Oil shale, thermal processing waste

Genetic toxicity: in vitro

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:

2010-03-05 - 2010-04-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

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:

Oil shale, thermal processing residue

IUPAC Name:

Oil shale, thermal processing residue

Constituent 2

Reference substance name:

Oil shale, thermal processing waste

EC Number:

297-648-1

EC Name:

Oil shale, thermal processing waste

Cas Number:

93685-99-5

Molecular formula:

Not applicable (UVCB substance)

IUPAC Name:

tricalcium oxo[(oxoalumanyl)oxy]alumane oxo[(oxoferrio)oxy]iron silanedione carbonate oxidandiide sulfate

Details on test material:

- Name of test material (as cited in study report): Burnt Oil shale (BOS)
- Physical state: greish-reddish fine powder
- Analytical purity: no data
- Lot/batch No.: P.1082
- Expiration date of the lot/batch: not applicable
- Storage condition of test material: room temperature, avoid moisture
- Density: ca. 2.8 [g/m³]

Method

Target gene:

his-operon

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix (phenobarbital and β-naphthoflavone induced)

Test concentrations with justification for top dose:

0 (solvent control), 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

Aqua dest.

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: -S9: 4-nitroquinoline-N-oxide (10 µg/plate for TA 98, 40 µg/plate for Ta 1537), sodium azide (10 µg/plate), methylmethanesulfonate (1 µl/plate), +S9: 2-aminoanthracene (2.5 µg/plate; 10 µg/plate for TA 102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: Two experiments were performed: in agar (plate incorporation) (experiment 1) and preincubation (Experiment 2)

DURATION
- Preincubation period: 60 min (experiment 1)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: background lawn or reduction in the number of revertants

Evaluation criteria:

Evaluation of cytotoxicity:
Cytotoxicity can be detected bay a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 to the solvent control.

Evaluation of mutagenicity:
The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control.
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if a tester strains TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 98, TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.

Statistics:

No data

Results and discussion

Test results

Species / strain:

S. typhimurium, other:

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

Remarks on result:

other: strain/cell type:

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Ames Test Results - Experiment 1

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate
		Base-pair substitution type			Frameshift type
		TA 98	TA 100	TA 1535	TA 1537	TA 102
-	Neg. control (a. dest.)	22	115	17	11	301
-	31.6	21	98	14	14	290
-	100	20	95	11	9	288
-	316	23	111	16	12	367
-	1000	20	117	17	12	372
-	2500	27	138	14	15	396
-	5000	27	134	12	12	380
Positive controls - S9	Name	4-NOPD	NaN3	NaN3	4-NOPD	MMS
	Concentrations (μg/plate)	10	10	10	40	1 µl/plate
	Number of colonies/plate	418	815	921	206	1666
		TA 98	TA 100	TA 1535	TA 1537	TA 102
+	Neg. control (a. dest.)	28	131	10	9	204
+	31.6	35	124	10	18	212
+	100	29	117	10	13	211
+	316	30	125	10	11	176
+	1000	35	122	8	14	203
+	2500	28	141	11	13	250
+	5000	23	134	6	17	213
Positive controls + S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5	2.5	2.5	2.5	10
	Number of colonies/plate	2484	2251	201	439	1692

Table 2: Ames Test Results - Experiment 2

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate
		Base-pair substitution type			Frameshift type
		TA 98	TA 100	TA 1535	TA 1537	TA 102
-	Neg. control (a. dest.)	19	117	10	6	325
-	31.6	26	128	11	7	331
-	100	31	138	12	12	348
-	316	32	137	12	5	354
-	1000	36	153	10	11	362
-	2500	27	152	7	12	407
-	5000	28	128	10	11	452
Positive controls - S9	Name	4-NOPD	NaN3	NaN3	4-NOPD	MMS
	Concentrations (μg/plate)	10	10	10	40	1 µl/plate
	Number of colonies/plate	578	994	989	134	1678
		TA 98	TA 100	TA 1535	TA 1537	TA 102
+	Neg. control (a. dest.)	37	115	8	7	290
+	31.6	31	117	6	10	280
+	100	28	120	13	11	310
+	316	35	116	8	11	268
+	1000	26	114	8	12	323
+	2500	33	113	7	9	326
+	5000	34	139	10	14	305
Positive controls + S9	Name	2-AA	2-AA	2-AA	2-AA	2-AA
	Concentrations (μg/plate)	2.5	2.5	2.5	2.5	10
	Number of colonies/plate	2432	1411	95	207	1079

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions, Oil shale, thermal processing waste did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therfore, Oil shale, thermal processing waste is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of Oil shale, thermal processing waste for its ability to induce gene mutations the plate incorporation test (experiment 1) and the pre-incubation test (experiment 2) were performed with Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102. In 2 independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate. Precipitation of the test item was observed in all tester strains used in experiment 1 and 2 (with and without metabolic activation).

No toxic effect of the test item were noted in any of the 5 tester strains used up to the highest dose group evaluated in both experiments with the exception of a toxic effect noted in strain TA 100 at a concentration of 5000 µg/plate in experiment 2.

No biologically relevant increases in revertant colony numbers of any of the 5 tester strains were observed following treatment with Oil shale, thermal processing waste at any concentration level, neither in the presence nor absence of metabolic activation.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiment.