Dicopper hydroxide phosphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro and in vivo studies are available. The in vitro data performed on dicopper hydroxide phosphate has been conducted in accordance with recommended guidelines and under the conditions of GLP and is therefore considered to be reliable for use in assessing the genotoxic potential of the material to be registered. In addition two in vivo studies on the analogous material copper sulphate are also available and as such the genotoxicity endpoint has been fulfilled by applying a weight of evidence approach.
All available studies indicate that copper and copper compounds are not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 November 2010 - 17 December 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Specific details on test material used for the study:

Sponsor's identification: Dicopper hydroxide phosphate
Description: light green powder
CAS number: 12158-74-6
Lot number: 90101
Date received: 28 August 2009
Storage conditions: room temperature in the dark

Target gene:

Histidine for Salmonella.

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102, TA 1535

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Conc 1: 50 mg/ml
Conc 2: 15.82 mg/ml
Conc 3: 5.01 mg/ml
Conc 4: 1.58 mg/ml
Conc 5: 0.5 mg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: phosphate buffered saline
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: Daunomycin, ICR 191

Remarks:

See table 1 for details on the use of positive controls

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: incubation period was 48 hours.

Key result

Species / strain:

S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102, TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The results of the negative and positive controls confirm the sensitivity and accuracy of the test system, according to the requirements of the Moltox-kit. In general, the positive control frequencies should be at least 2.5 times the negative counts (spontaneous frequency).

RESULTS

Table 2: Experiments with test strain TA 97a

Control mutagen: ICR 191 acridine

Experiment without S9-mix		Experiment with S9-mix
Mutagen µg/ml	Number of revertants	Mutagen µg/ml	Number of revertants
10	1191	100	493
Spontaneous revertants:131		Spontaneous revertants:127
Test preparation concentration	Number of revertants	Test preparation concentration	Number of revertants
50 mg/ml	94	50 mg/ml	80
15.82 mg/ml	127	15.82 mg/ml	93
5.01 mg/ml	132	5.01 mg/ml	88
1.58 mg/ml	93	1.58 mg/ml	109
0.5 mg/ml	68	0.5 mg/ml	76

 

No genotoxic activity observed.

Table 3: Experiments with test strain TA 98

Control mutagen: Daunomycin

Experiment without S9-mix		Experiment with S9-mix
Mutagen µg/ml	Number of revertants	Mutagen µg/ml	Number of revertants
60	555	100	338
Spontaneous revertants:21		Spontaneous revertants:21
Test preparation concentration	Number of revertants	Test preparation concentration	Number of revertants
50 mg/ml	31	50 mg/ml	33
15.82 mg/ml	27	15.82 mg/ml	25
5.01 mg/ml	27	5.01 mg/ml	21
1.58 mg/ml	15	1.58 mg/ml	25
0.5 mg/ml	16	0.5 mg/ml	20

No genotoxic activity observed.

 

Table 4: Experiments with test strain TA 100

Control mutagen: Sodium azide

Experiment without S9-mix		Experiment with S9-mix
Mutagen µg/ml	Number of revertants	Mutagen µg/ml	Number of revertants
15	>300	100	>300
Spontaneous revertants:65		Spontaneous revertants:79
Test preparation concentration	Number of revertants	Test preparation concentration	Number of revertants
50 mg/ml	63	50 mg/ml	68
15.82 mg/ml	50	15.82 mg/ml	60
5.01 mg/ml	36	5.01 mg/ml	41
1.58 mg/ml	31	1.58 mg/ml	44
0.5 mg/ml	17	0.5 mg/ml	47

No genotoxic activity observed.

 

Table 5: Experiments with test strain TA 102

Control mutagen: Mytomycin C

Experiment without S9-mix		Experiment with S9-mix
Mutagen µg/ml	Number of revertants	Mutagen µg/ml	Number of revertants
5	754	100	874
Spontaneous revertants:221		Spontaneous revertants:277
Test preparation concentration	Number of revertants	Test preparation concentration	Number of revertants
50 mg/ml	213	50 mg/ml	216
15.82 mg/ml	192	15.82 mg/ml	250
5.01 mg/ml	211	5.01 mg/ml	256
1.58 mg/ml	205	1.58 mg/ml	272
0.5 mg/ml	145	0.5 mg/ml	217

No genotoxic activity observed.

 

Table 6: Experiments with test strain TA 1535

Control mutagen: sodium azide

Experiment without S9-mix		Experiment with S9-mix
Mutagen µg/ml	Number of revertants	Mutagen µg/ml	Number of revertants
15	339	100	381
Spontaneous revertants:7		Spontaneous revertants:8
Test preparation concentration	Number of revertants	Test preparation concentration	Number of revertants
50 mg/ml	10	50 mg/ml	7
15.82 mg/ml	7	15.82 mg/ml	6
5.01 mg/ml	7	5.01 mg/ml	6
1.58 mg/ml	7	1.58 mg/ml	10
0.5 mg/ml	4	0.5 mg/ml	6

 

No genotoxic activity observed.

Conclusions:

The test material was determined to be non-genotoxic under the conditions of the study.

Executive summary:

The test material was determined to be non-genotoxic under the conditions of the study.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-28 to 2016-08-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

LPT Laboratory of Pharmacology and toxicology GmbH & Co. KG, Redderweg 8, 21147 Hamburg

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Batch no.: 9000023447

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Human peripheral blood was obtained by venipuncture from young (approximately 18 to 35 years of age), healthy, non-smoking male or female individuals with no known recent exposures to genotoxic chemicals or radiation.

Cytokinesis block (if used):

CytoB (Cytochalasin B) 5 μg/mL

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial fraction (S9 fraction) from rats treated with Aroclor 1254

Test concentrations with justification for top dose:

6.25, 12.5, 25, 50 and 100 μg/mL
In a preliminary experiment precipitate and/or cytotoxicity were detected with higher concentrations.

Vehicle / solvent:

- Solvent used: 0.05 M HCl solution and culture medium
- Justification for choice of solvent: The test item was completely soluble at 1.25 mg per mL 0.05 M HCl solution. This solution was diluted with culture medium to obtain a final concentration of 100 μg/mL medium and was then further diluted to the appropriate lower concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine 0.02 μg/mL without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h
- Exposure duration: 4 and 24 h exposure
- Incubation time with inhibitor: 20 hours
- Fixation time: 24 or 44 h

SPINDLE INHIBITOR: CytoB (Cytochalasin B) 5 μg/mL

STAIN: 10 % Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The cultures were centrifuged for 10 minutes at 800 rpm, the supernatant was discarded and the cells resuspended in KCl (0.56%). After incubation for 17 minutes at 37°C, the cell suspensions were centrifuged for 10 minutes at 800 rpm. The supernatant was discarded and 5 mL of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added. The cells were left in fixative for 30 minutes followed by centrifugation at 800 rpm. The supernatant was carefully removed and discarded, and the cell pellet was resuspended in about 0.5 mL of fresh fixative and 30% glacial acetic acid by repeated aspiration through a Pasteur pipette. Two drops of this cell suspension were dropped onto a prewarmed, pre-cleaned microscope slide and left to air-dry at room temperature.

NUMBER OF CELLS EVALUATED: at least 500 cells per replicate cell culture

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: micronucleus frequencies were analysed in at least 2000 binucleated cells per concentration

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Care was taken not to score binucleate cells with irregular shapes or where the two nuclei differ greatly in size; neither would binucleate cells be confused with poorly spread multi-nucleate cells. Cells containing more than two main nuclei were not analysed for micronuclei, as the baseline micronucleus frequency might be higher in these cells. Scoring of mononucleate cells is acceptable if the test item is shown to interfere with CytoB activity.

