Frits, chemicals and bismuth oxide

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:

13 Oct - 04 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP-Landesleitstelle Bayern, Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Schwabach, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Frits, chemicals

EC Number:

266-047-6

EC Name:

Frits, chemicals

Cas Number:

65997-18-4

Molecular formula:

not applicable

IUPAC Name:

Frits, chemicals

Method

Target gene:

His operon

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/β-naphthoflavone-induced male rats

Test concentrations with justification for top dose:

Pre-experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
Main experiment: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 10 µg/plate for TA 98, 40 µg/plate for TA 1537, without metabolic activation; 2-aminoanthracene, 2.5 µg/plate for TA 98, TA 100, TA 1535, TA 1537, 10 µg/plate for TA 102, with metabolic activation

Remarks:

The profiency of S9-mix was characterised with benzo(a)pyrene (mutagen that requires metabolic activation by microsomal enzymes) before the experiment.

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1: plate incorporation; Experiemnt 2: pre-incubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 – 72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: the diminution of the background lawn or a significant reduction in the number of revertants in solvent control.

Evaluation criteria:

Negative control data within historical range. Positive controls show a distinct enhancement of revertant rates over the control plate. At least five different concentrations of each tester strain are analyzable.
Positive response: 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:
- if in tester strains TA 98 and TA 100 the number of reversions is at least and twice as high
- if in tester strains TA 102 the number of reversions is at least 1.5-fold higher
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
Negative response: neither a dose related increase in the number of revertants nor a reproducible biologically relevant increase.

Results and discussion

Test results

Key result

Species / strain:

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

RANGE-FINDING/SCREENING STUDIES: In the pre-experiment no toxic effects were observed at any concentration of the substance.

HISTORICAL CONTROL DATA
- Positive historical control data: Positive controls met the criteria of validity.
- Negative (solvent/vehicle) historical control data: Negative controls were within historical norms.

Any other information on results incl. tables

 Tester Strain: TA 98  Experiment: 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.		16			19
		14	19	7.6	22	32	20.6	0.8	1.7
		28			56
DMSO		17			20
		18	24	10.7	23	19	4.6	1.0	1.0
		36			14
Test Item	31.6 µg	18			13
		22	23	6.1	17	17	3.5	1.0	0.9
		30			20
Test Item	100 µg	55			33
		30	33	20.2	20	24	7.8	1.4	1.3
		15			19
Test Item	316 µg	24			29
		22	23	1.0	23	27	3.5	1.0	1.4
		23			29
Test Item	1000 µg	20			15
		19	17	4.4	22	18	3.5	0.7	1.0
		12			18
Test Item	2500 µg	27			34
		26	27	0.6	28	29	4.6	1.1	1.5
		27			25
Test Item	5000 µg	12 P			42 P
		14 P	14	1.5	20 P	28	12.2	0.6	1.5
		15 P			22 P
4-NOPD	10 µg	461			/
		543	515	46.8	/	/	/	21.8	/
		541			/
2-AA	2.5 µg	/			1957
		/	/	/	2402	2273	275.2	/	119.6
		/			2460

SD: Standard-deviation P: Precipitation

B: Background lawn reduced C: Contamination

N: No background lawn

Mutationfactor = mean revertants (test item)/mean revertants (vehicle control)

 

Tester Strain: TA 100  Experiment: 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.		92			86
		87	92	5.0	80	85	4.2	1.2	1.1
		97			88
DMSO		92			81
		75	77	14.6	81	75	11.0	1.0	1.0
		63			62
Test Item	31.6 µg	58			80
		81	66	13.0	57	69	11.6	0.9	0.9
		59			71
Test Item	100 µg	62			77
		158	107	48.3	98	83	13.1	1.4	1.1
		100			74
Test Item	316 µg	65			82
		63	57	11.6	74	89	20.0	0.7	1.2
		44			112
Test Item	1000 µg	70			66
		52	61	9.0	88	79	11.5	0.8	1.1
		61			83
Test Item	2500 µg	91			60
		72	83	9.7	95	83	20.2	1.1	1.1
		85			95
Test Item	5000 µg	69 P			82 P
		63 P	74	14.2	66 P	77	9.9	1.0	1.0
		90 P			84 P
NaN3	10 µg	796			/
		845	837	37.2	/	/	/	10.9	/
		869			/
2-AA	2.5 µg	/			2252
		/	/	/	2181	2261	84.3	/	30.3
		/			2349

 

