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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three key studies are available: a bacterial reverse mutation test (OECD guideline 471), a chromosome abberration study and a CHO hprt test. The test substance was found to be not mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 04-NOV-2005 to 06-JAN-2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Species / strain / cell type:
bacteria, other: Salmonella 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 / beta-Naphthoflavone + cofactors (= S9 mix)
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
for details see table 1 below
Vehicle / solvent:
> Vehicle(s)/solvent(s) used: deionised water
> Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties
> Vehicle controls tested: medium with solvent or vehicle alone
> volume of vehicle/solvent in the medium: 100 µL/2600 µL medium)
Untreated negative controls:
yes
Remarks:
concurrent untreatment and solvent controls
Negative solvent / vehicle controls:
yes
Remarks:
medium with solvent or vehicle alone
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: (NaN3, 10 µg/pl) in TA1535 and TA100 without S9 mix
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD, 10 and 50 µg/pl) in TA98 and TA1537, without S9 mix, respectively
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: (MMS, 4 µg/pl) in TA 102 without S9 mix
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA, 2.5 µg/pl, 10 µg/pl with TA 102) in TA1535, TA1537, TA98, TA100 and TA102 with S9 mix
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 cleaning of the bacterial background lawn.
- Other: Scoring method: The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK).
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
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
cytotoxicity observed in TA 98 without S9-mix (experiment II) at 2500 and 5000 µg/pl, and in TA 1537 and TA 98 at 5000 µg/pl with S9-mix
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
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 from 1000 µg/plate to 5000 µg/plate in each experiment and in each strain.

- Comparison with historical control data: The laboratory's historical control range was exceeded in the untreated and solvent control of strain TA 102 with and without 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

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)

- MA

+ MA

Precipit.
(yes/no)

- MA

+ MA

Precipit.
(yes/no)

- MA

+ MA

Precipit.
(yes/no)

- MA

+ MA

Precipit.
(yes/no)

0*

16

26

no

10

17

no

33

44

no

139

163

no

449

596

no

Untreated

21

24

no

10

18

no

40

56

no

140

174

no

447

600

no

3

16

27

no

12

13

no

34

42

no

147

169

no

455

594

no

10

21

23

no

13

14

no

30

49

no

162

166

no

468

606

no

33

25

27

no

7

20

no

31

44

no

143

161

no

435

620

no

100

21

25

no

6

20

no

27

49

no

121

140

no

469

607

no

333

20

18

no

14

18

no

36

50

no

145

154

no

449

618

no

1000

21

21

yes

7

14

yes

33

40

yes

137

165

yes

443

619

yes

2500

17

18

yes

6

9

yes

23

33

yes

126

137

yes

406

498

yes

5000

18

19

yes

7

7

yes

23

22

yes

98

119

yes

321

386

yes

NaN3

1533

2196

4-NOPD

97

418

MMS

5592

2-AA

306

229

1666

2657

2836

*solvent control with water

MA: metabolic activation

NaN3: sodium azide

2 -AA: 2 -aminoanthracene

MMS: methyl methane sulfonate

4 -NOPD: 4 -nitro-o-phenylene-diamine


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)

- MA

+ MA

Precipit.
(yes/no)

- MA

+ MA

Precipit.
(yes/no)

- MA

+ MA

Precipit.
(yes/no)

- MA

+ MA

Precipit.
(yes/no)

0*

22

31

no

13

13

no

46

41

no

171

217

no

459

693

no

Untreated

17

37

no

14

16

no

32

43

no

185

225

no

482

676

no

33

20

38

no

4

16

no

24

41

no

188

192

no

482

661

no

100

17

26

no

12

17

no

22

41

no

160

217

no

484

708

no

333

23

24

no

10

16

no

31

40

no

177

196

no

494

708

no

1000

20

28

yes

9

15

yes

32

47

yes

180

213

yes

475

668

yes

2500

28

22

yes

11

6

yes

19

40

yes

166

177

yes

512

713

yes

5000

11

20

yes

12

3

yes

18

10

yes

171

191

yes

494

623

yes

NaN3

1518

4-NOPD

92

423

2148

MMS

4775

2-AA

316

209

2249

2765

3081

*solvent control with water

MA: metabolic activation

NaN3: sodium azide

2 -AA: 2 -aminoanthracene

MMS: methyl methane sulfonate

4 -NOPD: 4 -nitro-o-phenylene-diamine

Conclusions:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with cerium trinitrate at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
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.
Executive summary:

In a reverse gene mutation assay in bacteria (Sokolowski A, 2006), strains TA1535, 1537, 98, 100 and 102 of S. typhimurium were exposed to cerium trinitrate, (75.03 % a.i.), at concentrations of 0 - 5000 µg/plate in the presence and absence of mammalian metabolic activation [plate co-incubation and pre-incubation].

Cerium trinitrate 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 with cerium trinitrate in each strain with and without metabolic 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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 02 August 2012 and 03 January 2013
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT locus of Chinese Hamster Ovary (CHO) cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were routinely cultured in Hams F12 medium, supplemented with 5% foetal bovine serum and antibiotics (Penicillin/Streptomycin at 100 units/100 µg per mL) at 37°C with 5% CO2 in air.
- Periodically "cleansed" against high spontaneous background: yes: Cell stocks spontaneously mutate at a low but significant rate. Before stocks of cells were frozen down they were cleansed of HPRT-mutants by culturing in HAT medium for 4 days. This is Hams F12 growth medium supplemented with hypoxanthine, aminopterin and thymidine. After 4 days in medium containing HAT, the cells were passaged into HAT-free medium and grown for 4 to 7 days. Bulk frozen stocks of HAT cleansed cells were frozen down, with fresh cultures being recovered from frozen before each experiment.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:
4-hour without S9: 76.41, 152.81, 305.63, 611.25, 916.88, 1222.5 µg/mL
4-hour with S9 (2%): 76.41, 152.81, 305.63, 611.25, 1222.5, 2445 µg/mL
24-hour without S9: 19.1, 38.2, 76.41, 152.81, 305.63, 458.44, 611.25, 1222.5 µg/mL
4-hour with S9 (1%): 76.41, 152.81, 305.36, 611.25, 1222.5, 2445 µg/mL

The concentrations of the test item were calculated based on the anhydrous form.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Hams F12 medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
ethyl methane sulphonate used as positive control at 500 and 750 µg/mL without S9; dimethyl benzanthracene used as positive control at 0.5 and 1.0 µg/mL in cultures with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: Experiment 1: 4 hours exposure in the presence of S9 and 4 hours in the absence of S9; in experiment 2: 4 hours exposure in the presence of S9 and 24 hours of exposure in the absence of S9
- Expression time (cells in growth medium): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): Cytotoxicity flasks were incubated for 6 to 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.

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

NUMBER OF REPLICATIONS: triplicate for determination of cloning efficiency; 5 replicates per group for determination of the mutant frequency

NUMBER OF CELLS EVALUATED: 200 cells/25 m² (cloning efficiency); 2 x 1E05 cells/75 cm² (mutant frequency)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:
For a test item dose level to be considered as positive the mutant frequency value must exceed the vehicle control value by 20 x 1E-06 per survivor in order to compensate for random fluctuation in the 0 to 25 x 1E-06 background mutant frequencies that is typical for this assay. A single dose level that meets the minimum criterion for a positive response within a range of assayed concentrations is not sufficient to evaluate the test item as a mutagen. Test results to be obtained for either with or without-metabolic activation conditions before this conclusion can be made, are detailed in the field 'Any other information on materials and methods incl. tables'.
Statistics:
If a test item gives a marked and dose-related increase in the mutant frequency over the vehicle controls it will be designated as mutagenic and statistical analysis will not be required. However, if weaker responses are observed then statistical analysis be performed using the SPSS program or a suitable alternative. All weak responses wil be assessed by the Study Director for biological relevance and justified in the report.
CALCULATIONS
The cloning efficiency (CE), mean plate counts, % control, mutant plate counts, mutant frequency/1E06 and mutant frequency/1E06 survival rate were calculated using the following formulae:
CE% = (mean CE counts/200) x 100
% control = (CE% of dose IDx/CE%) of dose ID0) x 100
MF1E-06 for each dose = total mutant plate counts
MFSV for each dose = (MF 1E-06/CE) x 100
Dose ID0 = vehicle control values
Dose IDx = dose level values
The calculations were performed using an Excel spreadsheet which may result in minor variations in the calculated values when compared to manual calculations due to rounding up differences.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Experiment 1: a precipitate of the test item was seen at the end of exposure at and above 76.41 µg/mL in the absence of S9 and at and above 611.25 µg/mL in the presence of S9. In the absence of S9 the precipitate became greasy/oily at and above 152.81 µg/mL; Experiment 2: a precipitate of the test item was seen at the end of the exposure period at and above 152.81 µg/mL in both exposure groups.
- Other confounding effects: The osmolality did not increase by more than 50 mOsm when the test item was dosed into media at the dose levels investigated. However, there was a decrease in the pH value of greater than one pH unit when the test item was dosed into media. To help reduce this difference HEPES was added (supplement with buffering capacity) to the culture media at a final concentration of 20mM and this brought the pH readings into an acceptable range (6.21- 6.98).

