Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 21-OCT-2005 to 06-JAN-2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to EU guidelines and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain / cell type:
bacteria, other: 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 below
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
Untreated negative controls:
yes
Remarks:
concurrent untreated
Negative solvent / vehicle controls:
yes
Remarks:
medium with solvent or vehicle alone
Positive controls:
yes
Positive control substance:
other: for details see below in : other information on materials and methods
Details on test system and experimental conditions:
- METHOD OF APPLICATION:
> in agar (plate incorporation) in experiment I,
> preincubation in experiment II
- DURATION:
> Preincubation 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 TA 98, TA 100, and TA 102) or thirce (strains TA 1535 and TA 1537) 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
Species / strain:
bacteria, other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, and TA 102
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: 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 TA 1537, TA 98, TA 100 and TA 102.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A minor toxic effect (below the indication factor of 0.5) was observed in strain TA 102 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 TA 102 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 TABLES 2 AND 3 BELOW
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported , the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, YTTRIUM OXIDE is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria (Sokolowski A, 2006), strains TA1535, TA1537, TA98, TA100 and TA102 of S. typhimurium were exposed to yttrium oxide (99.36 %), at concentrations of 0 - 5000 µg/plate in the presence and absence of mammalian metabolic activation [plate co-incubation and pre-incubation].

Yttrium oxide was tested up to limit concentration (5000 µg/plate). 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 métabolic activation.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline EU B.13/14 for in vitro mutagenicity (bacterial reverse gene mutation) data.