Hydrocarbons, C9-C10, aromatics, >1% naphthalene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

 

Hydrocarbons, C9 Aromatics:

Genetic Toxicity in vitro - Bacterial reverse mutation assay (OECD TG 471)

Genetic Toxicity in vitro - In vitro Mammalian Chromosome Aberration Test (OECD TG 473)

Genetic Toxicity in vitro - In vitro Mammalian Cell Gene Mutation Test (OECD TG 476)

Genetic Toxicity in vitro - In vitro Sister Chromatid Exchange Assay in Mammalian Cells (OECD TG 479) 

 

Hydrocarbons, C10-C12 Aromatics:

Genetic Toxicity in vitro - Bacterial reverse mutation assay (OECD TG 471)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

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.

Justification for type of information:

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

Reason / purpose for cross-reference:

read-across: supporting information

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

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 / 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

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.

Key result

Species / strain:

S. typhimurium TA 1535

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium 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

Key result

Species / strain:

S. typhimurium TA 102

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'.

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

Conclusions:

Interpretation of results: 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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD 471 guidelines. GLP.

Justification for type of information:

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fractions from Aroclor exposed rats

Test concentrations with justification for top dose:

Tests (done in triplicate) with and without Metabolic Activation: 0, 32, 63, 125, 250, 500 ug/plate
Positive controls:
TA 1535 (S9-: sodium azide 1.0 ug/plate) (S9+: 2-aminoanthracene: 2.0 ug/plate)
TA 1537 (S9-: 9-aminoacridine 80 ug/plate) (S9+: benzo(a)pyrene: 4.0 ug/plate)
TA 98 (S9-: 2-nitrofluorene 2.0 ug/plate) (S9+: 2-aminoanthracene: 2.0 ug/plate)
TA 100 (S9-: sodium azide 1.0 ug/plate) (S9+: 2-aminoanthracene: 2.0 ug/plate)
WP2 uvr A (S9-: N-ethyl-N-nitrosourea 100 ug/plate) (S9+: 2-aminoanthracene: 80 ug/plate)

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Remarks:

non treated

Positive controls:

yes

Positive control substance:

other: See Test Concentrations

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar
DURATION
- Exposure duration: 48 hours


NUMBER OF REPLICATIONS:
- triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants and/or clearing of the background lawn of bacterial growth

Evaluation criteria:

The mutagenicity study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the positive controls meet the criteria for a positive response and if no more than 5% of the plates are lost through contamination or other unforeseen events.

A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates increase in a concentration-related manner and/or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test substance is considered negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitution or frameshifts in the genome of Salmonella typhimurium. Negative results indicate that under the test conditions, the test substance is not mutagenic.

Statistics:

The mean plate count and standard deviation for each dose point were determined. Any test value that was equal to or greater than two times the mean value of the concurrent vehicle control was considered to be a positive dose.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity pretest was performed from doses of 556, 1667, 5000 ug/plate using all strains. Toxicity was observed at all of these doses and in phase 2 of the study, the doses were reduced to 0, 32, 63, 125, 250, 500 ug/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

In all cases, the test material did not induce any significant changes in the number of revertant colonies. It is concluded in this study that the test material is not a mutagenic agent and is not classified under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.

Executive summary:

Test material was examined for mutagenic activity in the bacterial reverse mutation test using histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 98, and 100, and theE. colistrain WP2 uvr A in the absence and presence of a liver S9 fraction for metabolic activation. Concentrations above 500 µg/plate were found to be cytotoxic and so the test was performed in triplicate using doses of 0, 32, 63, 125, 250, 500 µg/plate. 

 

In all cases, the test material did not induce any significant changes in the number of revertant colonies.  

 

It is concluded in this study that the test material is not a mutagenic agent and is not classified under the new Regulation (EC) 1272/2008 on classification, labelling, and packaging of substances and mixtures (CLP).  

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1991/01/28-1991/03/07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD 471 guidelines. GLP.

