Tar bases, coal, lutidine fraction

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:

Experimental start: 05 May 2016, Experimental completion: 20 May 2016; Final Report: 01 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

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:

Tar bases, coal, lutidine fraction

EC Number:

293-766-2

EC Name:

Tar bases, coal, lutidine fraction

Cas Number:

91082-52-9

IUPAC Name:

Tar bases, coal, lutidine fraction

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from male Sprague-Dawley derived rats

Test concentrations with justification for top dose:

5, 15, 50, 150, 500, 1500 and 5000 µg/plate in the absence and presence of metabolic activation. The maximum concentration was selected based on the standard limit concentration recommended in the regulatory guidelines that the assay follows.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO, ACS reagent grade
- Justification for choice of solvent/vehicle: the test substances dissolved completely in the vehicle at the highest dose tested

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding (if applicable): at least 10^9 per mL at test begin

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 to 72 hours
- Expression time (cells in growth medium): 10 hours

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity: toxicity was observed as a thin background lawn of non-revertant colonies and/or a reduction in the number of revertant colonies

NUMBER OF REPLICATIONS: 3

Rationale for test conditions:

Following relevant test guideline

Evaluation criteria:

Criteria for valid test:
- the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory (maintained as a rolling record over two years or a minimum of 20 data sets)
- the positive control compounds must induce an increase in mean revertant colony numbers of at least twice that of the concurrent vehicle control
- mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9 per mL
- a minimum of five analysable concentrations must be present with at least four showing no signs of toxic effects, evident as bacterial inhibition and/or a reduction in the number of revertants below the indication factor of 0.5

If exposure to a test substance produces a reproducible increase in mean revertant colony numbers of at least twice that of the vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic effects.
If exposure to a test substance does not produce a reproducible increase in mean revertant colony numbers, it is considered to show no evidence of mutagenic activity.
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. In general, treatment-associated increases in mean revertant colony numbers below two or three times those of the vehicle controls are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.

Statistics:

The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett's test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no fluctuations in pH of the medium were observed at 2000 μg/mL of more than 1.0 unit compared with the vehicle control
- Effects of osmolality: no fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control
- Evaporation from medium: not reported
- Precipitation: no precipitation was observed

RANGE-FINDING/SCREENING STUDIES:


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- See under any other information on results

