Cerium terbium magnesium undecaaluminum nonadecaoxide (hexagonal) ((Ce, Tb)MgAl11O19) and aluminium oxide (Al2O3) from calcination of Al2O3, CeO2, MgO and Tb4O7

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in vitro assays (AMES, chromosome aberration test and an in vitro mammalian cell gene mutation test) were performed according to current OECD/EC guidelines and GLP principles to study the mutagenic potential of CAT. None of the studies showed mutagenic effects of the substance with or 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:

05 Oct 2011- 22 Dec 2011

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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

Test concentrations with justification for top dose:

Experiment 1
Preliminary test: TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (without and with S9)
Main study: TA1535, TA1537 and TA98: 33, 100, 333, 1000 and 3330 µg/plate (without and with S9-mix)
Experiment 2: 33, 100, 333, 1000 and 3330 µg/plate (without and with S9-mix)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle:
A homogeneous suspension could be obtained in DMSO/ DMSO is accepted and approved by authorities and international guidelines.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 at 2.5 µg/plate in DMSO for TA1537

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO at 2.5 µg/plate for TA1535; 2.5 or 5 µg/plate for TA1537; 1 µg/plate for TA98; 1 or 2.5 µg/plate for TA100; 10 µg/plate for WP2uvrA

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

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

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

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

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Key result

Species / strain:

S. typhimurium TA 1535

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:

S. typhimurium TA 1537

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:

S. typhimurium TA 98

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:

S. typhimurium 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: Precipitation was observed at dose levels of 3330 and 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within 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 top dose of 5000 µg/plate

Conclusions:

CAT is shown to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without S9-mix.

Executive summary:

CAT was tested in accordance with the most recent OECD and EC guidelines in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor). CAT did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four 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. No cytotoxicity was observed, but the substance was tested up to precipitating concentrations (at 3330 μg/plate and above).

Based on the results of this study it is concluded that CAT is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or 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:

20 Oct 2011 - 07 Jan 2012

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:

1997

Deviations:

yes

Remarks:

Study integrity was not adversely affected by the deviations.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Principles of method if other than guideline:

In the second cytogenetic assay the temperature was below the range of 37.0 ± 1.0°C with a minimum of 34.5°C for appr. 0.5 hour. In the dose range finding study at 3 h exposure, no duplicates or positive controls were included.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture 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.
- 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:

Dose range finding test:
Without and with S9-mix, 3hr exposure: 3, 10, 33, 100 and 333 μg CAT/mL
Without S9-mix, 24/48hr exposure: 3, 10, 33, 100, 333, 1000 and 3330 μg CAT/mL
First cytogenetic test:
Without and with S9-mix, 3 h exposure time: 10, 33 and 100 μg/mL
Second cytogenetic test:
Without S9-mix, 24/ 48 hr exposure: 10, 33 and 100 μg/mL
With S9-mix, 3 hr exposure: 10, 33 and 100 μg/mL

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: A homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Remarks:

DMSO at 1.0% (v/v)

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9

Negative solvent / vehicle controls:

yes

Remarks:

DMSO at 1.0% (v/v)

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

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 (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

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) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

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

Remarks:

at dose levels of 100 μg/ml and above.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- Precipitation: Precipitation in the exposure medium was observed at dose levels of 100 μg/ml and above.
- No toxicity was observed up to and including the highest precipitating tested dose
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses. The effect of the highest concentration of mitomycin C was not evaluated, since the lower concentration gave acceptable results.

Conclusions:

A chromosome aberration study with CAT was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments with and without metabolic activation. It is concluded that CAT is not clastogenic in human lymphocytes.

Executive summary:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of CAT (dissolved in DMSO), in the presence and absence of S9 -mix according to the most recent OECD and EC guidelines. In the first cytogenetic assay, CAT was tested up to 100 μg/ml for a 3 h exposure time with a 24 h fixation time (with CAT precipitating in the culture medium at this dose level). In the second cytogenetic assay, CAT was tested up to 100 μg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. In the presence of S9-mix CAT was also tested up to 100 μg/ml for a 3 h exposure time with a 48 h fixation time. CAT precipitated in the culture medium at this dose level. CAT 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 CAT on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that CAT does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19-03-2012 to 29-03-2012

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)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007) were followed.

Deviations:

no

GLP compliance:

yes

Type of assay:

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):

- Type and identity of media:
- 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 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

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:
Without and with S9-mix, 3 hours treatment: 3.3-333 µg/mL
Without S9-mix, 24 hours treatment: 3.3-333 µg/ml
Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.03, 0.1, 0.3, 1, 3, 10, 33 and 100 μg/ml
Experiment 2
Without and with S9-mix, 24 hours treatment: 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 μg/ml

Vehicle / solvent:

- Solvent used: DMSO (final conc. 0.8% (v/v))

Negative solvent / vehicle controls:

yes

Remarks:

DMSO at 0.8% (v/v)

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9

Negative solvent / vehicle controls:

yes

Remarks:

DMSO at 0.8% (v/v)

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

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 (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be 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 mutation frequency of MMS should not be below 500 per 10^6 survivors, and for CP not below 700 per 10^6 survivors.

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 a 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:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTGP 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

Remarks:

at 100 µg/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
- Effects of osmolarity: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 100 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to the precipitating dose levels of 100 µg/mL in the absence and presence of S9-mix.

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 and within the acceptability criteria of this assay.

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

Conclusions:

In a mouse lymphoma assay that was conducted according to OECD 476 guideline and GLP principles, CAT was tested up to precipitating concentrations. Based on the results of two independent experiments, it was concluded that CAT was not mutagenic with or without metabolic activation.

Executive summary:

A mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, CAT 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, CAT 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 concentration of the S9 for metabolic activation. CAT was tested up to precipitating concentrations. Based on these data, it is concluded that CAT is not mutagenic in the mouse lymphoma L5178Y test system with or without metabolic activation under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

AMES-test:

CAT was tested in accordance with the most recent OECD and EC guidelines in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix. CAT did not induce a significant dose-related increase in the number of revertant (His+) colonies in any of the four 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. No cytotoxicity was observed, but the substance was tested up to precipitating concentrations (at 3330 μg/plate and above). Based on the results of this study it is concluded that CAT is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.

Chromosome aberration study:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of CAT, in the presence and absence of S9 -mix according to the most recent OECD and EC guidelines. CAT was tested up to 100 μg/ml (precipitating concentration) in two independent experiments. CAT 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 CAT on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that CAT does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.

In vitro mammalian cell gene mutation test:

A mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles. The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, CAT 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, CAT 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 concentration of the S9 for metabolic activation. CAT was tested up to precipitating concentrations. Based on these data, it is concluded that CAT is not mutagenic in the mouse lymphoma L5178Y test system with or without metabolic activation under the experimental conditions described in this report.

Justification for classification or non-classification

Based on the available data, CAT does not have to be classified for mutagenicity according to CLP Regulation (EC) No 1272/2008.