Justification for type of information:

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fractions from Aroclor exposed rats

Test concentrations with justification for top dose:

Tests (done in triplicate) with and without Metabolic Activation: 0 (DMSO or Acetone), 3.2, 10, 32, 100, 320 ug/plate
Vehicle control: 0.1 ml/plate DMSO (positive controls) or 0.1 ml/plate acetone (test material)
Positive controls: 5ug/plate 2AA, 10ug/plate MNNG, 100ug/plate 9AA, 5ug/plate 2NF

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO/ acetone
- Justification for choice of solvent/vehicle: Positive controls dissolved into DMSO, Test substance soluble in acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

0.1 ml/plate DMSO; 0.1 ml/plate Acetone

True negative controls:

yes

Remarks:

non treated

Positive controls:

yes

Positive control substance:

other: TA 1537 (+S9 2-aminoanthracene) TA 1537 (-S9 9-aminoacridine); TA 98 (-S9 2-nitrofluorene) (+S9 2-aminoanthracene); TA100 (-S9 MNNG) (+S9 2-aminoanthracene); TA1535 (-S9 MNNG) (+S9 2-aminoanthracene); TA138(-S9 2-Nitrofluorene) (+S9 2-aminoanthracene)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar
DURATION
- Exposure duration: 48 hours


NUMBER OF REPLICATIONS:
- triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants and/or clearing of the background lawn of bacterial growth

Evaluation criteria:

The mutagenicity study is considered valid if the mean colony counts of the control values of the strains are within acceptable ranges, if the positive controls meet the criteria for a positive response and if no more than 5% of the plates are lost through contamination or other unforeseen events.

A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates increases in a concentration-related manner and/or if a reproducible two-fold or more increase is observed compared to that on the negative control plates.

A test substance is considered negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Positive results from the bacterial reverse mutation test indicate that a substance induces point mutations by base substitution for frameshifts in the genome of Salmonella typhimurium. Negative results indicate that under the test conditions, the test substance is not mutagenic.

Statistics:

The mean plate count and standard deviation for each dose point were determined. Any test value that was equal to or greater than two times the mean value of the concurrent vehicle control was considered to be a positive dose.

Key result

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

Remarks:

not cytotoxic up to 10,000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

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

Remarks:

not cytotoxic up to 10,000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

not cytotoxic up to 10,000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Remarks:

not cytotoxic up to 10,000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

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

Remarks:

not cytotoxic up to 10,000 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity pretest was performed from doses of 1 to 10000ug/plate using TA98. Toxicity was observed and a maximum dose of 320 ug/plate was selected.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

In all cases, MRD-90-884 did not induce any significant changes in the number of revertant colonies. It is concluded in this study that MRD-90-884 is not a mutagenic agent and is not classified under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations

Executive summary:

MRD-90-884 was examined for mutagenic activity in the bacterial reverse mutation test using histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 1538, 98, and 100 in the absence and presence of a liver S9 fraction for metabolic activation. Concentrations above 320 µg/plate were found to be cytotoxic and so the test was performed in triplicate using doses of 0, 3.2, 10, 32, 100, 320 µg/plate. In all cases, MRD-90-884 did not induce any significant changes in the number of revertant colonies.

 

It is concluded in this study that MRD-90-884 is not a mutagenic agent and is not classified under the new Regulation (EC) 1272/2008 on classification, labelling, and packaging of substances and mixtures (CLP).

 

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented publication which meets basic scientific principles.

Justification for type of information:

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

Method was according to:
Hsie, AW, Brimer, PA, Mitchell, TJ, and Gosslee, DG. (1975) Dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxyanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells. Somat. Cell Genet. 1:247-261
Hsie, AW, Casciano, DA, Couch, DB, Krahn, DF, O'Neill, JP, and Whitfield, BL. (1981). The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals. Mutation Res. 86:193-214.
Myhr, BC and DiPaolo, JA. (1978). Mutagenesis of Chinese hamster cells in vitro by combination treatments with methyl methanosulfonate and N-acetoxy-2-acetylamino-fluorene. Cancer Res. 38: 2539-2543.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1-BH4 clone

Metabolic activation:

with and without

Metabolic activation system:

S9 from livers of aroclor-induced Sprague-Dawley rats

Test concentrations with justification for top dose:

0.01, 0.02, 0.04, 0.06, 0.07, 0.08, 0.1, 0.13 µl/ml without metabolic activation
0.02, 0.04, 0.06, 0.08, 0.1, 0.13, 0.16, 0.2 µl/ml with metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 10 µl/m

True negative controls:

other: not applicable

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate at 15 and 20 µl/ml, 5-bromo-2'-deoxyuridine at 50 µl/ml

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

10 µl/ml DMSO

True negative controls:

other: not applicable

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

Remarks:

with metabolic activation Migrated to IUCLID6: 5 µl/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days

SELECTION AGENT (mutation assays): 4 micrograms/ml 6-thioguanine

Evaluation criteria:

Treatment groups were evaulated for relative survival, relative population growth, absolute cloning efficiency, and mutation frequency.

