Cerium trinitrate

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 04-NOV-2005 to 06-JAN-2006

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:

Cerium trinitrate

EC Number:

233-297-2

EC Name:

Cerium trinitrate

Cas Number:

10108-73-3

Molecular formula:

Ce.3HNO3

IUPAC Name:

cerium trinitrate

Test material form:

solid: crystalline

Method

Species / strain

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)

Controls

Controls 1

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

Controls 2

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

Controls 3

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: (MMS, 4 µg/pl) in TA 102 without S9 mix

Controls 4

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

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)	- 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

Applicant's summary and conclusion

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.