inducing a 42% decrease in the PD and higher dose-levels being too cytotoxic.

A slight increase in the frequency of micronucleated cells was observed in the first experiment at the highest tested dose-level of 50 μg/mL. This increase exceeded the positive threshold of 2.5-fold the vehicle control value (5.5-fold), and was statistically significant in comparison to the vehicle control (p < 0.05). Since the frequency of micronucleated cells remained close to the historical data range (6‰ versus [0.5‰ - 3.5‰]), these results were considered to be equivocal and the second experiment was undertaken using longer treatment duration. No noteworthy increase in the frequency of micronucleated cells was observed in the second experiment. Since the equivocal result observed in the first experiment was not confirmed during the second experiment (using a longer treatment duration), its reproducibility was checked in a third experiment. No increase in the frequency of micronucleated cells was observed in the third experiment, despite that a higher cytotoxicity level was reached at the highest selected dose-level (42% decrease in the PD in comparison to the vehicle control). Since the equivocal results obtained in the first experiment were neither confirmed in the second experiment, nor reproduced in the third experiment, they were not considered to be biologically relevant.

Experiments with S9 mix The dose-levels selected for treatments were 12.5, 25, 37.5, 50, 62.5, 75, 87.5 and 100 μg/mL for both experiments. Cytotoxicity In the first experiment, a slight to severe toxicity was noted at dose-levels ≥ 25 μg/mL as shown by a 26 to 100% decrease in the PD. In the second experiment, a moderate to severe toxicity was noted at dose-levels ≥ 62.5 μg/mL as shown by a 60 to 100% decrease in the PD. Micronucleus analysis The dose-levels selected for micronucleus analysis were as follows: . 12.5, 25 and 50 μg/mL for the first experiment, the latter induced only 28% decrease in the PD but was just above a dose-level inducing a 50% decrease in the PD, . 25, 50 and 62.5 μg/mL for the second experiment, the latter inducing a 60% decrease in the PD.

No increase in the frequency of micronucleated cells was observed in the first experiment. In the second experiment, slight increases in the frequency of micronucleated cells were observed, exceeding the 2.5-fold threshold at all analysed dose-levels (up to 7-fold). At the highest analyzed dose-level, the increase in the frequency of micronucleated cells was statistically significant (p <0.05).

Since this effect was not reproducible (no similar increase being observed in the first experiment) and since the highest frequency obtained (i.e. 4‰ at 62.5 μg/mL) remained within the corresponding historical data range [0.5‰ –5‰], it was not considered to be biologically relevant, but only attributed to the particularly low frequency obtained in the vehicle control (i.e. 0‰).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 11 - September 16 , 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Only duplicates and no justification for it. No statistical analysis and no standard deviation. Justification of solution stability is not tested adequately.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only duplicate plates were used intead of triplicates. No statistical analysis and no standard deviation. Justification of solution stability is not tested adequately.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared from the liver of rats. The S9 was stored below -80°C until use and thawed just before use. S9 mix (1 ml) = 8 μmol MgCl2+33 μmol KCl+5 µmol Glucose-6-phosphate+4 μmol NADPH+4 μmNADH+100 μmol sodiumphosphate buffer (pH 7.4)+0.1 mL S9

Test concentrations with justification for top dose:

The final numbers of prepared viable cells are (×109cells/mL):
TA100: Dose-range finding test: 2.6; Main test: 2.6
TA1535: Dose-range finding test: 2.6; Main test: 2.6
WP2uvrA: Dose-range finding test: 4.1; Main test: 4.0
TA98: Dose-range finding test: 2.6; Main test: 2.5
TA1537: Dose-range finding test: 2.5; Main test: 2.4

Dose-range finding test:
A total of six doses including 5000 μg/plate as the highest dose and five lower doses diluted with a geometric progression of 4 were set (1250; 313; 78.1; 19.5; 4.88 µg/plate).
Main test:
The highest dose was selected at 5000 μg/plate at which the bacterial growth inhibition was observed and five lower doses of 2500, 1250, 625, 313 and
156 μg/plate diluted with a geometric progression of 2 were employed in the main test.