Any other information on results incl. tables

Table 1: Results of Experiment 1, plate incorporation

Experiment 1, plate incorporation, no metabolic activation
Strain	Addition		Concentration per plate [µg]		Mean revertant number per plate			Standard deviation				Fold increase over vehicle					Individual revertant counts
TA98	DMSO				31.0			1.7									32			29			32
	Test substance		5		26.7			6.5				0.9					27			33			20
			15		24.3			1.5				0.8					24			23			26
			50		12.7			4.6				0.4					18			10			10
			150		7.7			2.3				0.2					9			9			5
			500		9.0			4.4				0.3					4			12			11
			1500		12.7			2.1				0.4					15S			11S			12S
			5000		8.7			2.1				0.3					7S			11S			8S
TA100	DMSO				141.0			6.9									137			149			137
	Test substance		5		133.0			11.4				0.9					120			138			141
			15		149.7			2.5				1.1					152			150			147
			50		107.3			27.6				0.8					139			88			95
			150		104.7			5.8				0.7					108			98			108
			500		93.0			11.8				0.7					106			90			83
			1500		114.7			4.0				0.8					117			110			117
			5000		111.7			7.0				0.8					119			105			111
TA1535	DMSO				22.3			9.5									13			32			22
	Test substance		5		21.0			1.0				0.9					21			22			20
			15		18.0			3.5				0.8					16			16			22
			50		18.0			8.0				0.8					26			10			18
			150		14.0			6.1				0.6					11			10			21
			500		18.7			4.7				0.8					17			15			24
			1500		17.0			4.6				0.8					21			12			18
			5000		21.7			7.8				1.0					24			28			13
TA1537	DMSO				17.7			4.5									13			18			22
	Test substance		5		16.7			7.1				0.9					18			9			23
			15		19.0			5.6				1.1					24			13			20
			50		12.3			3.1				0.7					15			9			13
			150		12.3			3.2				0.7					11			10			16
			500		12.7			2.1				0.7					12			15			11
			1500		11.7			1.5				0.7					10			13			12
			5000		17.7			9.0				1.0					13			28			12
WP2 uvrA (pKM101)	DMSO				156.7			17.7									177			148			145
	Test substance		5		161.3			9.7				1.0					153			159			172
			15		164.0			5.6				1.0					169			165			158
			50		128.0			13.0				0.8					143			120			121
			150		121.7			11.2				0.8					109			130			126
			500		125.7			9.1				0.8					116			127			134
			1500		162.0			10.6				1.0					174			158			154
			5000		124.3			16.3				0.8					142			110			121
S: slight thinning of background lawn
Experiment 1, plate incorporation, no metabolic activation, positive controls
TA98	2-nitrofluorene		2		243.3			9.2				7.8					238			238			254
TA100	Sodium azide		2		540.0			6.2				3.8					542			533			545
TA1535	Sodium azide		2		828.3			44.5				37.1					821			876			788
TA1537	9-aminoacridine		50		202.0			19.5				11.4					182			221			203
WP2 uvrA	4-nitroquinoline-1-oxide		2		847.7			95.1				5.4					740			920			883
Experiment 1, plate incorporation, viability
			Strain					Mean counts per plate				Standard deviation					Individual counts (100 µL aliquots of 10^-6 dilution of 10-hour culture)
			TA98		Viability			244.7				17.0					225			255			254
			TA100		Viability			327.0				12.3					332			313			336
			TA1535		Viability			351.0				89.7					331			449			273
			TA1537		Viability			242.7				9.1					251			233			244
			WP2 uvrA		Viability			330.0				13.2					345			320			324
Experiment 1, plate incorporation, with metabolic activation
Strain	Addition			Concentration per plate [µg]			Mean revertant number per plate		Standard deviation				Fold increase over vehicle			Individual revertant counts
TA98	DMSO						31.7		6.4							39					27			29
	Test substance			5			20.0		3.6				0.6			23					21			16