Statistics:

Kastenbaum, MA, Bowman, KO. (1970). Tables for determining the statistical significance of mutation frequencies. Mutat. Res. 9: 527-549.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic at concentrations =>0.07 µl/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There was no evidence of mutagenicity as compared to controls.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

CHO/HGPRT Forward Mutation Suspension Assay without Metabolic Activation

	Mean Colony Number	Relative Population Growth (%)	Mutant Frequency in 10^-6 units
DMSO	202.7 ± 7.6	111.0	1.0
DMSO	190.0 ±17.8	89.0	2.2
5-bromo-2’-deoxyuridine	161.3 ±11.2	114.1	14.0
Methyl methanesulfonate 15 µl/ml	83.0 ±7.0	63.5	59.2
Methyl methanesulfonate 20 µl/ml	41.0 ±7.2	38.3	59.4
0.01 µl/ml	185.0 ±10.6	176.6	1.1
0.02 µl/ml	204.7± 1.5	148.6	0.0
0.04 µl/ml	204.3 ±2.5	147.5	1.9
0.06 µl/ml	202.7± 20.5	107.5	0.6
0.07 µl/ml	77.3 ±7.0	35.2	1.4
0.08 µl/ml	5.7 ±1.5	10.7	0.9
0.1 µl/ml	0.0± 0.0	ND	ND
0.13 µl/ml	0.0 ±0.0	ND	ND

CHO/HGPRT Forward Mutation Suspension Assay with Metabolic Activation

	Mean Colony Number	Relative Population Growth (%)	Mutant Frequency in 10^-6 units
DMSO	203.7 ± 16.9	90.5	0.9
DMSO	201.0 ± 12.5	109.5	4.4
3-methylcholanthene	201.0 ± 7.8	77.1	161.3
0.02 µl/ml	185.3 ± 3.5	119.7	2.6
0.04 µl/ml	205.3 ± 21.1	111.7	3.4
0.06 µl/ml	196.7 ± 22.0	110.0	3.4
0.07 µl/ml	3.3 ± 1.5	4.0	1.3
0.08 µl/ml	0.0 ± 0.0	ND	ND
0.1 µl/ml	0.0 ± 0.0	ND	ND
0.13 µl/ml	0.0 ± 0.0	ND	ND
0.16 µl/ml	0.0 ± 0.0	ND	ND
0.2 µl/ml	0.0 ± 0.0	ND	ND

Chromosome Aberrations in CHO Cells Exposed to High-Flash Aromatic Naphtha

Conclusions:

Interpretation of results: negative

The test material is not mutagenic, however, it is cytotoxic at concentrations of 0.07 µL/mL or above.

Executive summary:

A mammalian cell gene mutation assay was performed on Chinese hamster ovary cells to assess the mutagenicity of high flash aromatic naphtha to mammalian cells. The test was performed both with (0.02, 0.04, 0.06, 0.08, 0.1, 0.13, 0.16, 0.2 microliters/mL) and without (0.01, 0.02, 0.04, 0.06, 0.07, 0.08, 0.1, 0.13 microliters/mL) metabolic activation. Cells were then examined for mutation frequency.

Only positive controls showed a significant increase in mutation frequency. No increase in mutation frequency was seen at any concentration either with or without metabolic activation. An analysis of relative population growth showed a reduction in population growth to 35.2% or greater of negative controls at exposures to concentrations or 0.07 microliters/mL or above. The test substance is therefore not mutagenic, however, it is cytotoxic at concentrations of 0.07 µL/mL or above.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Source of data is from peer reviewed literature. Acceptable well-documented study report which meets basic scientific principles: GLP.