Vehicle / solvent:

DMSO (Dimethyl sulfoxide)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide 5AF-2), 2-Methoxy-6-chloro-9-[3-(2-chloroethyl)aminopropylamino]acridine·2HCl (ICR-191), 2-Aminoanthracene (2AA)

Remarks:

2AA was the only one positive controle for all strains with S9 activation.

Evaluation criteria:

The numbers of the revertant colonies above twice that in the negative controls to be a positive results.

Statistics:

No statistics was used.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

yes, but only at the highest concentration (5mg/plate)

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

Additional information on results:

The mutagenicity of the test substance was judged to be negative because the number of revertant colonies in the test substance treatment groups was less than twice that in the negative control for all tester strains regardless of the absence or presence of S9 mix.
The numbers of the revertant colonies in the positive controls were above twice that in the negative controls. The test results showed that the numbers of revertant colonies in the negative control and the positive controls were within the range of the historical data at the testing facility. It was also confirmed that no bacterial contamination was observed,
which indicates the test results to be valid.
Therefore, it was concluded that CuGUN had no ability to induce mutations under the
present test conditions.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Dose-finding range test		Mean number of revertant colonies per plate
	Test substance dose (µg/plate)	TA100	TA15535	WP2uvrA	TA98	TA1537
-S9	Negativecontrol	107	9	19	16	8
-S9	4.88	99	5	17	17	8
-S9	19.5	96	11	22	10	5
-S9	78.1	111	8	19	9	6
-S9	313	99	9	17	12	3
-S9	1250	116	6	15	12	3
-S9	5000	131	9	13	14	13
+S9	Negativecontrol	104	10	27	27	15
+S9	4.88	104	12	24	23	17
+S9	19.5	108	7	28	26	15
+S9	78.1	103	15	20	22	10
+S9	313	102	8	10	12	7
+S9	1250	119	11	12	13	4
+S9	5000	99	6	15	5	1
	Positive control (µg/plate)	AF-2 (0.01)	NaN3 (0.5)	AF-2 (0.01)	AF-2 (0.1)	ICR-191 (0.5)
-S9	Number of revertants colonies/plate	705	269	373	386	1886
	Positive control (µg/plate)	2AA (1)	2AA (2)	2AA (10)	2AA (0.5)	2AA (2)
+S9	Number of revertants colonies/plate	1011	274	521	337	419

Main test		Mean number of revertant colonies per plate
	Test substance dose (µg/plate)	TA100	TA15535	WP2uvrA	TA98	TA1537
-S9	Negativecontrol	107	13	25	17	11
-S9	156	101	6	20	17	6
-S9	313	120	7	17	16	6
-S9	625	99	5	19	24	8
-S9	1250	94	8	18	17	9
-S9	2500	95	10	22	21	14
-S9	5000	120	4	13	23	8
+S9	Negativecontrol	101	10	25	24	15
+S9	156	117	14	25	28	9
+S9	313	105	9	23	28	14
+S9	625	89	9	30	22	13
+S9	1250	109	9	17	24	6
+S9	2500	101	8	14	15	5
+S9	5000	85	8	14	18	3
	Positive control (µg/plate)	AF-2 (0.01)	NaN3 (0.5)	AF-2 (0.01)	AF-2 (0.1)	ICR-191 (0.5)
-S9	Number of revertants colonies/plate	681	288	365	468	1909
	Positive control (µg/plate)	2AA (1)	2AA (2)	2AA (10)	2AA (0.5)	2AA (2)
+S9	Number of revertants colonies/plate	1189	304	576	274	436

Conclusions:

Interpretation of results (migrated information):
negative

The mutagenicity of the test substance was judged to be negative because the number of revertant colonies in the test substance treatment groups was less than twice that in the negative control for all tester strains regardless of the absence or presence of S9 mix.
The numbers of the revertant colonies in the positive controls were above twice that in the negative controls. The test results showed that the numbers of revertant colonies in the negative control and the positive controls were within the range of the historical data at the testing facility. It was also confirmed that no bacterial contamination was observed, which indicates the test results to be valid. Therefore, it was concluded that CuGUN had no ability to induce mutations under the present test conditions.

