Aromatic hydrocarbons, C9-12, benzene distn.

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:

06/11/1997-01/13/1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study done according to standard method.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hydrocarbons, C9, aromatics

EC Number:

918-668-5

IUPAC Name:

Hydrocarbons, C9, aromatics

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine and biotin requiring

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: histidine requiring

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Range finding: 8, 40, 200, 1000, 5000 μg/plate
Mutation Experiment 1 without metabolic activation: 0.5333, 2.67, 13.3, 66.7, 333.0 μg/plate
Mutation Experiment 1 with metabolic activation: 2.67, 8, 40, 200, 1000 μg/plate
Mutation Experiment 2 without metabolic activation: 31.25, 62.5, 125, 250, 500 μg/plate
Mutation Experiment 2 with metabolic activation, strains TA98 and TA100 initial treatments: 12.5, 25, 50, 100, 200 μg/plate
Mutation Experiment 2 with metabolic activation, strains TA1535, TA1537 and TA102 initial treatments: 62.5, 125, 250, 500, 1000 μg/plate
Mutation Experiment 2 with metabolic activation, strains TA1535 and TA102 repeat treatments: 7.8125, 15.625, 31.25, 62.5, 125, 250 μg/plate
Mutation Experiment 2 with metabolic activation, strain TA1535: 3.90625, 7.8125, 15.625, 31.25, 62.5, 125 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

other: not applicable

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene, sodium azide, 9-aminoacridine, gluteraldehyde, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) - Platings were made by adding 0.1 ml bacterial culture, 0.1 ml test article solution, and 0.5 ml 10% S-9 mix or buffer to 2.5 ml molten agar at 46°C. The plating mixture was rapidly mixed and poured onto Minimal Davis agar plates. After setting, the plates were inverted and incubated for 3 days at 37°C in the dark. Plates were then examined for toxicity. In experiment 2, the test solution was reduced to 0.05 ml due to the toxicity of the solvent, DMSO.


DURATION
- Preincubation period: Experiment 2 - 1 hour
- Exposure duration: 3 days
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

NUMBER OF REPLICATIONS: 3

OTHER: Colonies were counted using a Seescan Colony Counter or manually when automatic counts could not be obtained.

Evaluation criteria:

Mean control counts fell within normal ranges.
Positive controls induced clear increases in revertant numbers.
No more than 5% of the plates lost through contamination.
Dunnett's test gave a significant response (p <= 0.01), and the data set showed a significant dose-correlation.
The positive responses were reproducible.

Statistics:

Individual plate counts were averaged. Dunnett's test was used to calculate significant responses.

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:

200, 1000, 5000 μg/plate in TA100

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mean revertant colonies (-S-9) - Experiment 1

Substance	Dose level (µg/plate)	TA98	TA100	TA1535	TA1537	TA102
		Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
DMSO	100 µl	28 ± 6	109 ± 6	19 ± 5	12 ± 3	271 ± 18
SHELLSOL A	0.533	26 ± 6	101 ± 10	14 ± 2	10 ± 8	251 ± 10
	2.67	31 ± 1	108 ± 10	14 ± 2	9 ± 3	255 ± 15
	13.3	26 ± 6	98 ± 6	17 ± 1	10 ± 1	277 ± 4
	66.7	24 ± 5	91 ± 14	18 ± 6	9 ± 2	214 ± 2
	333 (V+Ppn)	21 ± 4 (V+Ppn)	86 ± 0 (V+Ppn)	13 ± 2 (V+Ppn)	6 ± 2 (S+Ppn)	184 ± 3 (S+Ppn)
Positive Controls	Compound	2NF	NaN3	NaN3	AAC	GLU
	Dose Level	5 µg	2 µg	2 µg	50 µg	25 µg
	Mean ± SD	754 ± 130	509 ± 29	312 ± 27	376 ± 27	435 ± 28

Mean Revertant Colonies (+S-9) - Experiment 1

Substance	Dose level (µg/plate)	TA98	TA100	TA1535	TA1537	TA102
		Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
DMSO	100 µl	32 ± 8	134 ± 9	17 ± 6	14 ± 5	318 ± 21
SHELLSOL A	2.67	37 ± 5	143 ± 11	17 ± 7	11 ± 1	335 ± 45
	8	41 ± 9	131 ± 7	21 ± 2	14 ± 3	315 ± 15
	40	33 ± 6	133 ± 20	17 ± 3	15 ± 4	323 ± 12
	200	34 ± 10 S	125 ± 2 S	23 ± 5	12 ± 2	275 ± 19
	1000	31 ± 1 S	112 ± 6 S	15 ± 4 S	9 ± 2 S	252 ± 17 S
Positive Controls	Compound	AAN	AAN	-	-	-
	Dose Level	5 µg	5 µg	-	-	-
	Mean ± SD	925 ± 121	1226 ± 104	-	-	-

Mean Revertant Colonies (-S-9) - Experiment 2

Substance	Dose level (µg/plate)	TA98	TA100	TA1535	TA1537	TA102
		Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
DMSO	100 µl	29 ± 7	117 ± 7	22 ± 5	5 ± 3	331 ± 19
SHELLSOL A	31.25	26 ± 6	131 ± 11	20 ± 7	10 ± 2	304 ± 6
	62.5	30 ± 6	113 ± 7	20 ± 4	8 ± 2	276 ± 9
	125	27 ± 5 S	113 ± 21 S	18 ± 3	10 ± 2	278 ± 28
	250	16 ± 1 S	111 ± 19 S	16 ± 1 S	7 ± 2 S	180 ± 6 S
	500	15 ± 2 M+Ppn+V	100 ± 19 M+Ppn+S/V	17 ± 6 M+S	5 ± 2 S	199 ± 11 S/V
Positive Controls	Compound	2NF	NaN3	NaN3	AAC	GLU
	Dose Level	5 µg	2 µg	2 µg	50 µg	25 µg
	Mean ± SD	1004 ± 95	722 ± 15	438 ± 30	410 ± 112	595 ± 11

Mean Revertant Colonies (+S-9) Experiment 2

Substance	Dose level (µg/plate)	TA98	TA100	TA1535	TA1537	TA102
		Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD	Mean ± SD
DMSO	100 µl	38 ± 4	140 ± 7	18 ± 5	7 ± 3	360 ± 19
SHELLSOL A	3.90625	-	-	22 ± 2 M	-	-
	7.8125	-	-	19 ± 2	10 ± 2	344 ± 13
	12.5	36 ± 7	143 ± 9	-	-	-
	15.625	-	-	12 ± 4 M	6 ± 3	323 ± 35
	25	32 ± 9	133 ± 4	-	-	-
	31.25	-	-	17 ± 5	9 ± 1	287 ± 66
	50	34 ± 9	134 ± 3	-	-	-
	62.5	-	-	17 ± 0	10 ± 1	307 ± 23
	100	24 ± 3 S	133 ± 4 S	-	-	-
	125	-	-	17 ± 5 S	4 ± 3 S	242 ± 15 S
	200	26 ± 0 S	131 ± 4 S	-	-	-
	250	-	-	-	5 ± 3 S	209 ± 27 S
Positive Controls	Compound	AAN	AAN	AAN	AAN	-
	Dose Level	5 µg	5 µg	5 µg	5 µg	-
	Mean ± SD	1109 ± 28	1045 ± 16	68 ± 3	63 ± 4	-

S - Slight thinning of background lawn

M - Plate counted manually

Ppn - Precipitation observed

V - Very thin background lawn

AAN - 2 -aminoanthracene

2NF - 2 -nitrofluorene

NaN3 - sodium azide

AAC - 9 -aminoacridine

GLU - gluteraldehyde

Summary of other bacterial mutagenicity studies.

End Point

Study Reference

REACH requirement

IUCLID Section

Study Name

Data Waiving

Waiving Justification

Species

Study Result Type

Test Guideline/Qualifier

Test Guideline/Guideline

Test Guideline/Deviations

Reliability

Rational For Reliability

GLP Compliance

Test Materials/Identity

Study Result

Reference Type

Reference Author

Reference Year

Reference Title

Bibliographic Source

Testing Laboratory

Reference Report No.

Owner Company

Company Study No.

Report Date

Data access

8.4.1 In vitro bacterial mutagenicity

7.6.1

The mutagenic potential of high flash aromatic naptha

Salmonella typhimurium

experimental result

According to

Ames, B.N., McCann, J., and Yamasaki, E. (1975).  Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutat. Res.31: 347-364.

No

1

Well-documented journal article.

No

Test material was specifically prepared to meet the composition requirements for High Flash Aromatic Naptha, Type 1

not mutagenic

study report

Schreiner, CA, Edwards, DA, McKee, RH, Swanson, M, Wong, ZA, Schmitt, S, Beatty, P

1989

The mutagenic potential of high flash aromatic naptha

Cell Biology and Toxicology, Vol. 5, No. 2, 169-188

yes

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It was concluded that the substance did not induce mutation in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537, and TA102 ) when tested under the conditions employed in this study, which included treatment at concentrations up to the lower limit of toxicity, both in the absence and in the presence of rat liver metabolic activation system (S-9).

Executive summary:

A bacterial reverse mutation assay was performed on five strains of Salmonella typhimurium. The study was performed both with (maximum concentration of 1000 microgram/ml) and without metabolic activation (maximum concentration of 500 microgram/ml). After 3 days of exposure to the test substance, the number of mean revertants per plate was calculated. Toxicity was seen at the highest doses. Most notably in the strains TA1535, TA1537, and TA102 during Experiment 2. These strains were retested at lower doses. There was no significant increase in the number of revertants in any of the test strains. There was a significant increase in the number of revertants in the positive control treatment. The study is therefore valid, and the test substance is not mutagenic.