				15			19.3		3.1				0.6			22					20			16
				50			20.3		3.8				0.6			16					23			22
				150			21.3		0.6				0.7			22					21			21
				500			26.3		2.1				0.8			27					28			24
				1500			21.7		2.5				0.7			24S					22S			19S
				5000			22.7		3.8				0.7			21S					20S			27S
TA100	DMSO						150.0		14.9							133					161			156
	Test substance			5			109.3		9.2				0.7			104					120			104
				15			108.0		8.0				0.7			116					108			100
				50			110.0		6.6				0.7			111					116			103
				150			108.0		15.9				0.7			114					90			120
				500			126.3		10.2				0.8			138					119			122
				1500			131.3		5.0				0.9			136					126			132
				5000			134.7		9.0				0.9			134					126			144
TA1535	DMSO						14.0		2.6							15					16			11
	Test substance			5			14.0		3.5				1.0			12					12			18
				15			10.7		4.5				0.8			11					15			6
				50			14.7		7.5				1.0			7					22			15
				150			9.3		3.8				0.7			12					11			5
				500			16.7		4.5				1.2			12					21			17
				1500			20.3		2.9				1.5			22					17			22
				5000			13.3		8.1				1.0			12					22			6
TA1537	DMSO						19.7		6.7							23					24			12
	Test substance			5			17.0		6.9				0.9			21					21			9
				15			14.7		1.5				0.7			13					15			16
				50			15.0		4.4				0.8			18					10			17
				150			15.7		6.4				0.8			23					12			12
				500			14.3		3.8				0.7			10					16			17
				1500			18.0		5.0				0.9			13					23			18
				5000			17.0		5.3				0.9			13					15			23
WP2 uvrA (pKM101)	DMSO						199.0		14.0							189					215			193
	Test substance			5			152.3		4.7				0.8			154					147			156
				15			167.7		17.8				0.8			160					155			188
				50			184.0		19.0				0.9			189					200			163
				150			174.3		3.8				0.9			176					170			177
				500			182.3		5.9				0.9			178					180			189
				1500			199.7		20.6				1.0			178					202			219
				5000			152.3		22.3				0.8			178					138			141
S: slight thinning of background lawn
Experiment 1, plate incorporation, with metabolic activation, positive control
TA98	Benzo[a]pyrene			5			139.0		7.2				4.4			141					131			145
TA100	2-aminoanthracene			5			817.7		388.6				5.5			1266					577			610
TA1535	2-aminoanthracene			5			542.0		50.9				38.7			488					589			549
TA1537	Benzo[a]pyrene			5			737		2.9				3.7			77					72			72
WP2 uvrA	2-aminoanthracene			10			834.0		35.0				4.2			874					819			809
Additional experiment 1, plate incorporation, no metabolic activation
Strain	Addition			Concentration per plate [µg]			Mean revertant number per plate			Standard deviation				Fold increase over vehicle				Individual revertant counts
TA98	DMSO						21.7			0.6								22			21			22
	Test substance			0.5			17.3			0.6				0.8				17			17			18
				1.0			17.3			3.2				0.8				15			16			21
				5			12.0			1.7				0.6				10			13			13
				15			19.3			3.1				0.9				22			16			20
				50			21.0			0.0				1.0				21			21			21
				150			20.0			7.0				0.9				17			28			15
				500			13.3			3.5				0.6				13			10			17
				1500			8.3			0.6				0.4				8S			9S			8S
				5000			6.0			1.0				0.3				6S			7S			5S
S: slight thinning of background lawn
Additional experiment 1, plate incorporation, no metabolic activation, positive control
TA98	2-nitrofluorene			2			262.7			16.9				12.1				255			282			251
Additional experiment 1, plate incorporation, no metabolic activation, viability
Strain						Mean counts per plate					Standard deviation				Individual counts (100 µL aliquots of 10^-6 dilution of 10-hour culture)
TA98		Viability				211.0					29.5				177				226			230