Executive summary:

The ability of CuGUN to induce mutations was investigated using Salmonella typhimurium strains TA100, TA1535, TA98 and TA1537 and Escherichia coli strain WP2uvrA with a pre-incubation method in the presence and absence of a metabolic activation system (S9 mix). As a result, the mutagenicity of the test substance was judged to be negative because the numbers of revertant colonies in the test substance treatment groups were less than two times that in each negative control in all tester strains. Therefore, it is concluded that CuGUN has no ability to induce mutations under the present test conditions.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

According to 1907/2006 (REACh) regulation, as the In Vitro Mammalian Cell Gene Mutation Test (476) was positive, an in vivo genetic toxicity assay was performed : In Vivo Mammalian Cell Gene Mutation Assay (OECD guidelines 489) on male rats treated by oral gavage with CuGUN (at 200, 400 and 800 mg/kg/day (two daily treatments at 24 hour intervals or X2)).

 

Under these experimental conditions, CuGUN did not induce statistically or biologically significant increases in DNA strand breaks at all tested concentrations in male rat isolated glandular stomach cells after oral administration. Therefore, CuGUN does not have genotoxic activity in this organ.

Under these same ecperimental conditions, CuGUN induced dose-related statistically and biologically significant increases in DNA strand breaks at 400 and 800 mg/kg/day (x2) in male rat isolated liver cells after oral administration. Therefore, CuGUN produces genotoxic activity in this organ. Notably the magnitude of the observed result was 4.6-fold below the results for the methylmethane sulfonate positive control.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03/15/16 to 08/09/2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study realised according to OECD GD 489 (2014) and GLP but the determination of Copper Guanylurea nitrate (CuGUN) in treatment formulations was performed by a non-GLP-compliant laboratory (ESCOM, France) using a method that was similar in its sensitivity for the dose level of copper to be assayed, to the one validated and used by the GLP-compliant laboratory INERIS. Analysis were performed with the presence of GLP-compliant INERIS study personnel and under the supervision of INERIS quality assurance. The dosing results may thus be considered as reliable. However, the analytical part of the current study was not GLP-Compliant, and the results are presented as informative only. The results obtained for the concentration assay of Copper Guanylurea nitrate (CuGUN) in treatment formulations used in the main assays were considered as satisfactory.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 489 (In Vivo Mammalian Alkaline Comet Assay)

Deviations:

not applicable

GLP compliance:

yes

Remarks:

Study realised according to OECD GD 489 (2014) and GLP but the determination of Copper Guanylurea nitrate (CuGUN) in treatment formulations was performed by a non-GLP-compliant laboratory (ESCOM, France) using a method.

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

IPL REGISTRATION NUMBER : 160309
BATCH NUMBER : 40, INERIS ref12AE617*
EXPIRY DATE : September 30, 2018
APPEARANCE : blue powder
PURITY : 98.4% / copper, nitrate, guanylurea
SALT / BASE RATIO : unknown
WATER CONTENT : unknown
CORRECTION FACTOR : 1.016**
QUANTITY SUPPLIED : 27.63, 26.21 and 27.27 g
STORAGE CONDITIONS : room temperature (+20±5°C), protected from light and from humidity:
with humidity less than 60%. Vapour pressure should be lower than
9x10
-7
at 20 °C and away from any oxidizers.
STABILITY UNDER
STORAGE CONDITIONS : in process (no change since 2012 as indicated in the shelf life letter
(October 12, 2015)). Up to September 30, 2018 for batch 40

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

selection issued from preliminary toxicity and main assays

Sex:

male

Details on test animals or test system and environmental conditions:

Young adult 8 weeks old male OFA Sprague-Dawley rats (Charles River France origin, Saint-Germain-sur-l’Arbresle; FRANCE) were used for the study. Body weight in male rats of the main assay ranged between 177 g and 210 g.
The period of acclimatization was of 6 day for the preliminary toxicity assay and for the main assay. It was of 7 days for the confirmatory toxicity assay. The animals received a clinical examination in order to retain only those which were healthy. The animals were identified by numbered ear rings.
The bedding consists of dust-free, irradiated softwood pellets.
The animals were dispatched in polypropylene cages by random-distribution.
The cages were placed in a ventilated system in the animal room, which was also ventilated. A timer provides lighting 12 hours a day (8 a.m. - 8 p.m.) in all the animal room. The temperature in the ventilated animal cupboard was 22 ± 3 °C, and humidity was 55 ± 15 %, with minor exceptions. The animals were not fasted at the treatment time. Drinking water, softened by reverse osmosis and filtered on 0.22 µm membrane, was provided ad libitum. The feedstuff used was A04C-10 from SAFE (batch 15230).

