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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471, GLP): not mutagenic

HPRT test (OECD 476, GLP): not mutagenic

Link to relevant study records

Reference

Reference 1

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

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

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

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

Target gene:

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

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).

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.

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

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:

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.

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..

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 Jun 2020

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Aug 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 100 % (UVCB) (for details see analytical report No.: 21L00179)
Homogeneity: The test item was homogeneous by visual inspection.
Storage stability: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
Expiry date: April 26, 2023
Storage conditions: Room temperature
Physical state/ color: Solid / black

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from male Wistar rats having received phenobarbital i.p. and beta-naphthoflavone orally

24 hours after the last administration, the rats were sacrificed, and the induced livers were
prepared using sterile solvents and glassware at a temperature of +4°C. The livers were
washed with 150 mM KCl solution. Afterwards, the livers were weighed and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, appropriate portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

Test concentrations with justification for top dose:

1st Experiment - Standard plate test
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Reason: In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose.

2nd Experiment - Preincubation test
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Reason: No mutagenicity was observed in the standard plate test.

Vehicle / solvent:

DMSO

Justification for choice of solvent/vehicle: insolubility in water, good solubility in DMSO, availability of historical control data for DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (with metabolic activation /TA 1535, 100, 1537, 98, Escherichia coli WP2 uvrA), 4-nitro-o-phenylenediamine (without metabolic activation / TA 98); N-methyl-N'-nitro-N-nitrosoguanidine (without metabolic activation / TA 1535, 100)

Details on test system and experimental conditions:

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

- Test substance added in agar (plate incorporation) or preincubation

Standard Plate Test:
Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with external metabolic activation)
or
0.5 mL phosphate buffer (without external metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular
Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C (mean value ±2°C) for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). In several cases, colonies were counted manually, in
particular, if precipitation of the test substance hindered the counting using the Image Analysis System.

Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with external metabolic activation)
or
0.5 mL phosphate buffer (without external metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular
Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C (±2°C) for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer
Image Analysis System with the software program Ames Study Manager (Perceptive

Preincubation Test:
0.1 mL test solution, vehicle or positive control, 0.1 mL bacterial suspension and 0.5 mL S9 mix
(with external metabolic activation) or phosphate buffer (without external metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker.
Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C (±2°C) for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager. In several cases, colonies were counted manually, in particular, if precipitation of the test substance hindered the counting using the Image Analysis System.

Evaluation criteria:

Mutagenicity:
Individual plate counts, the mean number of revertant colonies per plate and the standard
deviations were given for all dose groups as well as for the positive and negative (vehicle)
controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.

Toxicity:
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Solubility:
If precipitation of the test material was observed, it would be recorded and indicated in the tables. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at
least in the 1st Experiment even in the case of relatively insoluble test compounds to detect
possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

SPT without metabolic activation: no SPT with metabolic activation: no PIT without metabolic activation: no PIT with metabolic activation: no

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

SPT without metabolic activation: no SPT with metabolic activation: no PIT without metabolic activation: no PIT with metabolic activation: no

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

SPT without metabolic activation: no SPT with metabolic activation: no PIT without metabolic activation: no PIT with metabolic activation: no

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

SPT without metabolic activation: no SPT with metabolic activation: no PIT without metabolic activation: no PIT with metabolic activation: no

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

SPT without metabolic activation: no SPT with metabolic activation: no PIT without metabolic activation: yes (5000 µg/plate) PIT with metabolic activation: yes (5000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY:
A bacteriotoxic effect (slight decrease in the number of trp+ revertants) was only observed in
the preincubation assay testing E.coli at 5000 μg/plate with and without S9 mix.

SOLUBILITY:
Test substance precipitation was observed at and above 2500 μg/plate with and without
S9 mix.

STERILITY CONTROL:
The additional treated plates for sterility control showed no contamination in all performed experiments.

The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria. The number of revertant colonies in the negative controls, with and without S9 mix, were within the range of the respective historical control data of each tester strain, except testing the tester strain E.coli in the absence and presence of S9 mix in in the standard plate test (see Appendix – historical control data). Although the two values exceeded the maximum value of the historical control data, the increases were still compatible. In addition, the positive control substances induced a significant increase in the number of revertant colonies with and without S9 mix. This increase was compatible to the range of the respective historical control data.

Conclusions:

The test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay). Therefore, the test susbtance is not mutagenic in the Salmonella typhimurium/E.coli bacterial reverse mutation test with and without metabolic activation under the conditions tested.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 Dec 2021 to 13 Jan 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 Jun 2020

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Aug 1998

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from male Wistar rats having received phenobarbital i.p. and beta-naphthoflavone orally

24 hours after the last administration, the rats were sacrificed, and the induced livers were
prepared using sterile solvents and glassware at a temperature of +4°C. The livers were
washed with 150 mM KCl solution. Afterwards, the livers were weighed and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4°C, appropriate portions of the supernatant (S9 fraction) were stored at -70°C to -80°C.