Justification for type of information:

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

Method was according to:
Hsie, AW, Brimer, PA, Mitchell, TJ, and Gosslee, DG. (1975) Dose-response relationship for ethyl methanesulfonate-induced mutations at the hypoxyanthine-guanine phosphoribosyl transferase locus in Chinese hamster overy cells. Somat. Cell Genet. 1:247-261
Hsie, AW, Casciano, DA, Couch, DB, Krahn, DF, O'Neill, JP, and Whitfield, BL. (1981). The use of Chinese hamster ovary cells to quantify specific locus mutation and to determine mutagenicity of chemicals. Mutation Res. 86:193-214.
Myhr, BC and DiPaolo, JA. (1978). Mutagenesis of Chinese hamster cells in vitro by combination treatments with methyl methanosulfonate and N-acetoxy-2-acetylamino-fluorene. Cancer Res. 38: 2539-2543.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1-BH4 clone

Metabolic activation:

with and without

Metabolic activation system:

S9 from livers of aroclor-induced Sprague-Dawley rats

Test concentrations with justification for top dose:

0.01, 0.02, 0.04, 0.06, 0.07, 0.08, 0.1, 0.13 μl/ml without metabolic activation
0.02, 0.04, 0.06, 0.08, 0.1, 0.13, 0.16, 0.2 μl/ml with metabolic activation

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO 10 μl/ml

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate at 15 and 20 μl/ml, 5-bromo-2'-deoxyuradine at 50 μl/ml, 3-methylcholanthrene 5 μl/ml

Remarks:

without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days

SELECTION AGENT (mutation assays): 4 micrograms/ml 6-thioguanine

Evaluation criteria:

Treatment groups were evaulated for relative survival, relative population growth, absolute cloning efficiency, and mutation frequency.

Statistics:

Kastenbaum, MA, Bowman, KO. (1970). Tables for determining the statistical significance of mutation frequencies. Mutat. Res. 9: 527-549.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic at concentrations =>0.1 microliters/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

There was no evidence of mutagenicity as compared to controls.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

CHO/HGPRT Forward Mutation Suspension Assay without Metabolic Activation

	Mean Colony Number	Relative Population Growth (%)	Mutant Frequency in 10-6 units
DMSO	202.7 ± 7.6	111.0	1.0
DMSO	190.0 ±17.8	89.0	2.2
5-bromo-2-deoxyuridine	161.3 ±11.2	114.1	14.0
Methyl methanesulfonate 15 µl/ml	83.0 ±7.0	63.5	59.2
Methyl methanesulfonate 20 µl/ml	41.0 ±7.2	38.3	59.4
0.01 µl/ml	185.0 ±10.6	176.6	1.1
0.02 µl/ml	204.7± 1.5	148.6	0.0
0.04 µl/ml	204.3 ±2.5	147.5	1.9
0.06 µl/ml	202.7± 20.5	107.5	0.6
0.07 µl/ml	77.3 ±7.0	35.2	1.4
0.08 µl/ml	5.7 ±1.5	10.7	0.9
0.1 µl/ml	0.0± 0.0	ND	ND
0.13 µl/ml	0.0 ±0.0	ND	ND

CHO/HGPRT Forward Mutation Suspension Assay with Metabolic Activation

	Mean Colony Number	Relative Population Growth (%)	Mutant Frequency in 10-6 units
DMSO	203.7 ± 16.9	90.5	0.9
DMSO	201.0 ± 12.5	109.5	4.4
3-methylcholanthene	201.0 ± 7.8	77.1	161.3
0.02 µl/ml	185.3 ± 3.5	119.7	2.6
0.04 µl/ml	205.3 ± 21.1	111.7	3.4
0.06 µl/ml	196.7 ± 22.0	110.0	3.4
0.07 µl/ml	3.3 ± 1.5	4.0	1.3
0.08 µl/ml	0.0 ± 0.0	ND	ND
0.1 µl/ml	0.0 ± 0.0	ND	ND
0.13 µl/ml	0.0 ± 0.0	ND	ND
0.16 µl/ml	0.0 ± 0.0	ND	ND
0.2 µl/ml	0.0 ± 0.0	ND	ND

 

Conclusions:

Interpretation of results: negative

The test material is not mutagenic, however, it is cytotoxic at concentrations of 0.1 μL/mL or above.