Route of administration:

oral: gavage

Vehicle:

Copper Guanylurea nitrate (CuGUN) was suspended in drinking water (provided by IPL under the form of osmosed water). The stability of the test item in suspension in drinking water at 2 and 200 mg/mL is of 7 days at room temperature protected from light (CiToxLAB Study No. 39705 AHS).

Details on exposure:

For the preliminary toxicity assay, suspensions at initial concentration of 200, 125 and 80 mg/mL were prepared and administered to the animals at the dose volume of 10 mL/kg, giving final doses of 2000, 1250 and 800 mg/kg, respectively. For the 80 and 125 mg/mL initial concentrations, only one formulation was prepared and used for both treatments while a suspension at 200 mg/mL was extemporaneously made for each treatment of the 2000 mg/kg treatment group,.
For the confirmatory toxicity assay, a suspension at an initial concentration of 80 mg/mL was prepared and administered to the animals at the dose volume of 10 mL/kg, giving a final dose of 800 mg/kg. A single suspension was prepared and used for both treatments.
In the main genotoxicity assay, three suspensions of the test item at the initial concentrations of 80 - 40 and 20 mg/mL were prepared, giving final doses of 800 - 400 and 200 mg/kg, respectively when administered at 10 mL/kg. For each concentration, a single suspension was prepared and used for both treatments.

Duration of treatment / exposure:

PRELIMINARY TOXICITY ASSAY: 2 daily treatments at 24-hour interval
CONFIRMATION TOXICITY ASSAY: 2 daily treatments at 24-hour intervals
COMET ASSAY: 2 daily treatments at 24-hour intervals

Frequency of treatment:

PRELIMINARY TOXICITY ASSAY: 2000 – 1250 – 800 mg/kg/day (x 2)
CONFIRMATION TOXICITY ASSAY: 800 mg/kg/day (x 2)
COMET ASSAY: 800 – 400 – 200 mg/kg/day (x 2)

Post exposure period:

48 hours after 2nd treatment

Remarks:

Doses / Concentrations:

Basis:

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Preliminary toxicity assay, 800 mg/kg/day (x 2)

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Remarks:

Preliminary toxicity assay, 1250 mg/kg/day (x 2)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

Preliminary toxicity assay, 2000 mg/kg/day (x 2)

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

Confirmatory toxicity assay, 800 mg/kg/day (x 2)

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

COMET ASSAY, 200 mg/kg/day (x 2)

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

COMET ASSAY, 400 mg/kg/day (x 2)

Dose / conc.:

800 mg/kg bw/day (nominal)

Remarks:

COMET ASSAY, 800 mg/kg/day (x 2)

No. of animals per sex per dose:

PRELIMINARY TOXICITY ASSAY: 2 males
CONFIRMATION TOXICITY ASSAY: 5 males
COMET ASSAY: 5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Methylmethane sulfonate (MMS; Aldrich, batch MKBL6789V in sterile water, Fresenius, batch 13KAP031), 100 mg/kg (x2), oral administration at 24-hour interval

Tissues and cell types examined:

Liver and glandular stomach

Details of tissue and slide preparation:

A portion of the liver and glandular stomach was removed (one part of each organ was preserved in formalin for eventual further analysis) and washed in the cold mincing buffer until as much blood as possible has been removed. The portion was minced with a pair of fine scissors to release the cells. The cell suspension was stored on ice for 15-30 seconds to allow large clumps to settle. The whole cell suspension was collected.
Cells were enumerated on a haemocytometer, and sufficient cells to obtain 20 x 10-3 viable cells per slide were harvested from each cell suspension for proceeding to slides preparation.
A statistically significant increase in the occurrence of hedgehogs in liver cells was noted at the highest dose of 800 mg/kg/day (x2), i.e. slightly above the maximal value already noted in historical data for negative control (i.e. 5.59%, see Appendix No. 7), but no dose-response relationship was noted and the effect was thus not biologically relevant.
No statistically significant increase in the occurrence of hedgehogs in glandular stomach cells was noted at any of the doses tested when compared to the respective control.