Table 2: Results of Experiment 2, pre-incubation

Experiment 2, pre-incubation, no metabolic activation
Strain	Addition	Concentration per plate [µg]	Mean revertant number per plate	Standard deviation	Fold increase over vehicle	Individual revertant counts
TA98	DMSO		50.3	12.7		65	43	43
	Test substance	0.5	39.7	2.9	0.8	38	38	43
		1.5	41.0	3.5	0.8	45	39	39
		5	45.7	9.3	0.9	38	43	56
		15	52.0	8.9	1.0	45	62	49
		50	43.0	0.0	0.9	53	43	43
		150	33.3	5.5	0.7	39	28	33
		500	19.3	1.2	0.4	20S	15S	20S
		1500	19.7	1.5	0.4	21T	20T	18T
		5000	20.3	3.1	0.4	17T	23T	21T
TA100	DMSO		146.0	9.5		137	156	145
	Test substance	5	152.7	5.5	1.0	158	147	153
		15	151.7	23.4	1.0	169	161	125
		50	109.7	26.1	0.8	139	89	101
		150	109.0	10.6	0.7	101	121	105
		500	107.0	5.6	0.7	108	101	112
		1500	111.7	13.5	0.8	98	112	125
		5000	121.7	24.6	0.8	94	141	130
TA1535	DMSO		16.3	5.7		21	18	10
	Test substance	5	13.3	2.5	0.8	11	13	16
		15	20.7	12.9	1.3	10	17	35
		50	14.3	5.8	0.9	21	11	11
		150	12.0	1.7	0.7	13	10	13
		500	13.3	2.3	0.8	16	12	12
		1500	18.3	2.5	1.1	21	16	18
		5000	15.3	5.8	0.9	22	12	12
TA1537	DMSO		10.0	2.6		11	12	7
	Test substance	5	13.7	8.5	1.4	17	4	20
		15	11.0	6.2	1.1	6	18	9
		50	6.0	3.5	0.6	4	10	4
		150	11.7	1.2	1.2	11	13	11
		500	6.0	3.6	0.6	2	7	9
		1500	6.7	2.5	0.7	4	7	9
		5000	5.7	2.9	0.6	4	9	4
WP2 uvrA (pKM101)	DMSO		163.7	24.0		177	136	178
	Test substance	5	168.0	6.6	1.0	161	174	169
		15	193.7	25.0	1.2	172	221	188
		50	135.0	17.4	0.8	155	123	127
		150	137.7	6.7	0.8	142	130	141
		500	138.3	10.2	0.8	134	131	150
		1500	136.0	3.0	0.8	136	139	133
		5000	103.7	9.2	0.6	93	109	109
Experiment 2, pre-incubation, no metabolic activation, positive control
TA98	2-nitrofluorene	2	237.2	42.9	4.7	189	271	252
TA100	Sodium azide	2	652.7	31.8	4.5	630	689	639
TA1535	Sodium azide	2	672.7	58.5	41.2	732	671	615
TA1537	9-aminoacridine	50	191.3	28.9	28.9	209	207	158
WP2 uvrA	4-nitroquinoline-1-oxide	2	1028.7	12.1	6.3	1038	1033	1015
S: slight thinning of background lawn; T: thinning of background lawn
Experiment 2, pre-incubation, with metabolic activation
Strain	Addition	Concentration per plate [µg]	Mean revertant number per plate	Standard deviation	Fold increase over vehicle	Individual revertant counts
TA98	DMSO		30.0	1.7		32	29	29
	Test substance	0.5	24.3	7.5	0.8	32	17	24
		1.5	25.0	5.2	0.8	22	22	31
		5	32.0	1.7	1.1	31	31	34
		15	33.0	3.5	1.1	31	37	31
		50	21.3	5.8	0.7	18	18	28
		150	21.0	0.0	1.7	21	21	21
		500	28.3	3.2	0.9	27S	32S	26S
		1500	24.0	2.0	0.8	26T	22T	24T
		5000	18.0	2.6	0.6	16T	17T	21T
TA100	DMSO		142.0	10.0		142	132	152
	Test substance	5	100.7	7.6	0.7	104	92	106
		15	110.0	4.6	0.8	106	109	115
		50	114.3	11.6	0.8	101	122	120
		150	124.3	13.1	0.9	138	112	123
		500	132.3	13.3	0.9	117	141	139
		1500	141.7	2.5	1.0	139	142	144
		5000	145.0	12.8	1.0	148	156	131
TA1535	DMSO		15.0	3.0		12	15	18
	Test substance	5	10.7	4.7	0.7	16	9	7
		15	13.7	1.2	0.9	15	13	13
		50	11.0	6.2	0.7	6	9	18
		150	12.3	7.1	0.8	6	11	20
		500	16.0	8.8	1.1	10	26	12
		1500	10.7	8.1	0.7	18	2	12
		5000	11.0	1.7	0.7	10	13	10
TA1537	DMSO		16.7	4.5		21	12	17
	Test substance	5	10.3	1.5	0.6	12	9	10
		15	10.7	4.5	0.6	15	11	6
		50	12.0	0.0	0.7	12	12	12
		150	13.7	2.3	0.8	15	15	11
		500	16.3	3.5	1.0	13	20	16
		1500	12.0	1.7	0.7	13	10	13
		5000	12.7	3.8	0.8	11	10	17
WP2 uvrA (pKM101)	DMSO		200.7	15.0		215	185	202
	Test substance	5	177.0	39.1	0.9	132	202	197
		15	173.3	15.4	0.9	166	191	163
		50	166.0	10.5	0.8	156	177	165
		150	176.3	11.7	0.9	163	185	181
		500	164.7	5.5	0.8	165	159	170
		1500	157.7	7.4	0.8	155	166	152
		5000	143.0	5.3	0.7	139	149	141
S: slight thinning of background lawn; T: thinning of background lawn
Experiment 2, pre-incubation, with metabolic activation, positive control
TA98	Benzo[a]pyrene	5	175.7	1.2	5.9	175	177	175
TA100	2-aminoanthracene	5	2141.7	377.8	15.1	1711	2297	2417
TA1535	2-aminoanthracene	5	361.0	19.5	24.1	346	354	383
TA1537	Benzo[a]pyrene	5	159.3	1.2	9.6	158	160	160
WP2 uvrA	2-aminoanthracene	10	1038.0	71.5	5.2	1109	1039	966
Experiment 2, pre-incubation, viability
		Strain		Mean counts per plate	Standard deviation	Individual colony counts (100 µL aliquots of 10^-6 dilution of 10-hour culture)
		TA98	Viability	150.0	9.8	142	147	161
		TA100	Viability	138.3	8.4	148	133	134
		TA1535	Viability	136.3	9.3	132	130	147
		TA1537	Viability	111.7	21.1	99	100	136
		WP2 uvrA (pKM101)	Viability	188.7	23.1	167	213	186

