Calcination products of BaCO3, SrCO3, MgO, Al2O3, Eu2O3 and MnCO3

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic activity of the substance was evaluated according to OECD 471, according to OECD 473 and according to OECD 490. The result of all 3 in vitro tests is negative, with and without metabolic activation.

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:

December 2003 - March 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles. Only the preparation of the test solutions is not clearly described.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(1997)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Specific gravity used: 3.83
- Solubility: Insoluble in water

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

First, a suspension was obtained by extracting the test substance at 500 mg/mL vehicle at 45 degrees Celsius for at least 72 hours prior to each of the 2 experiments. Part of this suspension was diluted and used for 2 of the 5 test solutions in the experiments. Subsequently, part of the 500 mg/mL suspension was centrifuged for 10 minutes at 800 rpm. The extract of that suspension was also diluted and used for 3 of the 5 test solutions.

Experiment 1 and 2:
All strains: Without and with S9-mix: 1.67 and 5 mg/plate and 5.56, 16.67 and 50 mg extract after centrifugation/plate

As precipitation was observed, the test substance concentration was sufficiently high.

Vehicle / solvent:

- Vehicle used: phosphate buffered saline adjusted to pH 6.55
- Justification for choice of vehicle: the test substance is insoluble in water and soluble in inorganic acids. Since the use of acid is not possible, the test substance was extracted in phosphate buffered saline, adjusted to slightly acidic conditions

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: preincubation

DURATION
- Exposure duration: 48 to 72 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered positive if a concentration-related increase over the range tested and/or a reproducible increase in at least one or more concentrations in the number of revertant colonies per plate in at least one strain (with or without metabolic activation). Biological relevance of the results will be considered first. The increase in mean revertants at the peak of the dose response is at least three times the mean vehicle control value for strains TA1535 and TA1537 and at least twice the value for strains TA98, TA100 and WP2uvrA.
A test substance is considered negative (not mutagenic) in the test if a result does not meet the above criteria.

Statistics:

Dunnett's analysis was used.

Key result

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation was observed at the 2 dose levels 1.67 and 5 mg/plate

RANGE-FINDING/SCREENING STUDIES:
Not applicable

COMPARISON WITH HISTORICAL CONTROL DATA:
- The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the highest dose level

Conclusions:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.

Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, both in the absence and presence of S9-mix. The substance was tested up to limit concentrations. No cytotoxicity was observed. Slight precipitation was observed at the 2 dose levels 1.67 and 5 mg/plate.

Adequate solvent and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 August 2015 - 16 October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(2014)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(2008)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- pH (1% in water, indicative range): 6.5 - 6.2

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood was collected from healthy adult, non-smoking volunteers (approx 18 to 35 years of age). Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

As the test substance precipitated at concentrations of 164 µg/mL and upwards, the following concentrations were tested:

- Dose range finding test:
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1.7, 5.4, 17, 52 and 164 µg/mL

- First cytogenetic assay:
Without S9-mix, 3hr exposure; 24 hr fixation: 17, 52 and 164 µg/mL
With S9-mix (1.8%), 3hr exposure; 24 hr fixation: 17, 52 and 164 µg/mL
(all dose levels were selected for scoring of chromosome aberrations)

- Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 17, 52 and 164 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 17, 52 and 164 µg/mL
(all dose levels were selected for scoring of chromosome aberrations)

Vehicle / solvent:

- Vehicle used: the test substance was suspended in dimethyl sulfoxide (DMSO) of spectroscopic quality. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Test substance concentrations were used within 2.5 hours after preparation. The final concentration of the solvent in the culture medium was 1.0% (v/v).

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO; 1.0% v/v)

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR: colchicine
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%).

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) Any of the results are outside the 95% control limits of the historical control data range.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) All results are inside the 95% control limits of the negative historical control data range.

In case the Fisher’s exact test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group a Cochran Armitage trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction.

Statistics:

See above

Key result

Species / strain:

lymphocytes: Cultured peripheral human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Cytotoxicity was not observed
- Both in the absence and presence of S9-mix L 125 PLUS did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.
- No effects of L 125 PLUS on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Conclusions:

A chromosome aberration study with the substance was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that the substance is not clastogenic in human lymphocytes.

Executive summary:

In accordance with OECD 473 (2014) and according to GLP principles, a chromosome aberration study with the substance was performed in cultured peripheral human lymphocytes in two independent experiments with and without metabolic activation. Adequate solvent and positive controls were included. The substance was tested up to and including precipitating concentrations.

The substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Based on the results of this study, it is concluded that the substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations. Based on these results, it is concluded that the substance is not clastogenic in human lymphocytes under the experimental conditions of the study.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 November 2015 - 14 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Exposure medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin; supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium) in case of 3 hour exposure and supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium) in case of 24 hour exposure
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
- Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 μg/mL trifluorothymidine (TFT)
- Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
As the test substance precipitated in the exposure medium at ≥164 µg/mL, the following concentrations were tested:
Without and with S9-mix (4% (v/v)), 3 hours treatment: 5.4, 17, 52, 164 and 512 µg/mL
Without S9-mix, 24 hours treatment: 5.4, 17, 52, 164 and 512 µg/mL

Experiment 1:
As no toxicity was observed in the dose range finding test, the following concentrations were tested in 2 experiments:
Without and with S9-mix (4% (v/v)), 3 hours treatment: 0.055, 0.17, 0.55, 1.7, 5.4, 17, 52 and 164 µg/mL
Experiment 2:
Without S9-mix, 24 hours treatment: 0.055, 0.17, 0.55, 1.7, 5.4, 17, 52 and 164 µg/mL

Vehicle / solvent:

- Vehicle: DMSO (recommended by guideline)
- Test substance concentrations were used within 1 hour of preparation.

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9; 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9; 7.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CE day2) is between 65 and 120% in order to have an acceptable number of surviving cells analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The positive control should demonstrate an absolute increase in the total mutation frequency above the spontaneous background MF (an induced MF (IMF) of at least 300 x 10^-6). At least 40% of the IMF should be reflected in the small colony MF. Furthermore, the positive control should have an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent/control (a small colony IMF of at least 150 x 10^-6).

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces an MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range. A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study. A test substance is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No data
- Effects of osmolality: No data
- Precipitation: Precipitation was observed at dose levels of 164 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed up to and including 512 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed up to and including the highest tested precipitated dose levels in both experiments in the absence and presence of S9-mix.

POSITIVE CONTROLS
- Mutation frequencies in cultures treated with positive control chemicals were increased 15- and 9-fold for MMS in the absence of S9-mix, and 21-fold for CP in the presence of S9-mix
- The mutation frequencies in cultures treated with positive control chemicals were between the minimum and maximum value of the historical control data range

Conclusions:

The mouse lymphoma assay was conducted according to OECD 490 guideline and GLP principles. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the absence and presence of S9 -mix.

Executive summary:

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles.

No toxicity was observed up to and including the highest tested precipitated dose levels in both experiments in the absence and presence of S9-mix. In the absence of S9-mix, the substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, the substance did not induce a significant increase in the mutation frequency in one experiment. Positive control chemicals induced appropriate responses. It is concluded that the substance is not mutagenic in the TK mutation test system under the experimental conditions described in the absence and presence of S9 -mix.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro gene mutation study in bacteria

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, both in the absence and presence of S9-mix. The substance was tested up to limit concentrations. No cytotoxicity was observed. Slight precipitation was observed at the 2 dose levels 1.67 and 5 mg/plate. Adequate solvent and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA, both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with and without metabolic activation.

In vitro mammalian chromosome aberration test

In accordance with OECD 473 (2014) and according to GLP principles, a chromosome aberration study with the substance was performed in cultured peripheral human lymphocytes in two independent experiments with and without metabolic activation. Adequate solvent and positive controls were included. The substance was tested up to and including precipitating concentrations. The substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments. No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Based on the results of this study, it is concluded that the substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations. Based on these results, it is concluded that the substance is not clastogenic in human lymphocytes under the experimental conditions of the study.

In vitro mammalian cell gene mutation assay

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles. No toxicity was observed up to and including the highest tested precipitated dose levels in both experiments in the absence and presence of S9-mix. In the absence of S9-mix, the substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time. In the presence of S9-mix, the substance did not induce a significant increase in the mutation frequency in one experiment. Positive control chemicals induced appropriate responses. It is concluded that the substance is not mutagenic in the TK mutation test system under the experimental conditions described in the absence and presence of S9-mix.

Justification for classification or non-classification

Based on the available information, the substance is not classified for genetic toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No 1272/2008 (CLP Regulation).