Evaluation criteria:

A study is accepted if the following criteria are fulfilled:
- Concurrent negative controls should be within the control limits of laboratory’s historical negative control database.
- The positive controls should induce responses that are comparable to the historical positive control data and produce a statistically significant increase compared with the negative control.
- The appropriate number of doses and cells must be analysed.

Moreover:
- In the vehicle group, for each organ, an eventual increase in the frequency of hedgehogs, must not be >50%.
- If death(s) is(are) observed at the tested doses, the mortality rate must be less than 20 % per group.

The validity criteria for the test were considered as fulfilled and the test was validated.

For a test item to be considered positive in the comet assay, it must be observed:
- At least one of the treatment groups exhibits a statistically significant increase in the mean of medians of percentage of DNA in tail compared with the negative control,
- This increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
- Any of these results are outside the distribution of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce DNA strand breakage in the tissues studied in this test system.

A test item is considered clearly negative if:
- none of the test doses exhibits a statistically significant increase compared with the negative control,
- there is no dose-related increase when evaluated with an appropriate trend test
- all results are inside the distribution of the historical negative control data for a given species, vehicle, route, tissue, and number of administrations
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) has been demonstrated.
The test chemical is then considered unable to induce DNA strand breakage in the tissues studied.

Statistics:

In order to quantify the test item effects on DNA, the following statistical analysis strategy was applied, using the statistical software Stat view®, version 5. As the median of percentage of DNA in tail and other tail parameters do not follow a Gaussian distribution (E. Bauer et al., 1998), the non-parametric, one-way Kruskall-Wallis test was performed. This method is based on the analysis of variance by ranks for testing equality of population medians among groups. The non-parametric Mann-Whitney U-test was applied to compare each of the doses tested with the vehicle control in order to determine statistical significance of differences in group median values between each group versus the vehicle control. This test was also used to compare vehicle control and positive control to determine acceptable criteria of a valid test.

Sex:

male

Genotoxicity:

negative

Remarks:

LIVER CELLS (200 mg/kg/day)

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

LIVER CELLS (400 mg/kg/day)

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

LIVER CELLS (800 mg/kg/day)

Toxicity:

yes

Remarks:

Diarrhea and slight decrease in spontaneous motor activity

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

GLANDULAR STOMACH CELLS (200 mg/kg/day)

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

GLANDULAR STOMACH CELLS (400 mg/kg/day)

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

GLANDULAR STOMACH CELLS (800 mg/kg/day)

Toxicity:

yes

Remarks:

Diarrhea and slight decrease in spontaneous motor activity

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

PRELIMINARY TOXICITY ASSAY

DOSES in mg/kg/day (x2)	DOSING VOLUME	NUMBER OF ANIMALS MALE RAT	% MORTALITY 24 HOURS AFTER FIRST TREATMENT	% MORTALITY 48 HOURS AFTER LAST TREATMENT
2000	10 mL/kg	2	0	50
1250	10 mL/kg	2	0	50
800	10 mL/kg	2	0	0

CONFIRMATORY TOXICITY ASSAY

DOSES in mg/kg/day (x2)	DOSING VOLUME	NUMBER OF ANIMALS MALE RAT	% MORTALITY 24 HOURS AFTER FIRST TREATMENT	% MORTALITY 48 HOURS AFTER LAST TREATMENT
800	10 mL/kg	5	0	0

COMET ASSAY IN LIVER CELLS

GROUP	TEST ITEM	DOSES in mg/kg/day (x2)	% of DNA in tail (mean of medians per animal (/5 animals))	NON PARAMETRIC statistical assessment (p Kruskall- Wallis)		NON PARAMETRIC statistical assessment (p Mann-Whitney)	Hedgehogs (Relative ratio of hedgehogs)	Hedgehogs (p)
Negative control	Drinking water	0	0.75	< 0.01		-	-	-
TREATED	CuGUN	800	10.45	< 0.01		< 0.01	1.68	< 0.05
TREATED	CuGUN	400	4.58	< 0.01		< 0.01	0.96	N.S.
TREATED	CuGUN	200	1.36	< 0.01		N.S.	1.15	N.S.
Positive control	Methylmethane sulfonate	100	48.35	-		< 0.01	1.03	N.S.

COMET ASSAY IN GLANDULAR STOMACH CELLS

GROUP	TEST ITEM	DOSES in mg/kg/day (x2)	% of DNA in tail (mean of medians per animal (/5 animals))	NON PARAMETRIC statistical assessment (p Kruskall- Wallis)		NON PARAMETRIC statistical assessment (p Mann-Whitney)	Hedgehogs (Relative ratio of hedgehogs)	Hedgehogs (p)
Negative control	Drinking water	0	22.38	< 0.01		-	-	-
TREATED	CuGUN	800	7.03	< 0.01		< 0.01	1.16	N.S.
TREATED	CuGUN	400	12.98	< 0.01		< 0.05	0.82	N.S.
TREATED	CuGUN	200	14.77	< 0.01		< 0.05	0.89	N.S.
Positive control	Methylmethane sulfonate	100	66.69	-		< 0.01	1.95	< 0.01

Conclusions:

Under these experimental conditions, Copper Guanylurea nitrate (CuGUN) induced no statistically or biologically significant increases in DNA strand breaks at 800, 400 and 200 mg/kg/day (x2) in male rat isolated glandular stomach cells after oral administration. Therefore, Copper Guanylurea nitrate (CuGUN) is considered having no genotoxic activity in this organ.
 In return, in these conditions, Copper Guanylurea nitrate (CuGUN) induced dose-related statistically and biologically significant increases in DNA strand breaks at 800 and 400 mg/kg/day (x2) in male rat isolated liver cells after oral administration. Therefore, Copper Guanylurea nitrate (CuGUN) has been considered having a genotoxic activity in this organ.

Executive summary:

The test item Copper Guanylurea nitrate (CuGUN) (batch 40) provided by LIVBAG SAS was investigated by the means of the in vivo comet assay under alkaline conditions (SCGE) in the liver and glandular stomach of male rat treated orally twice with 800, 400 and 200 mg/kg/day, with one sampling time 2 to 6 hours after the last treatment according to OECD guideline (OECD 489, 2014). The highest dose was set according to the toxicity induced by the test item.

 The determination of Copper Guanylurea nitrate (CuGUN) in treatment formulations was performed by a non-GLP-compliant laboratory (ESCOM, France) using a method that was similar in its sensitivity for the dose level of copper to be assayed, to the one validated and used by the GLP-compliant laboratory INERIS. Analysis were performed with the presence of GLP-compliant INERIS study personnel and under the supervision of INERIS quality assurance. The dosing results may thus be considered as reliable. However, the analytical part of the current study was not GLP-Compliant, and the results are presented as informative only.

 The results obtained for the concentration assay of Copper Guanylurea nitrate (CuGUN) in treatment formulations used in the main assays were considered as satisfactory.

 The validity criteria for the study were met. The current study in both organs is thus considered as valid.

 Under these experimental conditions, Copper Guanylurea nitrate (CuGUN) induced no statistically or biologically significant increases in DNA strand breaks at 800, 400 and 200 mg/kg/day (x2) in male rat isolated glandular stomach cells after oral administration. Therefore, Copper Guanylurea nitrate (CuGUN) is considered having no genotoxic activity in this organ.

 In return, in these conditions, Copper Guanylurea nitrate (CuGUN) induced dose-related statistically and biologically significant increases in DNA strand breaks at 800 and 400 mg/kg/day (x2) in male rat isolated liver cells after oral administration. Therefore, Copper Guanylurea nitrate (CuGUN) has been considered having a genotoxic activity in this organ.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

According to 1907/2006 (REACh) regulation, three in vitro and one in vivo genetic toxicity tests were performed in compliance with the International Standard and Good Laboratory Practices (Kimish 1 quotation):

- Bacterial Reverse Mutation Assay (OECD 471, EU method B.13/14) with Salmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100 and Escherichia colistrain WP2uvrA (with and without metabolic activation);

- In Vitro Mammalian Cell Micronucleus Test (OECD 487, EU method B.49) with mouse lymphoma L5178Y TK^+/-cells (with and without metabolic activation);

- In Vitro Mammalian Cell Gene Mutation Test (OECD 476, EU method B.17) with mouse lymphoma L5178Y TK^+/-cells (with and without metabolic activation);

- In Vivo Mammalian Cell Gene Mutation Assay (OECD guidelines 489) in the male rat treated by CuGUN oral gavage (200, 400 and 800 mg/kg/day (X2)).