Tester Strain: TA 1535  Experiment: 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.		12			17
		14	14	2.5	14	17	3.5	0.9	1.1
		17			21
DMSO		20			14
		12	16	4.0	15	16	3.2	1.0	1.0
		17			20
Test Item	31.6 µg	14			11
		15	13	2.1	10	13	3.8	0.8	0.8
		11			17
Test Item	100 µg	13			14
		14	13	1.5	17	14	2.5	0.8	0.9
		11			12
Test Item	316 µg	15			15
		17	16	1.0	20	16	4.0	1.0	1.0
		16			12
Test Item	1000 µg	15			12
		15	15	0.0	16	17	5.0	0.9	1.0
		15			22
Test Item	2500 µg	12			16
		12	14	2.9	16	14	3.5	0.8	0.9
		17			10
Test Item	5000 µg	14 P			16 P
		15 P	14	0.6	10 P	14	3.5	0.9	0.9
		14 P			16 P
NaN3	10 µg	1038			/
		1205	1147	94.7	/	/	/	70.2	/
		1199			/
2-AA	2.5 µg	/			127
		/	/	/	127	147	34.6	/	9.0
		/			187

 

Tester Strain: TA 1537  Experiment: 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.		8			9
		10	9	1.2	9	10	2.3	0.9	1.1
		8			13
DMSO		11			9
		7	10	2.3	12	9	2.5	1.0	1.0
		11			7
Test Item	31.6 µg	7			8
		13	10	3.0	9	8	1.5	1.0	0.8
		10			6
Test Item	100 µg	14			9
		14	12	3.5	9	9	0.6	1.2	0.9
		8			8
Test Item	316 µg	6			10
		11	9	2.6	8	8	1.5	0.9	0.9
		10			7
Test Item	1000 µg	10			8
		5	8	2.5	9	9	0.6	0.8	0.9
		8			9
Test Item	2500 µg	7			8
		10	8	1.5	9	8	1.0	0.9	0.9
		8			7
Test Item	5000 µg	6 P			7 P
		9 P	8	1.5	6 P	7	1.0	0.8	0.8
		8 P			8 P
4-NOPD	40 µg	160			/
		124	141	18.1	/	/	/	14.6	/
		139			/
2-AA	2.5 µg	/			181
		/	/	/	184	179	6.8	/	19.1
		/			171

 

Tester Strain: TA 102  Experiment: 1

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.		267			368
		285	280	11.7	398	362	39.3	1.2	1.1
		289			320
DMSO		227			300
		255	242	14.0	345	318	24.0	1.0	1.0
		243			308
Test Item	31.6 µg	231			288
		240	237	5.2	288	288	0.0	1.0	0.9
		240			288
Test Item	100 µg	351			235
		234	261	79.6	243	243	8.5	1.1	0.8
		199			252
Test Item	316 µg	214			256
		215	215	0.6	231	246	13.1	0.9	0.8
		215			250
Test Item	1000 µg	248			250
		249	247	3.2	321	266	49.0	1.0	0.8
		243			227
Test Item	2500 µg	281			222
		271	272	8.1	220	221	1.2	1.1	0.7
		265			222
Test Item	5000 µg	165 P			213 P
		190 P	210	58.2	267 P	245	28.5	0.9	0.8
		276 P			256 P
MMS	1 µL	1212			/
		1169	1298	186.9	/	/	/	5.4	/
		1512			/
2-AA	10 µg	/			847
		/	/	/	807	835	24.7	/	2.6
		/			852

 

Tester Strain: TA 98  Experiment: 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.		47			22
		51	48	3.1	37	30	7.5	1.1	1.2
		45			30
DMSO		47			26
		38	43	4.7	30	25	5.0	1.0	1.0
		45			20
Test Item	31.6 µg	57			30
		57	57	0.6	20	26	5.3	1.3	1.0
		58			28
Test Item	100 µg	51			18
		52	50	2.6	30	25	6.4	1.2	1.0
		47			28
Test Item	316 µg	46			23
		33	37	7.5	24	22	2.1	0.9	0.9
		33			20
Test Item	1000 µg	53			33
		44	43	11.1	24	29	4.5	1.0	1.1
		31			29
Test Item	2500 µg	52			38
		53	52	1.5	24	31	7.0	1.2	1.2
		50			32
Test Item	5000 µg	51 P			28 P
		50 P	50	0.6	31 P	31	3.5	1.2	1.2
		50 P			35 P
4-NOPD	10 µg	296			/
		312	302	8.7	/	/	/	7.0	/
		298			/
2-AA	2.5 µg	/			1529
		/	/	/	1861	1971	506.0	/	77.8
		/			2523

 

