Yttrium chloride hexahydrate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September 2016 - 3 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Yttrium chloride hexahydrate

Cas Number:

10025-94-2

Molecular formula:

YCl3.6H2O

IUPAC Name:

Yttrium chloride hexahydrate

Test material form:

solid: particulate/powder

Details on test material:

- Name of the test material (as cited in the report): yttrium trichloride hexahydrate
- Physical state: solid
- Appearance: white to yellowish crystalline powder
- Further information on test material confidential.

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Based on the results of the solubility test, a 100 mg/mL stock solution was prepared in distilled water, which was diluted by serial dilution to obtain dosing formulations for lower doses, spaced by factors of 2, 2.5 and approximately √10.
- No correction for purity of the test item was applied.

Method

Target gene:

Histidine locus (S. typhimurium strains TA98, TA100, TA1535 and TA1537); tryptophan locus (E. coli strain WP2uvrA)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

cofactor-supplemented post-mitochondrial S9 fraction (rat liver)

Test concentrations with justification for top dose:

Preliminary Concentration Range Finding Test: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate (TA98 and TA100).
Initial Mutation Test and Confirmatory Mutation Test: 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate (all strains).
Complementary Confirmatory Mutation Test in Salmonella typhimurium TA98 without metabolic activation: 15.81, 5, 1.581, 0.5, 0.1581, 0.05, 0.01581, 0.005 and 0.001581.
Complementary Confirmatory Mutation Test in Salmonella typhimurium TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA strains without metabolic activation: 15.81, 5, 1.581, 0.5, 0.1581, 0.05, 0.01581 and 0.005 µg/plate.

The selection of the doses was based on the results of the range finding study. No inhibitory, cytotoxic effect of the test item was observed in the range finding study. Test item precipitate and/or slight precipitate was observed on the plates at 5000, 2500, 1000 and 316 μg/plate concentrations in all examined strains with metabolic activation and at 5000, 2500, 1000, 316 and 100 μg/plate concentrations without metabolic activation in the preliminary experiment.

In the Confirmatory Mutation Test using the pre-incubation method, excessive cytotoxicity was observed in all examined bacterial strains without metabolic activation. In this case, the number of analysable doses did not meet the recommendations of the test guidelines. Therefore, an additional experiment (Complementary Confirmatory Mutation Test) was performed in these strains to complete the data. The experimental conditions were the same as in the Confirmatory Mutation Test, except of the tested concentrations.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using distilled water, dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). At a concentration of 100 mg/mL a clear solution was observed using distilled water as vehicle and after approximately 3 minutes vortex a clear solution was observed using DMSO and DMF as vehicles. Due to the better biocompatibility, distilled water was selected as vehicle (solvent) for the study.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-1,2-phenylenediamine

Remarks:

Salmonella TA98, non-activation, 4 µg

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Salmonella TA100 and TA1535, non-activation, 2 µg

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Salmonella TA1537, non-activation, 50 µg

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2 uvrA, non-activation, 2 µL

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All Salmonella strains, activation, 2 µg

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

E. coli WP2 uvrA, activation, 50 µg

Details on test system and experimental conditions:

METHOD OF APPLICATION
- Preliminary concentration range finding test / Initial mutation test: in agar (plate incorporation)
Bacteria (cultured in Nutrient Broth) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (3 per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C). The content of the tubes: top agar 2000 μL; vehicle or test item formulation (or reference controls) 50 μL; overnight culture of test strain 100 μL; phosphate buffer (pH 7.4) or S9 mix 500 μL. This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.
- Confirmatory mutation test and complementary confirmatory mutation test: pre-incubation method
Bacteria (cultured in Nutrient Broth) were exposed to the test item both in the presence and absence of an appropriate metabolic activation system. The equivalent number of minimal glucose agar plates was properly labelled. Molten top agar was prepared and kept at 45°C. Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer were added into appropriate tubes to provide direct contact between bacteria and the test item. The tubes (3 tubes per control and 3 tubes for each concentration level) were gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes, and then the content mixed and poured on the surface of minimal glucose agar plates. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative and positive controls. After preparation, the plates were incubated at 37°C for 48 ± 1 hours.