 

The results demonstrated that:

- The In Vitro Bacterial Reverse Mutation Assay (OECD 471) was negative (with and without metabolic activation) at the highest concentration of 5 mg CuGUN/plate.

- The In Vitro Mammalian Cell Micronucleus Test (OECD 487) was negative (with and without metabolic activation) at the highest concentration of 100 mg/mL.

- The In Vitro Mammalian Cell Gene Mutation Test (OECD 476) with mouse lymphoma L5178Y cells (with and without metabolic activation) up to 75 μg/mL (3-h treatment) or 18.8 μg/mL (24 -h treatment) without metabolic activation and up 150 μg/mL (3 -h treatment) or 200 µg/mL (24 -h treatment) with metabolic activation, showed mutagenic activity in the presence of a rat metabolizing system at concentration ≥ 75 μg/mL in both experiments and inconclusive results in the absence of a rat metabolizing system.

- As the In Vitro Mammalian Cell Gene Mutation (OECD 476) was positive with the rat metabolizing system, an In Vitro Mammalian Cell Gene Mutation Assay (OECD 489) in male rats treated by CuGUN oral gavage [200, 400 and 800 mg/kg/days (x2)] was conducted. Under these experimental conditions, CuGUN induced no statistically or biologically significant increases in DNA strand breaks at all tested concentrations in male rat isolated glandular stomach cells. However, in the same study CuGUN induced dose-related [at 400 and 800 mg/kg/day (x2)] statistically and biologically significant increases in DNA strand breaks in male rat isolated liver cells. Therefore, CuGUN is considered having genotoxic activity in the liver. Notably the magnitude of the observed result was 4.6-fold below the results for the methylmethane sulfonate positive control.

- As in vitro Mammalian Cell Gene Mutation is positive, an in vivo Mammalian Cell Gene Mutation assay (OECD guidelines 489) on rat treated by oral gavage (200, 400 and 800 mg/kg/days (X2)). Under these experimental conditions, Copper Guanylurea nitrate induced no statistically or biologically significant increases in DNA strand breaks at all tested concentrations in male rat isolated glandular stomach cells after oral administration. but induced dose-related statistically and biologically significant increases in DNA strand breaks at 800 and 400 mg/kg/day (x2) in male rat isolated liver cells after oral administration. Therefore, Copper Guanylurea nitrate (CuGUN) has been considered having a genotoxic activity in this organ. Notably the magnitude of the observed result was four times below positive control.

In Vitro Mammalian Cell Gene Mutation Assay (OECD 489)

i)                  Exposure Concentration Issues

The OECD 476 guidelines (adopted 21st July 1997 - which is the reference cited in the OECD 476 CuGUN report) under Section 17 (exposure concentrations) states : "Relatively insoluble substances should be tested up to or beyond their limit of solubility under culture conditions. Evidence of insolubility should be determined in the final treatment medium to which cells are exposed. It may be useful to assess solubility at the beginning and end of the treatment, as solubility can change during the course of exposure in the test system due to presence of cells, S9, serum etc. Insolubility can be detected by using the unaided eye. The precipitate should not interfere with the scoring.".

 As the tested substance (CuGUN) is of low solubility (< 0.01 g/L or < 10 μg/mL) this adaptation should have been considered in the actual OECD 476 CuGUN study. For example, the OECD 476 CuGUN report states: "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."

