Yttrium

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:

from 2005-10-21 to 2006-01-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Cited as Directive 2000/32/EC, B.13/14

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 oxide

EC Number:

215-233-5

EC Name:

Yttrium oxide

Cas Number:

1314-36-9

Molecular formula:

Y2O3

IUPAC Name:

Yttrium(III) oxide

Test material form:

solid: particulate/powder

Details on test material:

Name of test material: yttrium oxyde
Substance type: mono-constituent substance
Further information on test material confidential.

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Liver S9 fraction of rats induced with phenobarbital/ß-naphthoflavone

Test concentrations with justification for top dose:

Pre-experiment (Experiment I): 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate
Experiment II: 33, 100, 333, 1000, 2500, and 5000 µg/plate
See table 1 in "Any other information on materials and methods incl. tables".

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties and its relative non-toxicity to bacteria, and allows to obtain an homogeneous suspension.
- Vehicle controls tested: medium with solvent or vehicle alone
- Volume of vehicle/solvent in the medium: 100 µL/2600 µL medium

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Controls 4

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:

METHOD OF APPLICATION
- in agar (plate incorporation) in experiment I
- pre-incubation in experiment II

DURATION
- Pre-incubation period (experiment II): 60 minutes at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATES PER CONCENTRATION: 3

DETERMINATION OF CYTOTOXICITY: Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

OTHER
-Scoring method: The colonies were counted using Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager.

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA98, TA100 and TA102) or thrice (strains TA1535 and TA1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

not mandatory

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:

no cytotoxicity, but tested up to precipitating concentrations

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

a minor toxic effect (below the indication factor of 0.5) was observed at 5000 µg/plate with S9 mix in Experiment I

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
Precipitation was observed at 2500 and 5000 µg/plate, except in experiment II with metabolic activation, in which precipitation was observed only at 5000 µg/plate in strains TA1537, TA98, TA100 and TA102.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A minor toxic effect (below the indication factor of 0.5) was observed in strain TA102 at 5000 µg/plate with metabolic activation in experiment I. No toxic effects were observed in experiment II.

COMPARISON WITH HISTORICAL CONTROL DATA:
The laboratory´s historical control range was exceeded in the untreated and solvent control of strain TA102 without metabolic activation in experiment I and with metabolic activation in experiment II. These deviations are judged to be based on biologically irrelevant fluctuations in the number of colonies and have no impact on the outcome of the study.

See detailed results in Table 2 and 3 in the field "Any other information on results incl. tables".

Any other information on results incl. tables

Table 2: Number of revertants per plate in experiment I (mean of 3 plates) (plate incorporation)

	TA 1535				TA 1537				TA 98				TA 100				TA 102
Conc. [µg/plate]	- MA	+ MA	Precipit. (yes/no)	Cytotox. (yes/no)	- MA	+ MA	Precipit. (yes/no)	Cytotox. (yes/no)	- MA	+ MA	Precipit. (yes/no)	Cytotox. (yes/no)	- MA	+ MA	Precipit. (yes/no)	Cytotox. (yes/no)	- MA	+ MA	Precipit. (yes/no)	Cytotox. (yes/no)
0*	21	33	no	no	12	16	no	no	32	42	no	no	127	152	no	no	495	539	no	no
Untreated	17	25	no	no	7	16	no	no	34	39	no	no	136	165	no	no	536	593	no	no
3	19	34	no	no	7	19	no	no	35	40	no	no	140	147	no	no	463	585	no	no
10	25	33	no	no	14	18	no	no	33	37	no	no	171	151	no	no	443	561	no	no
33	28	25	no	no	12	21	no	no	36	41	no	no	143	151	no	no	465	557	no	no
100	23	25	no	no	12	19	no	no	34	40	no	no	146	163	no	no	451	553	no	no
333	20	28	no	no	12	22	no	no	31	43	no	no	135	151	no	no	456	552	no	no
1000	24	30	no	no	12	22	no	no	36	42	no	no	139	148	no	no	494	452	no	no
2500	19	25	yes	no	13	12	yes	no	26	26	yes	no	128	102	yes	no	468	273	yes	no
5000	19	18	yes	no	10	9	yes	no	23	21	yes	no	110	90	yes	no	346	219	yes	yes
NaN3	1446												1925
4-NOPD					119				444
MMS																	4779
2-AA		318				387				2306				3229				2600

*solvent control with DMSO

MA : metabolic activation

Table 3: Number of revertants per plate in experiment II (mean of 3 plates) (preincubation)

	TA 1535				TA 1537				TA 98				TA 100				TA 102
Conc. [µg/plate]	- MA	+ MA	Precipit. (yes/no)	Cytotox. (yes/no)	- MA	+ MA	Precipit. (yes/no)	Cytotox. (yes/no)	- MA	+ MA	Precipit. (yes/no)	Cytotox. (yes/no)	- MA	+ MA	Precipit. (yes/no)	Cytotox. (yes/no)	- MA	+ MA	Precipit. (yes/no)	Cytotox. (yes/no)
0*	17	25	no	no	8	17	no	no	29	30	no	no	124	158	no	no	446	536	no	no
Untreated	25	28	no	no	9	13	no	no	26	36	no	no	139	178	no	no	446	543	no	no
33	17	26	no	no	12	13	no	no	19	36	no	no	130	150	no	no	455	495	no	no
100	19	25	no	no	10	15	no	no	23	30	no	no	131	162	no	no	485	546	no	no
333	19	24	no	no	9	14	no	no	24	29	no	no	122	141	no	no	471	486	no	no
1000	25	26	no	no	10	16	no	no	23	33	no	no	125	140	no	no	452	447	no	no
2500	21	25	yes	no	6	9	yes/no	no	25	36	yes/no	no	122	153	yes/no	no	473	443	yes/no	no
5000	19	32	yes	no	6	10	yes	no	21	22	yes	no	118	128	yes	no	439	402	yes	no
NaN3	1393												1944
4-NOPD					101				364
MMS																	1597
2-AA		223				191				1154				1938				2533

*solvent control with DMSO

MA : metabolic activation

Applicant's summary and conclusion

Conclusions:

In this reverse gene mutation assay in bacteria, strains TA1535, TA1537, TA98, TA100 and TA102 of S. typhimurium were exposed to yttrium oxide at concentrations of 0 to 5000 µg/plate in the presence and absence of mammalian metabolic activation [plate co-incubation and pre-incubation]. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in each strain with and without metabolic activation.