DURATION
- Pre-incubation period: 48 h (confirmatory mutation test and complementary confirmatory mutation test)
- Exposure duration: 48 h
- Selection time (if incubation with a selection agent): 48 h (simultaneously with exposure duration)
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

SELECTION AGENT (mutation assays): histidine (S. typhimurium strains); tryptophan (E. coli strains)

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background inhibition; decrease in the number of revertant colonies

Evaluation criteria:

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a concentration-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response:
A test article was considered non-mutagenic if:
- the total number of revertants in tester strain Salmonella typhimurium TA98, TA100 or Escherichia coli WP2 uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strain Salmonella typhimurium TA1535 or TA1537 is not greater than three times the concurrent vehicle control;
- the negative response should be reproducible in at least one follow up experiment.

Statistics:

According to the guidelines, statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Test item precipitate and/or slight precipitate was detected in the Initial Mutation in Salmonella typhimurium TA98, TA100 and TA1537 at 5000, 1581, 500 and 158.1 μg/plate concentrations without metabolic activation, in Salmonella typhimurium TA1535 and Escherichia coli WP2 uvrA at 5000, 1581, 500 μg/plate concentrations without metabolic activation, in Salmonella typhimurium TA98, TA1537 and E. coli WP2 uvrA at 5000, 1581 and 500 μg/plate concentrations with metabolic activation, and in Salmonella typhimurium TA100 and TA1535 at 5000 and 1581 μg/plate concentrations with metabolic activation.
In the Confirmatory Mutation Test test item precipitate and/or slight precipitate was observed in Salmonella typhimurium TA1537 and E. coli WP2 uvrA with metabolic activation at 5000, 1581 and 500 μg/plate concentrations. In addition to these concentrations this effect was observed in Salmonella typhimurium TA100 and TA1535 on the plates at 158.1 μg/plate concentration and in Salmonella typhimurium TA98 on the plates at 158.1 and 50 μg/plate concentrations. In the Complementary Confirmatory Mutation Test the same effect was detected in all examined bacterial strains without metabolic activation on the plates at 15.81 and 5 μg/plate concentrations. In addition to these concentrations, this effect was observed at 1.581 μg/plate concentration in Salmonella typhimurium TA98 and TA1535.

CYTOTOXICITY
- In the Initial Mutation Test reduced and/or slightly reduced background lawn development was detected in Salmonella typhimurium TA98 and TA100 with and without metabolic activation on the plates at 5000 μg/plate concentration and on one plate at 1581 μg/plate concentration in Salmonella typhimurium TA100 with metabolic activation. Slightly reduced background lawn development was detected in Salmonella typhimurium TA1535 and TA1537 on the plates at 5000 μg/plate concentration with metabolic activation. No inhibitory, cytotoxic effect of the test item was observed on E. coli WP2 uvrA strain.
- The same effect was observed in the Confirmatory Mutation Test with metabolic activation in E. coli WP2 uvrA on the plates at 5000 μg/plate concentration, in Salmonella typhimurium TA100 and TA1535 on the plates at 5000 and 1581 μg/plate concentrations, and in Salmonella typhimurium TA98 and TA1537 on the plates at 5000, 1581 and 500 μg/plate concentrations.
- In the Complementary Confirmatory Mutation Test the same effect was detected without metabolic activation in Salmonella typhimurium TA100 and TA1537 and in E. coli WP2 uvrA on the plates at 15.81 and 5 μg/plate concentrations, in Salmonella typhimurium TA1535 on the plates at 15.81, 5 and 1.581 μg/plate concentrations, and in Salmonella typhimurium TA98 on the plates at 15.81, 5, 1.581 and 0.5 μg/plate concentrations.

RESULTS
- In the Initial Mutation Test, the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 50 μg/plate concentration with metabolic activation (the observed mutation factor value was 1.91). However, there was no clear dose-response relationship, the observed
mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- In the Confirmatory Mutation Test and in the Complementary Confirmatory Mutation Test, the highest revertant rate was observed in Salmonella typhimurium TA1535 at 1581, 50 and 5 μg/plate concentrations with metabolic activation (the observed mutation factor value was 1.95). However, there was no clear dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
- Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%))
- Positive historical control data: The reference mutagens showed a distinct increase of induced revertant colonies. The viability of the bacterial cells was checked by a plating experiment in each test. The tests were considered to be valid.
- Untreated, negative (solvent/vehicle) and positive control plates were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range.

Any other information on results incl. tables

Validity of the tests

Untreated, negative (solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range. At least five analysable concentrations were presented in all strains of the main tests. The selected dose range exhibited limited solubility as demonstrated by the preliminary range finding test and extended to 5 mg/plate. No more than 5% of the plates were lost through contamination or some other unforeseen event. The tests were considered to be valid.

Applicant's summary and conclusion

Conclusions:

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item yttrium trichloride hexahydrate had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.