RANGE-FINDING/SCREENING STUDIES:
A dose range of 9.55, 19.1, 38.2, 76.41, 152.81, 305.63, 611.25, 1222.5 and 2445 µg/mL was used in the preliminary cytotoxicity test. The maximum recommended dose level was 3260 µg/mL, but due to the water content of the test item not being corrected for, the maximum dose tested was 2445 µg/mL. However, since the main tests were limited by toxicity and precipitate this error was considered to be of little consequence and it is considered that the test item was adequately tested.
A precipitate of the test item was observed at the end of exposure at and above 38.2 µg/mL in the 4-hour exposure group in the absence of S9 and at and above 76.41 µg/mL in the 4-hour exposure group in the presence of S9 and in the 24-hour exposure group.

The results of the individual flask counts and their analysis: it can be seen that there was marked toxicity in the 4-hour exposure group in the absence of S9 and in the 24-hour exposure group where dose related reductions in the cloning efficiency can be seen. The 4-hour exposure group in the presence of S9 demonstrated no marked toxicity throughout the test item dose range.

The maximum dose level selected for the main experiments was based on the toxicity seen in the preliminary toxicity test and was 1225 µg/mL for the 4-hour exposure group in the absence of S9 and for the 24 hour exposure group of experiment 2. For the 4-hour exposure groups in the presence of S9 there was no marked toxicity observed throughout the dose range and therefore the maximum dose tested (2445 µg/mL) was selected for the main experiments.

COMPARISON WITH HISTORICAL CONTROL DATA: The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus.
The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test item was markedly toxic in the 4-hour exposures in the absence of S9 and in the 24-hour exposure in the absence of S9 but demonstrated only modest toxicity in the 4-hour exposure in the presence of S9.
In Experiment 1, dose related toxicity was achieved at day 0 in the absence of S9 and no marked toxicity was seen in the presence of S9 at day 0.
The 24-hour exposure group demonstrated marked toxicity at day 0 in Experiment 2 and the 4-hour exposure group in the presence of S9 again demonstrated only modest toxicity at day 0.

OTHER INFORMATION: The concentrations of the test item were originally calculated based on the anhydrous form of the test item and no correction for the water content of the molecule was made (25% water content). Although the test item has no longer been tested to the maximum recommended 10mM dose level, the presence of toxicity in the absence of S9 means that the test item has been adequately tested in these exposure groups. In the presence of S9 there was no marked toxicity at the dose levels tested. However it is considered that higher dose levels would have been affected by lowered pH greater than the acceptable 1 pH unit and would therefore have been excluded for this reason. Therefore, it is considered that the test item has been adequately tested.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Acceptance criteria:

An assay will normally be considered acceptable for the evaluation of the test results only if all the following criteria are satisfied. The with and without metabolic activation portions of mutation assays are usually performed concurrently, but each portion is, in fact, an independent assay with its own positive and negative controls. Activation or non-activation assays will be repeated independently, as needed, to satisfy the acceptance criteria.

1) The average absolute cloning efficiency of negative controls should be between 70 and 115% with allowances being made for errors in cell counts and dilutions during cloning and assay variables. Assays in the 50 to 70% range may be accepted but this will be dependent on the scientific judgement of the study director. All assays below 50% cloning efficiency will be unacceptable.