Test concentrations with justification for top dose:

1st Experiment - Standard plate test
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Reason: In agreement with the recommendations of current guidelines 5 mg/plate or 5 μL/plate were generally selected as maximum test dose.

2nd Experiment - Preincubation test
Doses: 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate (E. coli WP2 uvrA +/- S9 mix)
Doses: 0; 10; 33; 100; 333; 1000 and 2500 μg/plate (TA strains +/- S9 mix)
Reason: No mutagenicity was observed in the standard plate test. Due to toxicity testing all TA strains, the doses were adjusted in the preincubation test

3rd Experiment - Preincubation test
Doses: 0; 3.3; 10; 33; 100; 333 and 1000 μg/plate (TA 1535 - S9 mix, TA 98 -/+ S9 mix; TA 1537 + S9 mix)
Reason: Due to strong toxicity in the preincubation assay testing the tester
strains TA 1535 without S9 mix, TA 1537 with S9 mix and TA 98 with and without S9 mix, the experimental parts were repeated in a 3rd Experiment. The doses were adjusted.

Vehicle / solvent:

DMSO

Justification for choice of solvent/vehicle: insolubility in water, good solubility in DMSO, availability of historical control data for DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene (with metabolic activation /TA 1535, 100, 1537, 98, Escherichia coli WP2 uvrA), 4-nitro-o-phenylenediamine (without metabolic activation / TA 98); N-methyl-N'-nitro-N-nitrosoguanidine (without metabolic activation / TA 1535, 100)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 3

- Test substance added in agar (plate incorporation) or preincubation

Standard Plate Test:
Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with external metabolic activation)
or
0.5 mL phosphate buffer (without external metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular
Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C (mean value ±2°C) for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perceptive Instruments Ltd., Haverhill, UK). In several cases, colonies were counted manually, in
particular, if precipitation of the test substance hindered the counting using the Image Analysis System.

Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution, vehicle or positive control
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with external metabolic activation)
or
0.5 mL phosphate buffer (without external metabolic activation)
After mixing, the samples were poured onto Minimal glucose agar plates (Moltox Molecular
Toxicology, Inc.; Boone, NC 28607; USA) within approx. 30 seconds.
After incubation at 37°C (mean value ±2°C) for 48 – 72 hours in the dark, the bacterial colonies (trp+ revertants) were counted. The colonies were counted using the Sorcerer
Image Analysis System with the software program Ames Study Manager (Perceptive

Preincubation Test:
0.1 mL test solution, vehicle or positive control, 0.1 mL bacterial suspension and 0.5 mL S9 mix
(with external metabolic activation) or phosphate buffer (without external metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker.
Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C (mean value ±2°C) for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager. In several cases, colonies were counted manually, in particular, if precipitation of the test substance hindered the counting using the Image Analysis System.

Evaluation criteria:

Mutagenicity:
Individual plate counts, the mean number of revertant colonies per plate and the standard
deviations were given for all dose groups as well as for the positive and negative (vehicle)
controls in all experiments. In general, six doses of the test substance were tested with a maximum of 5 mg/plate, and triplicate plating was used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments were based on the findings of the 1st Experiment.

Toxicity:
Toxicity detected by a
• decrease in the number of revertants (factor ≤ 0.6)
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
was recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables. Single values with a factor ≤ 0.6 were not detected as toxicity in low dose groups.

Solubility:
If precipitation of the test material was observed, it would be recorded and indicated in the tables. As long as precipitation did not interfere with the colony scoring, 5 mg/plate was generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at
least in the 1st Experiment even in the case of relatively insoluble test compounds to detect
possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

SPT without metabolic activation: yes (>= 2500 µg/plate) SPT with metabolic activation: yes (>= 2500 µg/plate) PIT without metabolic activation: yes (>= 1000 µg/plate) PIT with metabolic activation: yes (>= 333 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

SPT without metabolic activation: yes (>= 2500 µg/plate) SPT with metabolic activation: yes (5000 µg/plate) PIT without metabolic activation: yes (25000 µg/plate) PIT with metabolic activation: yes (>= 1000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

SPT without metabolic activation: yes (>= 2500 µg/plate) SPT with metabolic activation: yes (>= 2500 µg/plate) PIT without metabolic activation: yes (>= 1000 µg/plate) PIT with metabolic activation: yes (>= 1000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

SPT without metabolic activation: yes (>= 2500 µg/plate) SPT with metabolic activation: yes (>= 2500 µg/plate) PIT without metabolic activation: yes (>= 1000 µg/plate) PIT with metabolic activation: yes (>= 333 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

SPT without metabolic activation: yes (>= 2500 µg/plate) SPT with metabolic activation: no PIT without metabolic activation: yes (5000 µg/plate) PIT with metabolic activation: yes (>= 2500 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY:
A bacteriotoxic effect (decrease in the number of his+ or trp+ revertants) was occasionally
observed in the standard plate test depending on the strain and test conditions at and above
2500 μg/plate.
In the preincubation assay bacteriotoxicity (decrease in the number of his+ or trp+ revertants) was occasionally observed depending on the strain and test conditions at and above 333 μg/plate.