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of cytotoxicity was based on the Cytokinesis-Block Proliferation Index (CBPI) or the Replicative Index (RI). The CBPI indicates the average number of cell cycles per cell during the period of exposure to CytoB, and is used to calculate cell proliferation. The RI indicates the relative number of nuclei in treated cultures compared to control cultures and can be used to calculate the % cytostasis.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is dose-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative control data (Poisson-based 95% control limits)
When all of these criteria are met, the test chemical is then considered able to induce chromosome breaks and/or gain or loss in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (Poisson-based 95% control limits).
The test chemical is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Statistics:

No details provided.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

100 μg/mL and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Precipitation: only in pre-experiment 316.2 and 1000 μg/mL

RANGE-FINDING/SCREENING STUDIES:
A preliminary experiment without and with metabolic activation concentrations of 1.0, 3.162, 10.0, 31.62, 100, 316.2 and 1000 μg/mL was done.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: Test substance: 3.0 to 11.5 micronucleated cells per 1000 binucleated cells without activation, 9.0 to 12.0 micronucleated cells per 1000 binucleated cells with metabolic activation; Vehicle controls: 13.0 or 6.5 micronucleated cells per 1000 binucleated cells for the 4-hour and 24- hour exposure without metabolic activation, 2.5 micronucleated cells per 1000 binucleated cells with metabolic activation; positive control: 59.5 or 42.0 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour without metabolic activation, 39.5 micronucleated cells per 1000 binucleate cells for the 4-hour exposure with metabolic activation

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI or RI

4 h exposure without metabolic activation

Concentration of test item [µg/mL]	CBPI	RI [%]	Number of binucleate cells scored	Number of micronucleated cells per 1000 binucleate cells
0.05 M HCl	1.43	100	2000	13
6.25	1.37	85	2000	11.5
12.5	1.4	93	2000	10.5
25	1.44	101	2000	9
50	1.24	55	2000	10.5
100	1.08	19	n.d.
Mitomycin (0.2)	1.27	62	2000	59.5

 

 

24 h exposure without metabolic activation

Concentration of test item [µg/mL]	CBPI	RI [%]	Number of binucleate cells scored	Number of micronucleated cells per 1000 binucleate cells
0	1.22	80	2000	5.5
0.05 M HCl	1.29	100	2000	6.5
6.25	1.32	110	2000	3
12.5	1.29	98	2000	3
25	1.28	96	2000	6.5
50	1.17	57	2000	5.5
100	n.d.	n.d.	n.d.
Colchicine (0.02)	1.25	88	2000	42

 

 

4 h exposure with metabolic activation

Concentration of test item [µg/mL]	CBPI	RI [%]	Number of binucleate cells scored	Number of micronucleated cells per 1000 binucleate cells
0.05 M HCl	1.26	100	2000	12.5
6.25	1.17	66	2000	12
12.5	1.21	79	2000	9
25	1.18	69	2000	10.5
50	1.14	52	2000	11
100	1.06	23	n.d.
Cyclophosphamide (20)	1.2	77	2000	39.5

 

n.d. = not determined due to cytotoxicity

Conclusions:

The test substance revealed no indications of chromosomal damage in the in vitro micronucleus test.

Executive summary:

