Alkaline co-precipitation products of soluble cobalt, manganese and nickel salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

Jul 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Test material information

Constituent 1

Reference substance name:

Alkaline co-precipitation products of soluble cobalt, manganese and nickel salts

Molecular formula:

Co(OH)2(0.005-0.1)Co2O3(0.03-0.3)Mn3O4(0.01-0.1)MnO2(0.02-0.25)Ni(OH)2(0.35-0.91) The stoichiometry of (OH)x+Oy per metal component in the substance cannot be determined unambiguously from solely analytical data, since the composition of the present UVCB substance is highly variable (i.e. complexly mixed and poorly ordered at the atomic level). The composition of the UVCB substance can be estimated based on analytical examination in combination with a calculation that is based on the average oxidation number (NOx) in the sum of the elements Ni, Co, Mn. As a result, the following constituents in concentration ranges as described below can be identified in the UVCB: The sum of all constituents equals 1 and ranges are: • Co(OH)2: 0.005-0.1 • Co2O3: 0.03-0.3 • Mn3O4: 0.01-0.1 • MnO2: 0.02-0.25 • Ni(OH)2: 0.35-0.91

IUPAC Name:

Alkaline co-precipitation products of soluble cobalt, manganese and nickel salts

Test material form:

solid

Specific details on test material used for the study:

Purity: 100% UVCB
Homogeneity: The homogeneity of the test substance, was guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance preparations.
Production date: 01 Mar 2019
Expiry date: 28 Feb 2024

Method

Target gene:

X-linked hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: rat liver induced phenobarbital and beta-naphthoflavone
- method of preparation of S9 mix: according to Ames et al. (1975)
- concentration or volume of S9 mix and S9 in the final culture medium: 1 part S9 fraction with 9 parts S9 supplement. S9 mix consisted of 10% S9 fraction.

Test concentrations with justification for top dose:

In the pre-test for toxicity based on the “SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 06 Mai 2018, 2.56 mg/mL test substance was used as stock dispersion. The highest tested concentration was 5000.0 μg/mL both with and without S9 mix at 4-hour exposure time.
The pre-test was performed following the method described for the main experiments. The Relative Survival (RS) was determined as a toxicity indicator for dose selection.
In the pre-test for dose selection the pH, osmolality and solubility were additionally determined
for selected doses.

Since the test substance is a nanomaterial, dose selection for genotoxicity testing was based on the SOP for "Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018. Furthermore, to fulfill the requirements of the OECD Guidelines for the HPRT assay, the
top concentrations in all main Experiments were defined as the highest homogenous
suspension. 0.05% w/v bovine serum albumin water (BSA-water) was used as vehicle.
1st Experiment (without S9 mix):
0; 3.0; 10.0; 30.0; 100.0; 350.0; 750.0; 2000.0; 5000.0 μg/mL
1st Experiment (with S9 mix):
0; 3.0; 10.0; 30.0; 100.0; 350.0; 750.0; 2000.0; 5000.0 μg/mL
2nd Experiment (without S9 mix):
0; 300.0; 600.0; 900.0; 1400.0; 2000.0 μg/mL

Vehicle / solvent:

- Vehicle used: bovine serum albumin water (BSA-water)

- Justification for choice of vehicle:
In accordance to the “SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 06 May 2018, 0.05% w/v bovine serum albumin water (BSA-water) was used as vehicle.
The final concentration of the vehicle 0.05% w/v BSA-water in culture medium was 10% (v/v).

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: single
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 20x10^6 cells in 40 ml
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Test substance incubation: 20-25 h after seeding
- Exposure duration/duration of treatment: 4 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 9 days
- Selection time (if incubation with a selective agent): 6-7 days, seeded in 20 ml selection medium (Ham's F12 with 6-thioguanine (10 µg/ml) and 10% FCS)
- Fixation time: Fixed with methanol, stained with Giemsa

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS), cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICIY
- Method: mutant frequency

Evaluation criteria:

A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative control value and the range of our laboratory’s historical negative control data (95% control limit)
Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit)

Statistics:

A linear dose-response was evaluated by testing for linear trend. The dependent variable was the corrected mutant frequency and the independent variable was the dose. The calculation was performed using EXCEL function RGP. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report.
A pair-wise comparison of each test group with the control group was carried out using Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using EXCEL function HYPGEOM.VERT.
If the results of these tests were statistically significant compared with the respective vehicle
control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.

Results and discussion

Test results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 2000 µg/ml and above

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 5000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
Not influenced by test substance treatment
- Data on osmolality:
Not influenced by test substance treatment
- Precipitation:
Test substance precipitation was observed microscopically in culture medium at the end of treatment at 3.0 μg/mL and above in the 1st Experiment. In the 2nd Experiment without S9 mix test substance precipitation occurred microscopically and macroscopically at 300.0 μg/mL and above.
With metabolic activation in the 1st Experiment test substance precipitation was observed microscopically in culture medium at the end of treatment at 3.0 μg/mL and above.

- Cell morphology:
After 4 hours treatment either in the absence or presence of metabolic activation, the cell morphology and attachment of the cells (grade 1) was not adversely influenced in any test group tested for gene mutations.

RESULTS OF MAIN STUDY:

Any other information on results incl. tables

Cytotoxicity data - 1st Experiment without S9 mix;
4-hour exposure period
* 0.05% w/v BSA-water

The results of the analysis showed that only percentages up to 37% of the dose are present as particles with less than 1 μm diameter and only up to 0% with up to 100 nm diameter. The test substance substantially leached metal ions into the cell culture medium. The ions could contribute to all observed effects.

According to the results of the present in vitro study, in two experiments performed independently of each other the test substance did not lead to a biologically relevant or dose-dependent increase the number of mutant colonies, either without S9 mix or after the addition of a metabolizing system. The mutant frequencies at any concentration were close to the range of the concurrent vehicle control values and within the 95% control limit of the historical negative control data.
Since the test substance is a nanomaterial the visual assessment of concentration of test substance precipitation is misleading. Thus, the documentation of test substance precipitation merely shows that the particulate matter could be observed visually and does not mean that the test substance at lower concentrations were necessarily dissolved.In the absence and presence of metabolic activation, neither a statistically significant nor dose related increase compared to the concurrent vehicle control value was found and all values were within the 95% control limit of the historical data.
The mutation frequencies of the vehicle control groups were within our historical negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this study. The proficiency of the laboratory to perform the HPRT assay in CHO cells was demonstrated by the laboratory’s historical control database on vehicle and positive controls and by X-bar chart to identify the variability of the vehicle control data.
The increase in the frequencies of mutant colonies induced by the positive control substances EMS and DMBA clearly demonstrated the sensitivity of the test method and/or of the metabolic activity of the S9 mix employed. The values were compatible with the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

Applicant's summary and conclusion

Conclusions:

Thus, in the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen..