Reaction mass of Dicerium trisulfide and Dilanthanum trisulfide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 20 May 1994 to 5 September 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: U.S.A./E.P.A./T.S.C.A. Federal Register, Vol. 50, No. 188, Subpart F, 27th September 1985

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Dicerium trisulphide

EC Number:

234-603-7

EC Name:

Dicerium trisulphide

Cas Number:

12014-93-6

Molecular formula:

Ce2S3

IUPAC Name:

Dicerium trisulphide

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Sulfure de cerium

Method

Target gene:

Each strain derived from Salmonella typhimurium LT2 contains one mutation in the histidine operon, resulting in a requirement for histidine. The strain of Escherichia coli contains one mutation in the tryptophan operon, resulting in a requirement for tryptophan. In addition, to increase their sensitivity to mutagenic substances, additional mutations have been added:
- the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules that do not penetrate the normal bacteira cell wall,
- the uvr (uvrB for S. typhimurium and uvrA for E; coli) mutation is a deletion of a gene code for the DNA excision repair system, which renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
- the addition of the pKM 101 ampicillin resistant plasmidic R-factor in the strains TA98 and TA100 enhances their detection sensitivity to some mutagens.

The TA1535, TA100 and WP2uvrA strains are reverted by base-pair substitution mutagens and the TA1537 and TA98 by frameshift mutagens.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial fraction (S9 fraction) obtained from a liver microsomal fraction of rats induced with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.

Test concentrations with justification for top dose:

0, 62.5, 125, 250, 500 and 1000 µg/plate for all mutagenicity experiments with and without S9 mix
The top dose was selected according to the results of the preliminary toxicity test and to the following criteria:
- for non-toxic, freely soluble test substances, the top dose is 5000 µg/plate, according to international regualtions,
- for non-toxic, poorly soluble test substances, the top dose is the lowest precipitating dose,
- for toxic test substances, irrespective of solubility, the top dose is based on the level of toxicity: moderately to markedly sparse bacterial lawn and/or decrease by approximately 50% of the number of revertants when compared to the controls. However, precipitation should not interface with the scoring of the test.

Vehicle / solvent:

Vehicle: distilled water

Controls

Untreated negative controls:

yes

Remarks:

without metabolic activation only

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: The preliminary experiment, the both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.

DURATION
- Preincubation period: The test substance solution, the S9 mix (0.5 mL) and the strain (0.1 mL) were incubated for 60 min at 37ºC before adding the overlay agar and pouring onto the surface of a minimum agar plate.
- Exposure duration: 48 to 72 hours
- Incubation: 37°C
- Expression time (cells in growth medium): the number of revertant colonies is determined at the end of the exposure time.

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: all revertants per plates are counted

ACCEPTANCE CRITERIA: This study was considered valid because the following criteria were fully met:
- the number of revertants of the controls was within the range of historical data,
- the number of revertants of the positive controls was higher than that of the controls and was within the range of our historical data.

Evaluation criteria:

The following criteria were used as an aid for determining a positive response:
- a reproducible and significant dose relationship,
and/or
- a reproducible and significant increase (i.e. a doubling in the number of revertants for at least one of the tested strains when compared to that of the controls) for at least one of the doses.

A test substance is considered as non-mutagenic in this test system if the above two criteria are not fully met.

Statistics:

Biological and statistical significances were considered during the evaluation.
No further information is available.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The control results were equivalent to those usually obtained in CIT laboratory. The number of revertants induced by the positive controls was higher than the controls, indicating the sensitivity of the test system.

Due to the poor solubility of the test substance, all tested doses precipitated.

The test substance did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the 5 strains.

