Tris[oxalato(2-)]digadolinium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro

In vitro mutagenicity in bacteria

A single reliable (Klimisch 1) key study is available (Varga-Kanizsai, 2016), performed according to OECD guideline 471 and conform GLP requirements. In this study, gadolinium oxalate did not show mutagenic activity in the applied bacterium tester strains in the absence or presence of metabolic activation under the conditions of the test system.

In vitro chromosome aberration study and in vitro gene mutation study in mammalian cells

Reliable (Klimisch 1) studies are available (Hargitai, 2017a,b). These studies were performed according to OECD guidelines 473 and 476 and conform GLP requirements. In these studies, gadolinium oxalate was not found to be clastogenic or mutagenic in the absence or presence of metabolic activation under the conditions of the test system. These studies are considered to be the key studies for endpoint coverage.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2016-06-20 to 2017-10-24

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:

2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature (15-25°C, below 70 RH %)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: insoluble, suspension only

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Stock formulations of 200 mg/mL anhydrous gadolinium oxalate in vehicle were prepared.

CORRECTION FACTOR
- A correction factor of 2.05 was applied during formulation to achieve the target concentration of the formulation expressed in anhydrous form.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cells Cultures)
- Sex: male
MEDIA USED
- Laboratory cultures were maintained in 150 cm2 plastic flasks at 37 +/- 0.5°C in a humidified atmosphere containing approximately 5% CO2 in air.
- V79 cells were grown in Dulbecco's Modified Eagle's Medium supplemented with 2 mM L-glutamine, 1% v/v antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphotericin-B) and 10% v/v heat-inactivated fetal bovine serum (DMEM-10, culture medium).
- During the treatments, the serum content of the medium was reduced to 5% v/v (DMEM-5).

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix, induced by a mixture of phenobarbital and beta-naphthoflavone

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assays were selected based on the results of a short preliminary test. Two concentration selection cytotoxicity assays were performed to establish an appropriate concentration range for the chromosome aberration assays (assay A: 3-h treatment with and without metabolic activation, 20-h harvesting time; assay B: 3-h treatment with metabolic activation or 20-h treatment without metabolic activation, 28-h harvesting time). A total of 10 concentrations between 2000 and 3906 µg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay. Treatment concentrations for the chromosome aberration assays were selected on the basis of results of these assays according to the OECD guideline instructions.

Chromosome Aberration Assay 1
3-hour treatment in the presence of S9-mix:
2000, 666.7, 222.2, 74.08 and 24.69 μg/mL
3-hour treatment in the absence of S9-mix:
2000, 666.7, 222.2, 74.08 and 24.69 μg/mL

Chromosome Aberration Assay 2
3-hour treatment in the presence of S9-mix:
2000, 666.7, 222.2, 74.08 and 24.69 μg/mL
20-hour treatment in the absence of S9-mix:
1500, 1000, 500, 250, 125 and 62.5 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 1% w/v methyl cellulose aqueous solution
- Justification for choice of solvent/vehicle: Based on available data (CiToxLAB study code 16/093-007M) and due to the chemical nature of the test item. The test item was insoluble in generally used vehicles (distilled water, DMSO, DMF, acetone), only a suspension could be achieved up to 100 mg/mL. To ensure suspension homogeneity (lower sedimentation speed), 1% w/v methyl cellulose aqueous solution was selected as vehicle of the study.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

1% methyl cellulose aqueous solution (negative (vehicle) control)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION
- Cell density at seeding (if applicable): 5 x 10^5 cells/dish

DURATION
- Exposure duration: 3 h (assay 1, with and without metabolic activation), 3 h (assay 2, with metabolic activation), 20 h (assay 2, without metabolic activation)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h (assay 1), 28 h (assay 2)

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.2 µg/mL)

STAIN (for cytogenetic assays): 5% Giemsa solution

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED
- 2 to 2.5 h prior to harvesting, cell cultures were treated with colchicine (0.2 µg/mL).
- Cells were swollen with 0.075 M KCl hypotonic solution for 8 min, then were washed in fixative (methanol:acetic acid 3:1 v/v mixture) until the preparation became plasma free (4 washes).
- A suspension of the fixed cells was dropped onto clean microscope slides and air-dried.
- Slides were stained with 5% Giemsa solution, air-dried and coverslips were mounted.
- At least three slides were prepared for each culture.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells)
- At least 150 metaphases with 22 +/- 2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible.
- Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.

DETERMINATION OF CYTOTOXICITY
- Method: At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC (Relative Increase in Cell Counts).

OTHER EXAMINATIONS
- Determination of polyploidy: Yes. Polyploid metaphases are defined as metaphases with approximate multiples of the haploid chromosome number (n), other than the diploid number (i.e. ca. 3n, 4n etc.).
- Determination of endoreplication: Yes. Endoreduplicated metaphases have chromosomes with 4, 8, etc. chromatids.

Rationale for test conditions:

Per Guideline

Evaluation criteria:

The assay is considered valid, if the following criteria are met:
- The negative (vehicle) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.

The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolality of the treated cultures.

Statistics:

Fisher's exact test. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was only observed at the highest concentration (1500 µg/mL) in assay 2 without metabolic activation (RICC = 51%). In both assays, the test item was tested up to precipitating concentrations.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In assay 1, insolubility (precipitation / minimal amount of precipitate) was detected at the end of the treatment period in the final treatment medium in the 2000-74.08 µg/mL concentration range with and without metabolic activation. There were no large changes in pH and osmolality. No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 666.7, 222.2, 74.08 and 24.69 µg/mL were chosen for evaluation in the experiment with and without metabolic activation.

In assay 2, insolubility was detected at the end of the treatment period in the final treatment medium in the 1500-62.5 µg/mL concentration range without metabolic activation and in the 2000-74.08 µg/mL concentration range with metabolic activation. There were no large changes in pH and osmolality. No cytotoxicity was observed in the experiment with metabolic activation, while cytotoxicity was detected in the experiment without metabolic activation (RICC value of the highest evluated concentration was 51%). Concentrations of 1500, 1000, 500, 250 and 125 µg/mL were evaluated in the experiment without metabolic activation, and concentrations of 666.7, 222.2, 74.08 and 24.69 µg/mL were evaluated in the experiment with metabolic activation.

The test item did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments without metabolic activation. In the experiments with metabolic activation, a marginal increase was seen at one concentration (222.2 µg/mL) in assay 1, however there was no dose response (no other increases were observed including the higher evaluated concentration in the same experiment), and the observed effect was not reproducible between replicates (increase was found only in one replicate of assay 1) nor between experiments using the same experimental conditions (no similar increase was seen in assay 2 with metabolic activation). Based on these facts, these data did not meet the criteria of a positive response, therefore, the test item was considered to have a negative response.

Polyploid metaphases (1-4) were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments. No endoreduplicated metaphases were detected in the performed experiments.

The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency and the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation, demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; at least four test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

Conclusions:

Gadolinium oxalate was considered not to be clastogenic in the absence and presence of metabolic activation under the conditions of the test system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-03-10 to 2017-10-19

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:

2016

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

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:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature (15-25°C, below 70 RH %)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: insoluble, suspension only

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Stock formulations of 200 mg/mL anhydrous gadolinium oxalate in vehicle were prepared.

CORRECTION FACTOR
- A correction factor of 2.05 was applied during formulation to achieve the target concentration of the formulation expressed in anhydrous form.

Target gene:

hprt (hypoxanthine-guanine phosphoribosyl transferase) enzyme locus located on the X chromosome of Chinese Hamster Ovary (CHO) cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

Sub-line K1

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection (Manassas, Virginia, United States)

MEDIA USED
- Periodically checked for Mycoplasma contamination: Yes, check carried out for each batch of frozen stock. Cell line tested was negative.
- Periodically 'cleansed' against high spontaneous background: Yes, prior to use in the test, the culture was cleansed of pre-existing mutant cells by culturing in HAT medium on 22 April 2016. Cells were stored as frozen stocks in a liquid nitrogen tank. For each experiment, one or more vials were thawed rapidly.
- Type and identity of media: When thawed, cells were diluted in F12-10 medium (culture medium) and incubated at 37°C, in a humidified atmosphere (5% CO2 in air). When cells were growing well, subcultures were established in an appropriate number of flasks. Trypsin-EDTA (0.25% Trypsin, 1 mM EDTA) solution was used for cell detachment to subculture.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9, induced by treatment with a mixture of phenobarbital and beta-naphthoflavone

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assays were selected based on the result of a short preliminary toxicity test. In this preliminary experiment, a 5-h treatment in the presence and absence of S9-mix and a 24-h treatment in the absence of S9-mix were performed with a range of test concentrations (highest concentration 2000 µg/mL) to determine toxicity immediately after the treatments.

The concentrations in the mutation assays were as follows:
Assay 1:
5-hour treatment in the presence of S9-mix:
2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL
5-hour treatment in the absence of S9-mix:
2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL
Assay 2:
5-hour treatment in the presence of S9-mix:
2000, 666.7, 222.2, 74.07, 24.69 and 8.23 μg/mL
24-hour treatment in the absence of S9-mix:
2000, 1333.4, 666.7, 222.2, 74.07, 24.69, 8.23 and 2.74 μg/mL

Vehicle / solvent:

1% (w/v) aqueous methyl cellulose solution was selected as vehicle for the test item in this study. Based on available data (CiToxLAB study code 16/093-007M, Ames test), due to the insolubility of the test item in commonly used vehicles (distilled water, DMSO, DMF, acetone), only suspensions could be prepared. To ensure suspension homogeneity (by lowering sedimentation rate), 1% (w/v) aqueous methyl cellulose solution was selected as appropriate vehicle. This vehicle was compatible with the survival of the mammalian cells and the metabolic activation system. Based on the information from another in vitro study (CiToxLAB study code 16/093-020C, in vitro CAT), a suspension at 200 mg/mL concentration was achievable using this vehicle.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

1% methyl cellulose aqueous solution (negative (vehicle) control for the test material) / DMSO (negative (vehicle) control for the positive control test materials)

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION
- In medium in plates.
- Cell density at seeding (if applicable): At least 2x10^6 cells were placed in each of a series of sterile dishes for 24 h incubation before treatment.

DURATION
- Pre-incubation period: Before treatment: 24 h (all treatments). After treatment and before start of expression period: 19 h (only for the 5-h treatments).
- Exposure duration: Assay 1: 5-h treatment with and without metabolic activation; Assay 2: 5-h treatment with metabolic activation and 24-h treatment wihtout metabolic activation.
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7-8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 15-16 days (for the 5-h treatments: 5 h treatment, 19 h pre-incubation, 7 days expression time, 7-8 days selection time; for the 24-h treatments: 24 h treatment, 7 days expression time, 7-8 days selection time)

SELECTION AGENT (mutation assays): 6-thioguanine, to determine mutation frequency

NUMBER OF REPLICATIONS:
- Duplicate cultures were tested in all cases (Assay 1: 2 cultures with and 2 without S9-mix, Assay 2: 2 cultures with and 2 without S9-mix).
- Triplicate dishes for survival check (after treatment and/or pre-incubation) and determination of viability (at the end of the expression period)
- 5 replicates plates per group for determination of mutation frequency

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED
After the growing or selection period, the culture medium was removed and colonies were fixed for 5 minutes with methanol. After fixation, colonies were stained using 10% Giemsa solution (diluted with distilled water) for 30 minutes, dried and manually counted.

NUMBER OF CELLS EVALUATED: 200 cells per dish (3 parallel dishes) for survival check (after treatment and/or pre-incubation) and determination of viability (at the end of the expression period), 4x10^5 cells/plate (5 replicate plates) for determination of mutation frequency

DETERMINATION OF MUTATION FREQUENCY
Mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected, corrected for the cloning efficiency of cells prior to mutant selection (viability) and were expressed as 6-TG resistant mutants per 10^6 clonable cells.

DETERMINATION OF CYTOTOXICITY
Method: cloning efficiency

Rationale for test conditions:

Per OECD guideline 476

Evaluation criteria:

The test item was considered to be mutagenic in this assay if the following criteria were met:
1. The assay was valid.
2. The mutant frequency at one or more doses was significantly greater than that of the relevant negative (vehicle) control (p<0.05).
3. Increase of the mutant frequency was reproducible.
4. There was a dose-response relationship.

The assay was considered valid if all of the following criteria were met:
1. The mutant frequency in the negative (vehicle/solvent) control cultures was in accordance with the historical control data.
2. The positive control chemicals induced a clear increase in mutant frequency.
3. The cloning efficiency of the negative controls was in the range of 60-140% on Day 1 and 70-130% on Day 8.
4. At least four test item concentrations in duplicate cultures were presented.

Statistics:

The mutation frequencies were statistically analysed. Statistical evaluation of data was performed with the SPSS PC+4.0 statistical program package (SPSS Hungary Ltd., Budapest, Hungary). The heterogeneity of variance between groups was checked by Bartlett`s test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained results were significant, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined using the Kolmogorow-Smirnow test. In the case of non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was applied. If a positive result was detected, the inter-group comparisons were performed using the Mann-Whitney U-test. Data also were checked for a trend in mutation frequency with treatment dose using Microsoft Excel 2007 software (R-squared values were calculated for the log concentration versus the mutation frequency).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

Sub-line K1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In Assay 1, insolubility (precipitate / minimal amount of precipitate) was detected in the final treatment medium at the end of the treatment in the 2000-24.69 µg/mL concentration range (experiment with S9-mix) and in the 2000-74.07 µg/mL concentration range (experiment without S9-mix). In Assay 2, insolubility was detected in the final treatment medium at the end of the treatment in the 2000-8.23 µg/mL concentration range (experiment with S9-mix) and in the 2000-2.74 µg/mL concentration range (experiment without S9-mix).
- No large changes in pH and osmolality were observed after treatment in any of the cases.

RANGE-FINDING/SCREENING STUDIES: Treatment concentrations for the mutation assay were selected based on the results of a preliminary experiment (5-h treatment with S9-mix and 24-h treatment without S9-mix). The concentrations in these experiments were 3.906, 7.813, 15.625, 31.25, 62.5, 126, 250, 500, 1000 and 2000 µg/mL. Insolubility and cytotoxicity were detected in the preliminary experiment. The concentrations selected for the main experiments were based on the recommended maximum concentration, but cytotoxicity data were also taken into account to cover the range from cytotoxicity to no or little cytotoxicity according to the OECD 476 guideline. Lower test concentrations were separated by a factor of 2, but more closely spaced concentrations were selected in the expected cytotoxic concentration range. At least 6 concentrations were selected for the main experiments.

HISTORICAL CONTROL DATA
- Positive historical control data: The anticipated increases in mutation frequency over the controls were observed. The increases were in good harmony with the historical data in all assays.
- Negative (solvent/vehicle) control: Spontaneous mutation frequency was in accordance with the general historical control range in all assays, and the observed values were in the expected range (5-20 x 10^-6).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Assay 1, with S9-mix (5-h treatment): No marked cytotoxicity observed (relative survival of 75% and 102% after treatment and on the survival plates, respectively, for the highest concentration of 2000 µg/mL).
- Assay 1, without S9-mix (5-h treatment): No marked cytotoxicity observed (relative survival of 30% and 52% after treatment and on the survival plates, respectively, for the highest concentration of 2000 µg/mL).
- Assay 2, with S9-mix (5-h treatment): No marked cytotoxicity observed (relative survival of 85% and 99% after treatment and on the survival plates, respectively, for the highest concentration of 2000 µg/mL).
- Assay 2, without S9-mix (24-h treatment): Cytotoxicity was observed, but the degree of cytotoxicity at the highest concentration of 2000 µg/mL did not acceed the acceptable level (relative survival of 27% and 78% after treatment and on the survival plates, respectively, and 15% of relative total growth).

Conclusions:

No mutagenic effect of gadolinium oxalate was observed in the presence or absence of metabolic activation under the conditions of this hprt assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2016-05-17 to 2016-09-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Final dilution of a dissolved solid, stock liquid or gel: Based on the results of the preliminary tests, a 100 mg/mL stock formulation was prepared in DMSO, which was diluted by serial dilutions in at least six steps to obtain at least seven dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate. The formulations were stirred with a magnetic stirrer in the main tests.
- Dimethyl sulfoxide (DMSO) was used as solvent to prepare the stock formulation of the test material. Test suspensions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock formulation using the selected solvent and were used within 4 hours after preparation.
- A correction factor of 2.05 was used for dose formulation preparation. Active ingredient content conversion was applied during the study. All concentrations and dose-levels were expressed as active ingredient content of the test item.

FORM AS APPLIED IN THE TEST (if different from that of starting material): suspension in dimethyl sulfoxide

Target gene:

histidine and tryptophan

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:

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

Test concentrations with justification for top dose:

100 mg/mL (5000 µg/plate), 31.62 mg/mL (1581 µg/plate), 10 mg/mL (500 µg/plate), 3.162 mg/mL (158.1 µg/plate), 1 mg/mL (50 µg/plate), 0.3162 mg/mL (15.81 µg/plate), 0.1 mg/mL (5 µg/plate), 0.03162 mg/mL (1.581 µg/plate)

Note: Concentrations 1-7 were examined in the Initial Mutation Test, concentrations 1-8 were examined in the Confirmatory Mutation Test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The appropriate vehicle (solvent) and the behaviour of the test item formulations with the solution of top agar and phosphate buffer were examined in a preliminary compatibility test. DMSO was used as solvent to prepare the stock formulation of the test material since better biocompatibility and homogeneity was observed in the preliminary compatibility test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-1,2-phenylenediamine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

INITIAL MUTATION TEST
- Method of application: plate incorporation method
- Bacteria (cultured in Nutrient Broth No.2) 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. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level). The equivalent number of minimal glucose agar plates 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
- A pre-incubation procedure was performed as a Confirmatory Mutation Test since no positive effect was observed in the Initial Mutation Test.
- Bacteria (cultured in Nutrient Broth No.2.) 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 was added into appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). 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 were 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.

EVALUATION OF EXPERIMENTAL DATA
- The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
* Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

In the main tests each sample (including the controls) was tested in triplicate.

Evaluation criteria:

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.

According to the guidelines, statistical method 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.

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.

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in the Confirmatory Mutation Test with metabolic activation at the 5000 µg/plate concentration - also tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

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

Untreated negative 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

Untreated negative 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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Preliminary Concentration Range Finding Test:
- The observed numbers of revertant colonies were in the normal range. Minor differences compared to the solvent control numbers were observed in some sporadic cases. However, they had no biological significance and were within the historical control range in all cases; thus, they were considered as reflecting the variability of the test system.
- Precipitate/slight precipitate was observed in Salmonella typhimurium TA98 strain with and without metabolic activation at 5000, 2500 and 1000 µg/plate concentrations and in Salmonella typhimurium TA100 strain with and without metabolic activation at 5000 and 2500 µg/plate concentrations. Note that due to the precipitate on the plates the assessment of the background lawn was difficult in all examined bacterial strains with and without metabolic activation at 5000 µg/plate concentration. Counting of colonies was not affected.
- Inhibitory or toxic effect of the test item was not detected in the Preliminary Concentration Range Finding Test.

ADDITIONAL INFORMATION:
Precipitate/slight precipitate was observed in the Initial Mutation Test and in the Confirmatory Mutation Test in all examined bacterial strains with and without metabolic activation at 5000 and 1581 µg/plate concentrations, furthermore in the Confirmatory Mutation Test in Salmonella typhimurium TA98 strain without metabolic activation at 500 µg/plate concentration.

In the Initial Mutation Test (using the plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 5000 and 50 μg/plate concentrations without metabolic activation (the observed mutation factor value was 1.82 in both cases). Higher numbers of revertant colonies compared to the solvent control plates were observed at some other tested concentrations in this strain without metabolic activation. However, there was no doseresponse 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 (using the pre-incubation method) the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 500 μg/plate concentration without metabolic activation (the observed mutation factor value was 2.00). Higher numbers of revertant colonies compared to the solvent control plates were observed at some other tested concentrations in this strain without metabolic activation. However, there was no 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.

Inhibitory, cytotoxic effect of the test item (slightly reduced background lawn development) was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA98 strain with metabolic activation at 5000 µg/plate concentration.

Lower revertant counts compared to the solvent control were observed in the Initial Mutation Test and Confirmatory Mutation Test in some cases. However, the mean numbers of revertant colonies were within the historical control range, thus they were considered as biological variability of the test system.

Slight increases in the numbers of revertant colonies compared to the solvent control were detected in some other sporadic cases. However, no dose-dependence was observed and they were below the biologically relevant threshold value and were within the historical control range, they were considered as reflecting the biological variability of the test.

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 included a clearly toxic concentration or exhibited limited solubility as demonstrated by the preliminary toxicity range-finding test or extended to 5 mg/plate. No more than 5% of the plates were lost through contamination or some other unforeseen event. 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.

*In the Confirmatory Mutation Test in case of Salmonella typhimurium TA100 strain without metabolic activation the positive control (SAZ) showed the expected positive response, however the actual number of revertant colonies was out of the historical control range (although it was close to the lower limit of the historical control range, the mean value of SAZ was 875.3). This fact was considered to have no impact on the results or integrity of the study.