Test samples of Dicopper hydroxide phosphate were assayed in an in vitro micronucleus test using human peripheral lymphocytes both in the presence and absence of metabolic activation by a rat liver post-mitochondrial fraction (S9 mix) from Aroclor 1254 induced animals. The test was carried out employing 2 exposure times without S9 mix: 4 and 24 hours, and 1 exposure time with S9 mix: 4 hours. The harvesting time was 20 hours after the end of exposure. The cytokinesis-block technique was applied. The test item was not soluble in any of the solvents recommended: aqua ad iniectabilia, dimethylsulfoxide (DMSO), ethanol or acetone. However, Dicopper hydroxide phosphate was completely soluble at 1.25 mg per mL 0.05 M HCl solution. This solution was diluted with culture medium to obtain a final concentration of 100 μg/mL medium and was then further diluted to the appropriate lower concentrations. 3.95 or 12.5 mg Dicopper hydroxide phosphate were suspended in 1 mL 0.05 M HCl solution. These suspensions were diluted with culture medium to obtain final concentrations of 316.2 or 1000 μg/mL medium, respectively, for the highest tested doses in the preliminary test for determination of cytotoxicity. The vehicle 0.05 M HCl solution served as the negative control. No correction factor was used. The concentrations employed were chosen based on the results of a cytotoxicity study. In this preliminary experiment without and with metabolic activation concentrations of 1.0, 3.162, 10.0, 31.62, 100, 316.2 and 1000 μg Dicopper hydroxide phosphate/mL medium were employed. Test item precipitation was noted at concentrations of 316.2 and 1000 μg/mL in both experiments. In the experiments without and with S9 mix (24- or 4-hour exposure, respectively) cytotoxicity was noted at concentrations of 100 μg/mL medium and higher. Hence, 100 μg/mL were employed as the top concentration for the genotoxicity tests without and with metabolic activation with a 4-hour or 24-hour exposure. In the main study pronounced to complete cytotoxicity was noted at the top concentration of 100 μg Dicopper hydroxide phosphate/mL medium in the experiments without and with metabolic activation (4- or 24-hour exposure). Hence, the top concentration of 100 μg/mL was not scored for micronuclei. Mitomycin C (at 0.2 μg/mL) and colchicine (at 0.02 μg/mL) were employed as positive controls in the absence and cyclophosphamide (at 20 μg/mL) in the presence of metabolic activation.

Tests without metabolic activation (4- and 24-hour exposure)

The micronucleus frequencies of cultures treated with the concentrations of 6.25, 12.5, 25 and 50 μg Dicopper hydroxide phosphate/mL medium in the absence of metabolic activation (4- and 24-hour exposure) ranged from 3.0 to 11.5 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 50 μg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test the following frequencies were observed: vehicle control: 13.0 or 6.5 micronucleated cells per 1000 binucleated cells for the 4-hour and 24- hour exposure, respectively. The vehicle result was within the historical control ranges. In the positive control cultures the micronucleus frequencies were increased to 59.5 or 42.0 micronucleated cells per 1000 binucleate cells for the 4-hour and 24-hour exposure, respectively. This demonstrated that Mitomycin C induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus.

Test with metabolic activation (4-hour exposure)

The micronucleus frequencies of cultures treated with the concentrations of 6.25, 12.5, 25 and 50 μg Dicopper hydroxide phosphate/mL medium (4-h exposure) in the presence of metabolic activation ranged from 9.0 to 12.0 micronucleated cells per 1000 binucleated cells. There was no dose-related increase in micronuclei up to the top concentration of 50 μg/mL medium. The frequency of micronucleated cells was within the historical control range of the untreated and vehicle controls. Vehicle controls should give reproducibly low and consistent micronucleus frequencies. In this test a mean frequency of 12.5 micronucleated cells per 1000 binucleated cells was observed. The vehicle result was within the historical control ranges. In the positive control culture the micronucleus frequency was increased to 39.5 micronucleated cells per 1000 binucleate cells for the 4-hour exposure. This demonstrated that cyclophosphamide induced significant chromosomal damage.

Conclusion

Under the present test conditions, Dicopper hydroxide phosphate tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times (without S9) and one exposure time (with S9) revealed no indications of chromosomal damage in the in vitro micronucleus test. The results for the vehicle controls were within historical control range. In the same test, Mitomycin C and cyclophosphamide induced significant chromosomal damage and colchicine induced significant damage to the cell division apparatus, respectively. Therefore, the test is considered valid.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2011 - 24 March 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DIN EN ISO 10993-3, 4-09-SOP-11-153

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

Guideline 490 was not available when the study was performed.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation

Specific details on test material used for the study:

Sponsor's identification: Dicopper hydroxide phosphate
Description: light green powder
CAS number: 12158-74-6
Lot number: 90101
Date received: 28 August 2009
Storage conditions: room temperature in the dark

Target gene:

Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI with 10% horse serum

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix. Supplier: Moltex USA

Test concentrations with justification for top dose:

Conc 1: 0.05 mg/ml
Conc 2: 0.025 mg/ml
Conc 3: 0.01 mg/ml
Conc 4: 0.005 mg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: cell culture medium
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Cell culture medium: RPMI = 10% horse serum

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Remarks:

- Methylmethane sufonate: 10 µg/ml without S9-mix, Benzo(a)pyrene: 1.5 µg/ml without S9-mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Cells were incubated with test material.