Tester Strain: TA 100  Experiment: 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.		76			88
		101	90	12.9	88	90	4.0	1.0	1.1
		94			95
DMSO		99			74
		95	94	5.0	93	81	10.2	1.0	1.0
		89			77
Test Item	31.6 µg	92			86
		90	92	2.5	76	80	5.5	1.0	1.0
		95			77
Test Item	100 µg	86			76
		90	85	5.0	92	83	8.1	0.9	1.0
		80			82
Test Item	316 µg	102			88
		79	87	13.3	89	88	1.5	0.9	1.1
		79			86
Test Item	1000 µg	78			93
		83	77	6.0	113	101	10.6	0.8	1.2
		71			97
Test Item	2500 µg	76			119
		85	85	9.0	99	109	10.0	0.9	1.3
		94			110
Test Item	5000 µg	112 P			77 P
		81 P	97	15.6	84 P	82	4.0	1.0	1.0
		99 P			84 P
NaN3	10 µg	925			/
		947	924	24.0	/	/	/	9.8	/
		899			/
2-AA	2.5 µg	/			1508
		/	/	/	1572	1485	101.0	/	18.3
		/			1374

 

Tester Strain: TA 1535  Experiment: 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.		24			18
		22	22	1.5	15	18	3.0	1.0	0.9
		21			21
DMSO		23			17
		24	22	2.1	18	19	2.6	1.0	1.0
		20			22
Test Item	31.6 µg	18			20
		30	23	6.2	23	21	1.5	1.0	1.1
		21			21
Test Item	100 µg	26			17
		21	23	2.5	18	18	0.6	1.0	0.9
		23			18
Test Item	316 µg	25			26
		24	23	2.6	21	22	3.6	1.0	1.2
		20			19
Test Item	1000 µg	26			20
		27	26	0.6	18	19	1.2	1.2	1.0
		26			18
Test Item	2500 µg	22			18
		25	26	5.1	19	19	1.0	1.2	1.0
		32			20
Test Item	5000 µg	31 P			17 P
		25 P	25	5.5	18 P	17	0.6	1.1	0.9
		20 P			17 P
NaN3	10 µg	1231			/
		1272	1211	72.5	/	/	/	54.2	/
		1131			/
2-AA	2.5 µg	/			254
		/	/	/	200	226	27.1	/	11.9
		/			223

 

Tester Strain: TA 1537  Experiment: 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.		17			15
		12	15	2.5	18	16	1.5	0.8	1.0
		15			16
DMSO		17			17
		19	18	1.0	15	16	1.0	1.0	1.0
		18			16
Test Item	31.6 µg	18			15
		16	18	1.5	14	15	1.5	1.0	1.0
		19			17
Test Item	100 µg	17			17
		15	16	1.0	18	17	1.5	0.9	1.0
		16			15
Test Item	316 µg	17			16
		20	18	1.7	17	16	1.0	1.0	1.0
		17			15
Test Item	1000 µg	15			16
		14	15	1.0	18	16	2.0	0.8	1.0
		16			14
Test Item	2500 µg	17			17
		15	17	1.5	18	18	1.0	0.9	1.1
		18			19
Test Item	5000 µg	12 P			14 P
		11 P	11	1.5	13 P	14	1.0	0.6	0.9
		9 P			15 P
4-NOPD	40 µg	164			/
		146	152	10.4	/	/	/	8.4	/
		146			/
2-AA	2.5 µg	/			251
		/	/	/	249	245	8.1	/	15.3
		/			236

 

Tester Strain: TA 102  Experiment: 2

Treatment	Dose/plate	REVERTANT COLONIES PER PLATE						MUTATION FACTOR
		Without activation (-S9)			With activation (+S9)
		Counts	Mean	SD	Counts	Mean	SD	-S9	+S9
A. dest.		305			385
		329	317	12.0	386	381	8.4	1.0	1.0
		318			371
DMSO		295			387
		313	313	18.0	357	385	26.6	1.0	1.0
		331			410
Test Item	31.6 µg	292			375
		299	295	3.6	379	378	2.6	0.9	1.0
		294			380
Test Item	100 µg	302			380
		313	312	9.5	354	365	13.3	1.0	0.9
		321			362
Test Item	316 µg	327			375
		307	316	10.1	365	367	7.6	1.0	1.0
		314			360
Test Item	1000 µg	274			405
		318	302	24.1	360	374	27.2	1.0	1.0
		313			356
Test Item	2500 µg	250			343
		276	276	26.0	366	363	18.2	0.9	0.9
		302			379
Test Item	5000 µg	308 P			301 P
		304 P	302	6.7	341 P	333	29.3	1.0	0.9
		295 P			358 P
MMS	1 µL	1129			/
		1315	1239	97.5	/	/	/	4.0	/
		1273			/
2-AA	10 µg	/			830
		/	/	/	911	899	64.3	/	2.3
		/			957

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of the conducted test the substance was not mutagenic in any of the five bacterial strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) tested with and without metabolic activation.