SOLUBILITY:
Test substance precipitation was observed at and above 2500 μg/plate with and without
S9 mix.

STERILITY CONTROL:
The additional treated plates for sterility control showed no contamination in all performed experiments.

Decreased revertant numbers were observed at following concentrations (μg/plate):

Experiment	S9	TA 1535	TA 100	TA 1537	TA 98	E.coli
1st-SPT	Without	≥2500	≥2500	≥2500	≥2500	≥2500
	With	≥2500	5000	≥2500	≥2500	-
2nd-PIT	Without	≥1000	2500	≥1000	≥1000	5000
	With	≥333	≥1000	≥1000	≥333	≥2500
3rd-PIT	Without	1000	n.t.	n.t.	1000	n.t.
	With	n.t.	n.t.	≥333	1000	n.t.

n.t. = not tested

 

The results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria. The number of revertant colonies in the negative controls, with and without S9 mix, were within the range of the respective historical control data of each tester strain (see Appendix – historical control data). In addition, the positive control substances induced a significant increase in the number of revertant colonies with and without S9 mix. This increase was compatible to the range of the respective historical control data.

Conclusions:

The test substance did not lead to a relevant increase in the number of revertant colonies without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard plate test and preincubation assay). Therefore, the test susbtance is not mutagenic in the Salmonella typhimurium/E.coli bacterial reverse mutation test with and without metabolic activation under the conditions tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The following data are available:

• In vitro: Ames test (OECD 471, GLP): not mutagenic


• In vitro: HPRT test (OECD 476, GLP): not mutagenic


• In vivo: no study available

 

Mutagenicity in bacteria: Ames test (OECD 471, GLP)

The key study for mutagenicity in bacteria was performed and reported according to the requirements of OECD testing guideline 471 and GLP.

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of five histidine-requiring bacterial strains, i.e. Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102) and Escherichia coli (WP2 uvrA), in a reverse mutation assay. The reverse mutation assay included both absence and presence of metabolic activation by phenobarbital-/beta-naphthoflavone -induced rat liver post-mitochondrial fraction (S9) in two independent experiments.

All bacterial strains of S. typhimurium as well as E. coli were treated with 33-5000 µg/plate in the standard plate test with or without metabolic activation. 

Test substance precipitation was observed at and above 2500 μg/plate with and without S9 mix.

No relevant increase in the number of revertants was observed in any of the conditions tested. 

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of external metabolic activation.

 

Mutagenicity in mammalian cells: HPRT test (OECD 476, GLP)

The key study for mutagenicity in mammalian cells was performed and reported according to the requirements of OECD testing guideline 476 and GLP.

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out, both with (1st Experiment only) and without the addition of liver S9 mix from phenobarbital- and ß-naphthoflavone induced rats (exogenous metabolic activation). 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.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.
In both experiments in the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations. However, cytotoxicity indicated by low cell counts were observed at 2000.0 and 5000.0 μg/mL without S9 mix or 5000.0 μg/mL with metabolic activation in Experiment 1.
Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other.
Under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

 

Since the registered substance Alkaline co-precipitation products of soluble cobalt, manganese and nickel salts and the different constituents of the UVCB release the same toxicological relevant unit under physiological relevant conditions, the overall toxicity of dissolved nickel, cobalt and manganese ions can be used to assess the toxicity of the registered substance for endpoints where substance specific data is not required. Therefore, data from Nickel dihydroxide, Cobalt dihydroxide, Tricobalt tetraoxide, Trimanganese tetraoxide and Manganese dioxide are used for hazard assessment based on a worst-case mixture approach.

Nickel dihydroxide is classified for genetic toxicity as Muta. 2 (H341) under the 1st ATP to the CLP. Based on the available data Cobalt dihydroxide, Tricobalt tetraoxide, Trimanganese tetraoxide and Manganese dioxide are not classified for genetic toxicity under Regulation (EC) No. 1272/2008.

Justification for classification or non-classification

Based on substance specific data and the mixture approach, the registered substance is classified for genetic toxicity as Muta. 2 (H341) under Regulation (EC) No. 1272/2008.