Gadoliniumsulfite trihydrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

19 February 2003 - 27 May 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

TA 98 (his D 3052, uvrB, rfa + R-factor)
TA 100 (his G 46, uvrB, rfa + R-factor)
TA 102 (his G 428, rfa + R-factor)
TA 1535 (his G 46, uvrB, rfa)
TA 1537 (his C 3076, uvrB, rfa)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

The Salmonella strains TA 98, TA 100, and TA 1535 were obtained from B.
N. Ames, University of Berkeley, California, USA, on August 15, 1985. Salmonella
typhimurium TA 102 was obtained from B. Diener, Institute of Toxicology,
University of Mainz, Germany, on June 24, 1992. Salmonella strain
TA 1537 was obtained from J. Hengstler, Institute of Toxicology, University
ofMainz, Germany, on December 8, 2000.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Escherichia coli WP2 uvrA was distributed by the National Collections of Industrial
& Marine Bacteria Ltd., Aberdeen, Scotland (NCIMB 11188 from
Aug. 18, 1977) and obtained from H. Träger, Knall AG, Ludwigshafen, Germany,
on December 23, 1994.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg per plate
The test material concentrations used were selected according to the EEC and OECD guidelines for this test system and the requirements ofthe Labor Ministry ofJapan. If the test material is soluble in the solvent of choice it should be investigated in the first experimental series at seven concentrations, separated by half-log intervals (i.e. a factor of root 10), ranging from 5 to 5000 µg per plate. In the second experimental series, usually 5 concentrations including at least 4 nontoxic concentrations should be tested. Signs of toxicity to the bacteria are a reduction in the number of spontaneaus revertants or a clearing of background lawn of non-revertant bacteria. Precipitation of the test material, if it occurs, should not interfere with scoring of the colonies. Depending upon the precipitation characteristics of the test material and toxicity data ascertained in the first series, high concentrations may be omitted or lower concentrations may additionally be tested in the second experimental series.

Vehicle / solvent:

0.5n HCl for the highest testmaterial concentration further diluted with distilled water for the next lower concentrations.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

cumene hydroperoxide

other: 2-aminoanthracene

Details on test system and experimental conditions:

Preparation of bacteria suspension
Fresh suspensions of the tester strains were prepared for each day's experiments. For each strain, 100 mL Standard I Nutrient Broth were inoculated by 500 µg/L of a frozen permanent culture and incubated overnight in a + 37°C shaking incubator. For the Refactor strains, ampicillin was added to the nutrient broth (25 µg/mL). S. typhimurium TA 102 was incubated overnight in the presence of 200 µg tetracycline per 100 mL nutrient broth. Cell density of the overnight cultures was checked by measuring the optical density of the cell suspensions and corrected if necessary. The suspensions were then maintained at ice-bath temperature until use.

Technique for testing
Salmonella typhimurium and Escherichia coli strains were tested in accordance with the plate incorporation method described by Ames et al. (1975) and the OECD and EEC guidelines for this test system. The methods were extensively harmonized to follow the requirements of the Labor Ministry of Japan - Notification dated June 13, 1979 and the Notification No. 1-24 of the Pharmaceutical Affairs bureau, Ministry of Health and Welfare, dated September 11, 1989.

Number of plates used:
3 parallel plates were used for each concentration step of the test material and the positive controls. Twice as many solvent control plates were used for each bacterial strain.
Type of plates used:
Commercially available Glucose-Agar plates were used for all strains except for S. typhimurium strain TA 102. Forthat strain, Minimal-Glucose plates were self-plated using DIFCO Agar (DIFCO 0140-01).

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Toxicity to the bacteria was observed at the highest concentration tested, i.e. 5000 µg/plate.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Toxicity to the bacteria was observed at the highest concentration tested, i.e. 5000 ug/plate.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

With and without addition of S9 mix as the external metabolizing system Gadoliniumsulfite trihydrate was not mutagenic under the experimental conditions described.

Executive summary:

The investigations for mutagenic potential were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated ratswas used. Two independent experimental series were performed. In the two series with S9 mix, 10 % and 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively. For the highest testmaterial concentration, Gadoliniumsulfite trihydrate was dissolved in 0.5n HCl, and it was further diluted with distilled water for the next lower concentrations. It was tested at concentrations ranging from 5.00 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations >= 1580 µg/plate or at 5000 µg/plate, depending upon the experimental condition. Taxicity to the bacteria was observed at the highest concentration tested, i.e. 5000 µg/plate. Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control test materials in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity ofthe S9 mix used. With and without addition of S9 mix as the external metabolizing system, Gadoliniumsulfite trihydrate was not mutagenic under the experimental conditions described.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 March 2003 - 11 August 2003

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)

GLP compliance:

yes

Type of assay:

other: mause Iymphoma L5178Y TK assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK^(-/-) mouse Iymphoma cells were stored as frozen stocks in liquid nitrogen. The original cultures were obtained from Dr. W. Muster, Hoffmann-La Roche, Basel, Switzerland on March 24, 1995. Each batch of frozen cells was purged of TK^(-/-) mutants, checked for spontaneaus mutant frequency and for absence of Mycoplasma.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 Mix

Test concentrations with justification for top dose:

2.81, 8.89, 28.1, 88.9 and 281 µg Gadoliniumsulfite trihydrate / mL medium
A reduction in the relative survival of the cells occurred at concentrations >= 281 and 889 µg/mL in the absence and presence of S9 mix, respectively. At the concentration of 889 µg/mL, precipitation of Gadoliniumsulfite trihydrate in the cell culture medium occurred and a clear change in the pH of the culture medium was seen.