Executive summary:

The test material is not mutagenic, however, it is cytotoxic at concentrations of 0.1 μL/mL or above.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented publication which meets basic scientific principles.

Justification for type of information:

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO-K1-BH4 clone

Metabolic activation:

with and without

Metabolic activation system:

S9 from livers of aroclor-induced Sprague-Dawley rats

Test concentrations with justification for top dose:

15.0, 30.1, 45.0, 60.0, 75.0, 90.0 µg/ml without metabolic activation
20.0, 25.0, 37.5, 40.1, 50.0, 60.1, 70.0, 80.2 µg/ml with metabolic activation

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

other: not applicable

True negative controls:

other: not applicable

Positive controls:

yes

Positive control substance:

other: MMC (unactivated cultures) and CP (metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 7 hrs (w/o activation); 2.5 hrs (w/ activation)
- Expression time (cells in growth medium): After 10 hour incubations, metaphase cells were collected by mitotic shake off
- Fixation: methanol:glacial acetic acid (3:1), stained with 5% Giemsa (pH 6.8)

Positive Controls: MMC (unactivated cultures) and CP (metabolic activation)

Evaluation criteria:

Treatment groups were evaulated for overall chromosomal aberration frequency, the percentage of cells with aberrations, the percentage of cells with more than one aberration, the presence or absence of a dose-response and the complexity of the aberrations.

Statistics:

Fisher’s exact test with an adjustment for multiple comparisons to compare the percentage of cells with aberrations in each treatment group with the results from the controls (Sokal and Rohlf, 1981).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic at 90.2 µg/mL with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There were no increases in chromosomal aberrations at any concentration level tested in either the presence or absence of metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

The test material is not clastogenic, however, it is cytotoxic at concentrations of 80.2 µg/mL or above.

Executive summary:

This study assessed the potential of high flash aromatic naphtha to cause chromosome aberrations in Chinese hamster Ovary (CHO) cells. Cells were exposed to concentrations of 15.0, 30.1, 45.0, 60.0, 75.0, 90.0 micrograms/mL without metabolic activation and 20.0, 25.0, 37.5, 40.1, 50.0, 60.1, 70.0, 80.2 micrograms/mL with metabolic activation. Cells were than evaluated for chromosomal aberration frequency, the percentage of cells with aberrations, the percentage of cells with more than one aberration, the presence or absence of a dose-response, and the complexity of the aberrations.

There were no increases in chromosomal aberrations at any concentration level tested in either the presence or absence of metabolic activation. The test material is not clastogenic, however, it is cytotoxic at concentrations of 80.2 micrograms/mL or above.

Reference 7

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented publication which meets basic scientific principles.

Justification for type of information:

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 from livers of aroclor-induced Sprague-Dawley rats

Test concentrations with justification for top dose:

2.0, 6.67, 20.0, 35.0, 50.1, 66.7 µg/ml without metabolic activation
0.667, 2.0, 6.67, 15.0, 20.0, 35.0, 50.1 µg/ml with metabolic activation

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

other: not applicable

True negative controls:

other: not applicable

Positive controls:

yes

Positive control substance:

other: MMC (unactivated cultures) and CP (metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 22.5 hrs (w/o activation); 2 hrs (w/ activation)
- Expression time (cells in growth medium): After 23 hour incubation with Brdu), metaphase cells were collected by mitotic shake off

Positive Controls: mitomycin C (MMC, unactivated cultures) and cyclophosphamide (CP, metabolic activation)

Evaluation criteria:

Total sister chromatid exchange (SCE), SCE per chromosome, SCE per cell in the M2 stage of mitosis, percentage of cells in the M1, M1+, or M2 stages of cell cycle.