2) The background (spontaneous) mutant frequency of the vehicle controls is generally in the range of 0 to 25 x 1E-06. The background values for the with and without-activation segments of a test may vary even though the same stock populations of cells may be used for concurrent assays. Assays with backgrounds greater than 35 x 1E-06 will not be used for the evaluation of a test item.

3) Assays will only be acceptable without positive control data (los due to contamination or technical error) if the test item clearly shows mutagenic activity. Negative or equivocal mutagenic responses by the test item must have a positive control mutant frequency that is markedly elevated over the concurrent negative control.

4) Test items with little or no mutagenic activity, should include an acceptable assay where concentrations of the test item have reduced the clonal survival to approximately 10 to 15% of the average of the negative controls, reached the maximum recommended dose (10 mM or 5 mg/mL) or twice the solubility limit of the test article in culture medium. Where a test item is excessively toxic, with a steep response curve, a concentration that is at least 75% of the toxic dose level should be used. There is no maximum toxicity requirement for test items that are clearly mutagenic.

5) Mutant frequencies are normally derived from sets of five dishes for mutant colony count and three dishes for variable colony counts. To allow for contamination losses it is acceptable to score a minimum of four mutant selection dishes and two viability dishes.

6) Five dose levels of test item, in duplicate, in each assay will normally be assessed for mutant frequency. A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.

Conclusions:
The test item did not induce any toxicologically significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation in either of the two experiments. The test item was therefore considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Between 30 August 2012 and 22 March 2013
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS), at approximately 37°C with 5% CO2 in humidified air. The lymhocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolising system (S9) induced by a mixture of phenobarbitone and beta-naphthoflavone
Test concentrations with justification for top dose:
Four hours exposure with 20-hour expression period without S9: 101.9, 203.75, 407.5, 815, 1222.5 and 1630 µg/mL
Four hours exposure with 20-hour expression period with S9 (2%): 203.75, 407.5, 815, 1630, 2445, 3260 µg/mL
24-hour exposure without S9: 101.9, 203.75, 407.5, 815, 1222.5 and 1630 µg/mL
Four hours exposure with 20-hour expression period with S9 (1%): 203.75, 407.5, 815, 1630, 2445, 3260 µg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item was insoluble in aqueous media at 32.6 mg/mL but was soluble in dimethyl sulphoxide at 326 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide at 326 mg/ml was, therefore, selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
0.4 and 0.2 µg/mL mitomycin C was used in experiment 1 and 2 respectively in the absence of S9. It was dissolved in Minimal Essential Medium. In the presence of S9, cyclophosphamide was used at 5 µg/mL in both experiments. It was dissolved in DMSO.
Details on test system and experimental conditions:
- Exposure duration: Four and 24 hours
- Expression time: Twenty hours

SPINDLE INHIBITOR: Colcemid
STAIN: Giemsa

NUMBER OF CELLS EVALUATED: 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Cells with 69 chromosomes or more were scored as polyploid.
Evaluation criteria:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides. A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploidy cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: Human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Qualitative observations in that inhibition of mitotic index was observed, & 52% and 82% mitotic inhibition was achieved at 815 and 1222.5 µg/mL, respectively, in the absence of S9. In the presence of S9, 26% mitotic inhibition was achieved at 2445 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Above 2445 µg/mL metaphases could not be accurately assessed due to the presence of excessive precipitate on the slides.

The maximum dose level selected for metaphase analysis was therefore 815 µg/mL and 2445 µg/mL in the absence and presence of S9, respectively. The toxicity observed at 1222.5 µg/mL, in the absence of S9, was considered to be excessive and precluded the dose from chromosome analysis.

Cerium trinitrate did not induce any statistically significant increases in the frequency of cells with aberrations in either exposure group, which included a dose level that was generally within the optimal 50% mitotic inhibition. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

All vehicle controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes, determined by the in-house historical range. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

Conclusions:
Cerium trinitrate was considered not to induce any statistically significant increases in the frequency of cells with aberrations and, therefore was considered to be non-clastogenic with and without metabolic activation.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo genetic toxicity study was performed

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro:

Bacterial reverse mutation assay:

Sokolowski A (2006) performed an Ames test (plate incorporation and pre-incubation test) with S. typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA102 with and without metabolic activation.