Therefore, positive genotoxic effects were observed at a maximum concentration 20-fold higher than the solubility limit and this major departure from the OECD 476 guidelines should be considered as an important deviation as all results obtained at the solubility limit (10 μg/mL) were negative.

ii)                Dose Response Issues

In the CuGUN OECD 489 study, CuGUN didn't induce statistical or biological significant DNA damage in rat isolated glandular stomach cells after oral exposure. However, an in the same study an increase in DNA strand breaks were observed at 400 and 800 mg/kg/day (x2) in male rat isolated liver cells. This liver cell positive result (10.45 % of DNA in tail at the highest tested dose was notably well below the methylmethane sulfonate positive control results (48.35 % of DNA in tail), however, no standard deviation is presented in the study to determine if there is an actual dose-response at 400 and 800 mg/kg/days.[CL1] 

As a consequence of the above discussed issues with the CuGUN OECD 489 study, as well as other genotoxicity and toxicity findings, the genetic toxicity properties of CuGUN have been reviewed and a self-classification as mutagen category 2 has been proposed.

Justification for classification or non-classification

According to 1272/2008 (CLP) regulation, as the In Vitro Mammalian Cell Gene Mutation Assay (OECD 476) is positive (at higher concentrations than the solubility limit concentration - as per guidance) with metabolic activation and the In Vivo Mammalian Cell Gene Mutation Assay (OECD 489) is positive (10.45 % of DNA in tail) on liver cells only, CuGUN should be considered as Mutagenic Category 2.

In the case where there are positive results for an in vitro test and an in vivo test on somatic cells, guidance R.7.a "Endpoint specific guidance" states that an expert judgement is needed to consider whether there is sufficient information to conclude that the substance poses a mutagenic hazard to germ cells. According to this guidance, the Mutagenic Category 2 listing best fulfilled the following consideration:

“Classification in Category 2 may be based on positive results of at least one in vivo valid mammalian somatic cell mutagenicity test, indicating mutagenic effects in somatic cells. A Category 2 mutagen classification may also be based on positive results of a least one in vivo valid mammalian somatic cell genotoxicity test, supported by positive in vitro mutagenicity results. Genetic damage to somatic cells in exposed humans shown to be caused by substance exposure supported by positive in vitro mutagenicity results may also offer information warranting classification as a Category 2 mutagen. In vitro results can only lead to a Category 2 mutagen classification in a case where there is support by chemical structure activity relationship to known germ cell mutagens. In the case where there are also negative or equivocal data, a weight of evidence approach using expert judgement has to be applied."

 

CuGUN was negative for two in vitro studies [In Vitro Bacterial Reverse Mutation Assay (OECD 471) and In Vitro Mammalian Cell Micronucleus Test (OECD 487)], had one positive result in vitro with metabolic activation but at CuGUN concentrations orders of magnitude above the known solubility limit [In Vitro Mammalian Cell Gene Mutation Test (OECD 476)] and a positive result on liver cells [In Vivo Mammalian Cell Gene Mutation Assay (OECD 489)] but not stomach glandular cells, all of which is best self-classified as Mutagenic Category 2, according to the guidance R.7.a. as stated below.

 

 “Evaluation of genotoxicity test data should be made with care. Regarding positive findings, particular points should be taken into account:

1. are the testing conditions (e.g.pH, osmolality, precipitates) in in vitro mammalian cell assays relevant to the conditions in vivo?

2. for studies in vitro, factors known to influence the specificity of mammalian cell assays such as the cell line used, the top concentration tested, the toxicity measure used or the metabolic activation system used, should be taken into consideration

3. responses generated only at highly toxic/cytotoxic doses or concentrations should be interpreted with caution (i.e. taking into account the criteria defined in OECD guidelines)

4. the presence or absence of a dose (concentration)-response relationship should be considered"

 

Considering the positive result obtained in vitro (OECD 476), and compared to the above R.7.a considerations, (i.e. Point 1.; are the testing conditions relevant?) all of the observed CuGUN effects in the OECD 476 study occurred at concentrations above the solubility limits. This means that in vitro cells were exposed to CuGUN concentrations (as much as 20-times higher) and therefore the positive results for the CuGUN OECD 476 study are questionable.

Considering the positive result obtained on liver cells only (negative result on glandular stomach cells) in the CuGUN in vivo study (OECD 489), the presence/absence of a dose-response relationship (Point 4: is there a true dose-response?) can not be determined since the effect at the highest CuGUN concentration is well below that for the positive control and that the standard deviation for CuGUN at each dose is not described.

As a consequence and according to 1272/2008 (CLP) regulation, CuGUN should be considered as Mutagenic Category 2.