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours

A part of the cells was used for the determination of cloning efficiency (CE 1). After 48 hours the cloning efficiency was determined again (CE 2). These determinations were made to give information on the cytotoxicity of the test material.
The mutation frequencies (MF) of each test group were defined after 14 days.

Cell culture conditions: 37°C, 5% CO2 , water saturated atmosphere

Evaluation criteria:

The following parameters were determined after incubation:
- number of wells without colonies
- number of wells without big colonies
- number of wells without small colonies

The mutation frequency was calculated using the equation in Attachment 1.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight decreases in CE were seen.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

A decrease in the cloning efficiency (CE 1) of test material solutions in comparison with negative controls was found in test solution concentrations 1, 2 and 3. The positive control shows a slight decrease of CE 1.
A decrease in the cloning efficiency (CE 2) of test material solutions in comparison with negative controls was found in test solution concentrations 1, 2 and 3. The positive control shows a slight decrease of CE 2.
A significant increase of mutation frequencies was found only I the positive controls.
The presence of liver extract as a metabolic activation system (S9) has no effect on the test results.
The distribution of small and big colonies shows neither chromosome nor gene mutations.
The results conform to literature data and test validation.

Table 1: Mean of CE 1 and CE 2

Test item	CE 1	CE 2
Positive control – S9	0.84	0.88
Positive control + S9	0.80	0.83
Negative control – S9	1.80	1.04
Negative control + S9	0.92	1.06
Concentration 1 – S9	0.56	0.71
Concentration 1 + S9	0.50	0.70
Concentration 2 – S9	0.54	0.83
Concentration 2 + S9	0.57	0.83
Concentration 3 – S9	0.82	0.94
Concentration 3 + S9	0.75	0.94
Concentration 4 – S9	1.06	0.99
Concentration 4 + S9	1.00	0.99

 

Table 2: Mutation Frequency (MF) for big and small colonies

Test item	MF (big colonies)	MF (small colonies)
Positive control – S9	343.08	301.34
Positive control + S9	355.65	336.14
Negative control – S9	47.37	50.14
Negative control + S9	50.69	51.39
Concentration 1 – S9	78.80	73.67
Concentration 1 + S9	77.32	76.30
Concentration 2 – S9	68.98	61.11
Concentration 2 + S9	62.42	56.39
Concentration 3 – S9	58.03	56.50
Concentration 3 + S9	56.05	49.94
Concentration 4 – S9	50.51	49.06
Concentration 4 + S9	49.07	50.51

 

Conclusions:

No genotoxic activity was observed under the test conditions of the mouse lymphoma assay. The results of the negative and positive controls confirm the validity of the test.

Executive summary:

No genotoxic activity was observed under the test conditions of the mouse lymphoma assay.  The results of the negative and positive controls confirm the validity of the test.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: OECD 487 (In Vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

Extended exposure, no metabolic activation system. Extracts of the test item were used.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Sponsor's identification: Dicopper hydroxide phosphate
Description: light green powder
CAS number: 12158-74-6
EC Number: 235-285-2
Sample number: 2011040713
Date received: 19 April 2011
Storage conditions: room temperature in the dark
Sample prepared under laminar flow.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

not applicable

Test concentrations with justification for top dose:

Dilutions of the test material extract: 8-fold, 24-fold and 72-fold

Vehicle / solvent:

- Preparation of extracts of test material (DIN ISO 10993-12)
0.563 g of test material was suspended in 13 ml extraction medium.
Extraction medium: Cell culture medium (RPMI1640 supplemented with 10% fetal calf serum) was used for extraction. The extraction was carried out by gentle agitation (about 20 rpm) in a shaker for 37°C for 24 hours. The saturated and particle containing suspension was centrifuged (3000 x g, 15 min), passed through a filter (0.2 µm pore size) and after finishing pH value of extract was controlled. Cell culture medium was used as a negative control and cell culture medium supplemented with mytomycin C acted as a positive control.