Vehicle / solvent:

0.16 N HCl for the highest test material concentration, diluted with Aqua bidest. (H20) for the next lower concentrations.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

7,12-dimethylbenzanthracene

Details on test system and experimental conditions:

Treatment of cell cultures
Each treatment, in the absence or presence of S9 mix, was performed in duplicate (single cultures only used for positive control treatments). On day 1 of the experiment, usually the following components (volumes in mL) were placed in each of a series of sterile disposable 50 mL centrifuge tubes (for the 3 hours incubations) or 75 cm^2 culture flasks (for the 24 hours incubations; any deviation from this, if it occurred, is mentioned in the results section):
After incubation for 3 hours in the presence and 24 hours (1st series) or 3 hours (2nd series) in the absence of S9 mix at 37°C, the cells were washed with tissue culture medium and resuspended further in 10 mL RPMI 10 per tube. Cell
densities were determined using a hemocytometer and the concentrations adjusted to 2 x 10^5 /mL. Cells were transferred to flasks for growth through the expression period or were diluted to be plated for survival.

Evaluation criteria:

The effects of the test material upon the mutation frequency are defined as
• "No effect" or "no increase" in the mutation frequency if the mean frequency of the parallel incubations of a given test material concentration is less than 2.0-fold above the mean of the actual negative controls or the mean mutationfalls within the historical range ofthe negative controls.
• "Clear effect" or "clear increase" in the mutation frequency ifthe test material induces at least a 3. 0-fold increase above the mean of the actual negative controls and the mean mutation frequency for a given test material concentration is at least 1.5-fold above the highest value ofthe historical negative controls.
• All other results are defined as a "weak effect" or a "weak increase" of the mutation frequency.

Testmaterials are assessed as negative or non-mutagenic in this test system if
• the assay is considered valid and
• no effect (no increase in the mutation frequency) occurs in the two experimental series performed or
• a weak effect (weak increase) occurs in one series and no effect (no increase) in the other series of experiments.

Test materials are assessed as positive or mutagenic in this test system if
• the assay is considered valid and
• a clear effect ( clear increase in the mutation frequency) occurs at similar concentrations of the test material in the two experimental series performed, or
• a clear effect (clear increase) occurs in one series and a weak effect (weak increase) in the other series of experiments at identical concentrations, or
• weak effects ( weak increases) occur dose-dependently ( over at least two test material concentrations) and reproducibly at identical concentrations in the two experimental series performed.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Clear cytotoxic effects, i.e. a relevant decrease in either the % relative survival or % total growth of the test cells, occurred in the concentration range from 8.89 to 281µg/mL, depending upon the experimental condition used.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test material in the incubation medium was observed at the highest concentration tested, i.e. 281 µg/mL. Clear cytotoxic effects, i.e. a relevant decrease in either the % relative survival or % total growth of the test cells, occurred in the concentration range from 8.89 to 281 µg/mL, depending upon the experimental condition used.

Conclusions:

No relevant increases in mutant frequency were observed following treatment with Gadoliniumsulfite trihydrate in the two experimental series in the absence and presence of S9 mix. It is therefore concluded that Gadoliniumsulfite trihydrate is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects.

Executive summary:

Gadoliniumsulfite trihydrate was assayed for its ability to induce mutations at the TK locus (5-trifluorothymidine resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of two independent experimental series, each conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pretreated with Aroclor 1254). The exposure times were 3 and 24 hours in the absence and 3 hours in the presence of S9 mix, respectively. Various Gadoliniumsulfite trihydrate concentrations ranging from 2.81 to 281 µg/mL were tested in the absence or presence of S9 mix. Precipitation of the test material in the incubation medium was observed at the highest concentration tested, i.e. 281 µg/mL. Clear cytotoxic effects, i.e. a relevant decrease in either the % relative survival or % total growth of the test cells, occurred in the concentration range from 8. 89 to 281 µg/mL, depending upon the experimental condition used. The doses tested were selected to determine viability and mutagenicity (5-trifluorothymidine

(TFT) resistance) 2 days after treatment. Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and 7, 12-dimethylbenz[a]anthracene (with S9 mix). Therefore, the study was accepted as valid. No relevant increases in mutant frequency were observed following treatment with Gadoliniumsulfite trihydrate in the two experimental series in the absence and presence of S9 mix. It is therefore concluded that Gadoliniumsulfite trihydrate is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available