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 had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro

In vitro mutagenicity in bacteria

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay (Varga-Kanizsai, 2016) according to OECD guideline 471 and conform GLP requirements. The experiments were carried out using histidine-requiring auxotrophic strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotrophic strain of E. coli (WP2 uvrA) in the presence and absence of metabolic activation (cofactor-supplemented post-mitochondrial S9 fraction (rat liver)) prepared from the livers of phenobarbital/beta-naphthoflavone-induced rats. The study included a preliminary compatibility test, a preliminary concentration range finding test, an initial mutation test and a confirmatory mutation test. Based on the results of a solubility test, the test item was formulated in DMSO. Based on the results of the range finding test, the test item concentrations in the initial mutation test (5 strains) were 5000, 1581, 500, 158.1, 50, 15.81 and 5 µg/plate, and the test item concentrations in the confirmatory mutation test (5 strains) were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 µg/plate. The tests were considered to be valid. The data demonstrate that the test item had no mutagenic activity in the applied bacterium tester strains in the absence or presence of metabolic activation under the test conditions used in this study. Untreated, negative (solvent) and positive controls were run concurrently. The results obtained in the control treatments were within the historical control range, except for those obtained in the confirmatory mutation test in the Salmonella typhimurium TA100 strain without metabolic activation, where the actual number of revertant colonies in the test with the positive control substance sodium azide was out of the historical control range (although it was close to the lower limit of the historical control range). This observation was however not considered to have impacted the results or integrity of the study. This study is considered reliable (Klimisch 1) and is considered as the key study for endpoint coverage.

In vitro chromosome aberration study in mammalian cells

The study of Hargitai (2017b) is performed according to OECD guideline 473 and conform GLP requirements. The experiments were carried out using Chinese hamster lung fibroblasts (V79) in the presence and absence of metabolic activation (rat liver S9-mix, induced by phenobarbital/beta-naphthoflavone). Treatment concentrations for the mutation assays were selected based on the results of a short preliminary test and ranged between 24.69 and 2000 µg/mL (Assay 1, 3-h treatment +/- S9-mix; Assay 2, 3-h treatment + S9-mix) or between 62.5 and 1500 µg/mL (Assay 2, 20-h treatment - S9-mix). The vehicle used was 1% w/v methyl cellulose aqueous solution (to ensure suspension homogeneity by lowering sedimentation speed). Cytotoxicity was only observed at the highest concentration (1500 µg/mL) in Assay 2 without metabolic activation (RICC = 51%). In both assays, the test item was tested up to precipitating concentrations. The test item did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments without metabolic activation. In the experiments with metabolic activation, a marginal increase was seen at one concentration (222.2 µg/mL) in Assay 1, however there was no dose response (no other increases were observed including the higher evaluated concentration in the same experiment), and the observed effect was not reproducible between replicates (increase was found only in one replicate of Assay 1) nor between experiments using the same experimental conditions (no similar increase was seen in Assay 2 with metabolic activation). Based on these facts, these data did not meet the criteria of a positive response, therefore, the test item was considered to have a negative response. The test was considered valid based on the results obtained in the negative (vehicle) and positive controls. In conclusion, gadolinium oxalate was not considered to be clastogenic in the absence and presence of metabolic activation under the conditions of the test system. This study is considered reliable (Klimisch 1) and is considered to be the key study for endpoint coverage.

In vitro mutagenicity study in mammalian cells (hprt assay)

The study of Hargitai (2017a) is performed according to OECD guideline 476 and conform GLP requirements. The experiments were carried out using Chinese Hamster Ovary (CHO) cells (sub-line K1) in the presence and absence of metabolic activation (rat liver S9-mix, induced by phenobarbital/beta-naphthoflavone). Treatment concentrations for the mutation assays were selected based on the results of a preliminary toxicity test and ranged between 8.23 and 2000 µg/mL (Assay 1, 5-h treatment +/- S9-mix; Assay 2, 5-h treatment + S9-mix) or between 2.74 and 2000 µg/mL (Assay 2, 24-h treatment - S9-mix). The vehicle used was 1% w/v methyl cellulose aqueous solution (to ensure suspension homogeneity by lowering sedimentation speed) and was compatible with the survival of the mammalian cells and the metabolic activation system. No cytotoxicity exceeding the acceptable level was observed, but the test item was tested up to precipitating concentrations in all assays. The mutant frequency was not significantly greater than that of the relevant negative (vehicle) control in any of the treatments in the different assays. The results obtained in the negative (vehicle) and positive controls were in line with historical control data. The test fulfilled all validity criteria. In conclusion, gadolinium oxalate was not considered to be mutagenic in the absence and presence or metabolic activation under the conditions of the test system. This study is considered reliable (Klimisch 1) and is considered to be the key study for endpoint coverage.

Justification for classification or non-classification

Based on the results of the available in vitro studies, gadolinium oxalate is considered not to be genotoxic and is not classified according to the CLP regulation.