Untreated negative controls:

yes

Remarks:

The negative controls (and the vehicle controls) were the cell cultures with cell culture medium RPMI 1640 supplemented with 10 % foetal calf serum.

Negative solvent / vehicle controls:

yes

Remarks:

The negative controls (and the vehicle controls) were the cell cultures with cell culture medium RPMI 1640 supplemented with 10 % foetal calf serum.

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: 1 day
- Exposure duration: 24 hours
Cells were harvested after a further day of incubation.


STAIN (for cytogenetic assays): Giemsa solution

NUMBER OF REPLICATIONS: no data

NUMBER OF CELLS EVALUATED: 2 x 1000 cells per extract concentration and controls.

DETERMINATION OF CYTOTOXICITY
- Method: no data. The 8-fold dilution of the extract caused a permitted cytotoxicity between 50-60%.

DETERMINATION OF COMPELTED CELL CYCLE
- The cells were counted by staff member (before start and after ending of the assay). At least a doubling of cell counts per day was observed and is an evidence of passing a complete cell cycle run (mitosis) per day.

Evaluation criteria:

The total number of cells and cells with none, one, two, three, four and more than four micronuclei were counted and logged. Cells incubated with the cell culture medium only (negative control) were the basis for the identification of a genotoxic potential. A 3-fold increase in the number of micronuclei in relation to the negative control was the threshold value.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the 8-fold concentration produced between 50-60% cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The acceptable rates of micronuclei in the negative controls show that the used cells at this time point were able to build micronuclei after an exemplary incubation with a genotoxic substance as such the results of this study are considered to be valid for use.

Table 1: Number of micronuclei detected

	0	1	2	3	4	>4	number/1000 cells
Negative Control (I)	974	21	5				26
Negative Control (I)	1039	23	3				25
Average of negative controls (I) and (II)							25.5
Threshold value (based on average of negative controls (I) and (II))							76.5
8-fold dilution (saturated) extract (I)	976	23	1				24
8-fold dilution (saturated) extract (II)	970	24	5	1			30
24-fold dilution (saturated) extract (I)	983	16	1				17
24-fold dilution (saturated) extract (II)	971	22	7				29
72-fold dilution (saturated) extract (I)	987	12	1				13
72-fold dilution (saturated) extract (II)	982	18	6				24
0.8 µM Mitomycin C (positive control) (I)	809	136	24	18	7	6	191
0.8 µM Mitomycin C (positive control) (II)	814	147	13	19	2	5	186

 

 

Table 2: number of cells counted

SAMPLE	NUMBER OF ALL CELLS COUNTED
Negative Control (I)	1000
Negative Control (I)	1065
8-fold dilution (saturated) extract (I)	1000
8-fold dilution (saturated) extract (II)	1000
24-fold dilution (saturated) extract (I)	1000
24-fold dilution (saturated) extract (II)	1000
72-fold dilution (saturated) extract (I)	1000
72-fold dilution (saturated) extract (II)	1006
0.8 µM Mitomycin C (positive control) (I)	1000
0.8 µM Mitomycin C (positive control) (II)	1000

Conclusions:

Under the conditions of the study, the extracts of dicopper hydroxide phosphate were found to be non-genotoxic.

Executive summary:

Under the conditions of the study, the extracts of dicopper hydroxide phosphate were found to be non-genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

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

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
For further information please refer to read across justification in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The data set is considered to be adequate and reliable for the purpose of classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP) and as such dicopper hydroxide phosphate is not considered to be genotoxic.