Any other information on results incl. tables

Table 1: Summary table of the first experiment

With (+) or without (-) S9 mix	Test substance concentration (µg/plate)	Number of revertants (number of colonies/plate)
		Base-pair substitution type												Frameshift type
		TA100				TA1535				WP2uvrA				TA98				TA1537
S9 mix (-)	SR	88	93	74	85	8	9	9	9	29	23	17	23	38	23	21	27	11	9	12	11
	0	91	65	75	77	8	14	6	9	31	19	22	24	30	26	25	27	11	5	10	9
	62.5	103	98	103	3+	12	11	17	13+	19	23	25	22+	27	25	16	23+	13	5	11	10+
	125	90	84	95	6+	8	10	10	9+	21	18	21	20+	25	21	28	25+	10	12	5	9+
	250	106	88	92	9+	13	8	13	11+	23	21	26	23+	27	25	29	27+	5	11	10	9+
	500	103	93	95	5++	20	14	10	15++	24	24	26	24++	25	29	26	27++	12	8	8	9++
	1000*	104	105	116	7++	18	10	13	14++	22	24	17	21++	20	31	30	27++	9	10	7	9++
S9 mix (+)	0	110	101	92	101	11	14	12	12	35	36	19	30	25	25	27	26	8	11	6	8
	62.5	105	89	97	97+	16	9	12	12+	21	35	27	28+	15	24	23	21+	8	10	11	10+
	125	96	101	110	102+	18	20	11	16+	30	30	20	27+	25	16	28	23+	9	6	12	9+
	250	106	89	96	97+	11	11	13	12+	17	17	32	22+	23	22	29	25+	9	8	7	8+
	500	109	91	95	98++	10	10	18	13++	32	31	35	33++	22	29	34	28++	5	10	9	8++
	1000*	93	89	93	92++	19	12	17	16++	22	30	34	29++	34	21	29	28++	10	8	11	10++
Positive control not requiring S9 mix	Name	Sodium azide				Sodium azide				N-ethyl-N-nitro-nitrosoguanidine				2-Nitrofluorene				9-Aminoacridine
	Concentration (µg/plate)	1				1				2				0.5				50
	Number of colonies/plate	607	467	577	550	459	475	460	465	889	913	824	875	157	123	122	134	135	261	130	175
Positive control requiring S9 mix	Name	2-Anthramine				2-Anthramine				2-Anthramine				2-Anthramine				2-Anthramine
	Concentration (µg/plate)	2				2				10				2				2
	Number of colonies/plate	3406	3355	3144	3302	375	346	349	357	807	827	865	833	2814	2972	3037	2941	460	596	591	549

SR: Spontaneous reversants: negative control

O: Vehicle control (distilled water)

*: Pink colouration

P: Precipitate

+: slight

++: moderate

Applicant's summary and conclusion

Conclusions:

Under these experimental conditions, the test substance Dicerium trisulphide did not show mutagenic activity in the reverse mutation assay on Salmonella typhimurium and Escherichia coli.

Executive summary:

The objective of this study was to evaluate the potential of the test substance Dicerium trisulphide to induce a reverse mutation in bacteria Salmonella typhimurium and Escherichia coli (Ames test). This test enables the detection of base-pair substitution or frameshift mutagens.

A preliminary toxicity test was performed to define the doses to be used for the mutagenicity study. The test substance was then tested in two independent tests, with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9) of rats induced with Aroclor 1254.

The tests were performed according to the direct plate incorporation method except the second test with S9 mix, according to the preincubation method (1 hour, 37°C).

Four strains of bacteria Salmonella typhimurium: TA1535, TA1537, TA98 and TA100 and one strain of Escherichia coli: WP2uvrA were used. Each strain was exposed to 5 doses of the test substance (3 plates/dose). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

The test substance Cerium sulphide was prepared in distilled water at 10 mg/mL.

The doses of Cerium sulphide were 62.5, 125, 250, 500 and 1000 µg/plate.

Due to the poor solubility of the test substance, all tested doses precipitated.

In both tests, the control results were equivalent to those usually obtained in CIT laboratory. The number of revertants induced by the positive controls was higher than the controls, indicating the sensitivity of the test system.

The test substance Cerium sulphide did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the 5 strains, in both of the 2 tests.

Under these experimental conditions, the test substance Dicerium trisulphide did not show mutagenic activity in the reverse mutation assay on Salmonella typhimurium and Escherichia coli.