Following test concentrations were applied in triplicate: 33, 100, 333, 1000, 2500, 5000 µg/plate (standard plate test and preincubation test). Solvent and positive controls were run in triplicate and were considered to be valid. According to the results of the study, the test substance is not mutagenic in the Ames test with and without metabolic activation.

Chromosome Aberration:

Bowles A (2013) performed an in vitro Chromosome Aberration test in human lymphocytes (OECD Guideline 473 and EU Method B10). Two experiments were performed using different test concentrations with and without S9 activation (4h or 24h exposure; 20h expression period). In both experiments cerium trinitrate exhibited a modest dose-related inhibition of mitotic index in the dose levels tested. However, the substance did not induce any statistically significant increases in the frequency of cells with aberrations in either exposure group, which included a dose level that was generally within the optimal 50% mitotic inhibition. The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups. Cerium trinitrate was considered to be non-clastogenic with and without metabolic activation.

In vitro mammalian cell gene mutation:

Morris (2013) performed a CHO hprt forward mutation assay,targeting the HPRT locus of Chinese hamster ovary (CHO) cells, according to OECD Guideline, EU Method B.17 and EPA OPPTS 870.5300. Chinese hamster ovary (CHO) cells were treated with the test item at up to eight dose levels, in duplicate, together with vehicle (dimethyl sulphoxide) and positive controls. Ethyl methane sulphonate (EMS) was used as the positive control for cultures in the absence of S9 and Dimethyl benzanthracene (DMBA) was used as the positive control in cultures with S9. Four treatment conditions were used for the test, i.e. In the Experiment 1, a 4 -hour exposure in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration and a 4 -hour exposure in the absence of metabolic activation (S9). In Experiment 2, the 4 -hour exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours. The dose ranges selected for Experiment 1 and Experiment 2 were based on the results of the preliminary cytotoxicity test and were as follows:

4 -hour exposure group without S9: final concentration of test item (µg/mL): 76.41, 152.81, 305.63, 611.25, 916.88, 1222.5

4 -hour with S9 (2%): final concentration of test item (µg/mL): 76.41, 152.81, 305.63, 611.25, 1222.5, 2445

24 -hour without S9: final concentration of test item (µg/mL):

19.1, 38.2, 76.41, 152.81, 305.63, 458.44, 611.25, 1222.5

4 -hour with S9 (1%): final concentration of test item (µg/mL): 76.41, 152.81, 305.63, 611.25, 1222.5, 2445

The vehicle (solvent) controls gave mutant frequencies within the range expected of CHO cells at the HPRT locus. The positive control treatments, both in the presence and absence of metabolic activation, gave significant increases in the mutant frequency indicating the satisfactory performance of the test and of the metabolising system.

The test item was considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Genetic toxicity in vivo:

According to REACH Annex IX section 8.4, column 2, no further in vivo testing is required as no positive results were obtained in any of the three in vitro studies performed according to REACH Annexes VII and VIII section 8.4.


Justification for selection of genetic toxicity endpoint
No study is selected as three key studies are used to cover this endpoint: Amest test, in vitro chromosome aberration test and in vitro gene mutation in mammalian cells.

Short description of key information:
Genetic toxicity in vitro:
Bacterial reverse mutation assay: K1 study performed according to EU method B13/14 in S. typhimurium TA 1535, TA 1537, TA 98 ,TA 100 and TA 102 (Sokolowski A, 2006) and in compliance with GLP. According to the results of the study, cerium trinitrate is not mutagenic in the Ames test with and without metabolic activation.

Chromosome aberration: K1 study performed according to OECD Guideline 473 and EU Method B10 in human lymphocytes (Bowles A, 2013) and in compliance with GLP. According to the results of the study, cerium trinitrate is considered to be non-clastogenic with and without metabolic activation.

CHO HPRT test: K1 study performed according to OECD Guideline 476, EU Method B.17 and EPA OPPTS 870.5300 (Morris A, 2013) and under GLP principles. According to the results of the study, cerium trinitrate is considered to be non-mutagenic to CHO cells at the HPRT locus under the conditions of this test.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data and according to the criteria of the CLP Regulation (EC) 1272/2008, cerium trinitrate should not be classified for mutagenicity.