Table 3: Historical control data

DMSO
	TA100		TA1535		WP2 uvrA (pKM101)		TA98		TA1537
S9Mix	-	+	-	+	-	+	-	+	-	+
Max	234	237	47	55	221	280	78	84	63	50
Min	103	91	11	11	54	56	24	27	8	15
Mean	158	168	26	23	193	193	42	55	21	32
No. of values	147	145	147	141	131	131	153	149	146	143
St.dev.	24	27	6	7	37	37	9	12	7	7
Upper 95% limit	206	222	38	36	266	266	59	80	35	46
Lower 95% limit	110	114	14	10	121	121	24	31	6	18
Positive controls
	TA100		TA1535		WP2 uvrA (pKM101)		TA98		TA1537
	NaN3	AAN	NaN3	AAN	NQO	AAN	2NF	B[a]P	AAC	B[a]P
S9Mix	-	+	-	+	-	+	-	+	-	+
Conc. (µg/plate)	2	5	2	5	2	10	2	5	50	5
Max	2776	4210	1255	716	3730	1923	802	648	2181	609
Min	396	425	209	147	639	352	82	90	87	84
Mean	1017	2017	799	378	2192	974	246	270	370	159
No. of values	198	196	199	194	189	186	205	202	196	193
St.dev.	292	763	219	127	651	368	123	94	270	58
NaN3: Sodium azide; 2NF: 2-nitrofluorene; AAC: 9-aminoacridine; B[a]P: Benzo[a]pyrene; AAN: 2-aminoanthracene

Applicant's summary and conclusion

Conclusions:

The test substance showed no evidence of mutagenic activity in the tested strains of S. typhimurium and E. coli in the absence or presence of metabolic activation.

Executive summary:

The mutagenic potential of the substance was studied in an in vitro bacterial reverse mutation (Ames) study in accordance with OECD TG 471 (1997) under GLP. The experiment is considered relevant, adequate and conclusive.

Experiments were conducted with the four histidine-dependent auxotrophic mutants of Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, and in addition with the tryptophan dependent mutant of Escherichia coli strain WP2 uvrA (pKM101) that were exposed to the test substance dissolved in dimethyl sulfoxide (DMSO).

Two independent mutation experiments were performed in the presence and absence of liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone. The first experiment was a standard plate incorporation assay; the second experiment included a pre-incubation stage. Concentrations of up to 5000 µg/plate were tested, which is the standard limit concentration recommended in the current test guideline. In the first experiment, toxicity occurred, observed as a thin background lawn of non-revertant colonies and/or a reduction in the number of revertant colonies, following exposure to the test substance in strain TA98 at 50 µg/plate and above in the absence of S9 mix and at 1500 µg/plate and above in the presence of S9 mix. Strain TA98, in the absence of S9 mix, did not fulfil the criteria of a valid experiment and an additional plain incorporation experiment was therefore with this strain. In the second experiment, a pre-incubation test, toxicity (observed as thinning of the background lawn of non-revertant colonies, together with a reduction in revertant colony numbers) was obtained in strain TA98 following exposure to 500 µg/plate and above in the absence and presence of S9 mix. No precipitate was observed on plates following exposure to the test substance in both experiments. The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the S9 mix. The mean revertant colony numbers for the vehicle controls were within or close to the historical control range for the lab. The concurrent sterility controls demonstrated the absence of microbial contamination of the S9 mix, buffer or test substance formulation. No evidence of mutagenic activity of the test substance was observed at any tested concentration in either experiments in the absence or presence of S9 mix, and it was concluded that the test substance was not mutagenic in this Ames test.