Statistics:

Evaluation of mutagenic responses was based on statistical comparisons of SCE frequencies in the C9 treated cultures with those in the negative (solvent) control cultures (Bancroft 1957, Hollander and Wolfe 1973).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic at 90.2 µg/mL with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There were no increases in SCE at any concentration level tested in either the presence or absence of metabolic activation. Cell-cycle delay was not observed at concentration levels below 66.7 µg/mL (w/o activation) or below 50.1 µg/mL (w/ activation). The positive controls produced significant increases in the % SCE as compared to their respective negative controls.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

	Dose	Cells	# Chromosomes	# SCE	SCE	SCE/Cell
	ug/mL	Scored			Chromosomes	(Mean +/- SE)
Assay #1: w/o metabolic activation
Negative Control	-	50	1044	443	0.42	8.86 (.36)
Solvent Control	11	50	1038	536	0.52	10.72 (.45)
Positive Control: MMC	0.005	20	420	570	1.36	28.5 (1.13)
C9 Aromatics	2	50	1037	530	0.51	10.6 (.43)
	6.67	50	1038	474	0.46	9.48 (.51)
	20	50	1044	480	0.46	9.6 (.44)
	66.67	50	1038	524	0.5	10.48 (.39)
	200	Toxic	-	-	-	-
Assay #2: w/o metabolic activation
Negative Control	-	50	1038	399	0.38	7.98 (.38)
Solvent Control	11	50	1047	432	0.41	8.64 (.5)
Positive Control: MMC	0.005	20	417	547	1.31	27.35 (1.49)
C9 Aromatics	35	50	1043	428	0.41	8.56 (.49)
	50.1	50	1042	461	0.44	9.22 (.36)
	66.7	50	1041	443	0.43	8.86 (.44)
	90.1	50	Toxic	-	-	-
	Dose	Cells	# Chromosomes	# SCE	SCE	SCE/Cell
	ug/mL	Scored			Chromosomes	(Mean +/- SE)
Assay #1: w/ metabolic activation
Negative Control	-	50	1037	443	0.43	8.86 (.43)
Solvent Control	11	50	1032	430	0.42	8.60 (.49)
Positive Control: MMC	1.5	20	415	379	0.91	18.95 (1.20)
C9 Aromatics	0.667	50	1038	449	0.43	8.98 (.34)
	2	50	1034	484	0.47	9.68 (.43)
	6.67	50	1045	474	0.45	9.48 (.46)
	20	50	1040	441	0.42	8.82 (.45)
	66.67	50	Toxic	-	-	-
Assay #2: w/ metabolic activation
Negative Control	-	50	1048	417	0.4	8.34 (.43)
Solvent Control	11	50	1046	398	0.38	7.96 (.38)
Positive Control: MMC	1.5	20	418	457	1.09	22.85 (.91)
C9 Aromatics	15	50	1043	372	0.36	7.44 (.40)
	20	50	1048	444	0.42	8.88 (0.44)
	35	50	1055	400	0.38	8.00 (.46)
	50.1	50	1047	420	0.4	8.4 (.48)
	66.7	50	Toxic	-	-	-

Conclusions:

Interpretation of results: negative

The test material did not cause sister chromatid exchange in CHO cells, however it is cytotoxic at the highest concentrations tested.

Executive summary:

The study examined the potential of high flash aromatic naphtha to cause sister chromatid exchange in Chinese hamster ovary (CHO) cells. CHO cells were exposed to test substance concentrations of 2.0, 6.67, 20.0, 35.0, 50.1, 66.7 micrograms/mL without metabolic activation, and 0.667, 2.0, 6.67, 15.0, 20.0, 35.0, 50.1 micrograms/mL with metabolic activation. The sister chromatid exchange frequency of cells in the treatment groups was then compared to the frequency in the control group.

There were no increases in SCE at any concentration level tested in either the presence or absence of metabolic activation.

The test material did not cause sister chromatid exchange in CHO cells, however it was cytotoxic at the highest concentrations tested.

Reference 8

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Source of data is from peer reviewed literature. Acceptable well-documented study report which meets basic scientific principles: GLP.

Justification for type of information:

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 from livers of aroclor-induced Sprague-Dawley rats

Test concentrations with justification for top dose:

2.0, 6.67, 20.0, 35.0, 50.1, 66.7 μg/ml without metabolic activation
0.667, 2.0, 6.67, 15.0, 20.0, 35.0, 50.1 μg/ml with metabolic activation

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: MMC (unactivated cultures) and CP (metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Exposure duration: 22.5 hrs (w/o activation); 2 hrs (w/ activation)
- Expression time (cells in growth medium): After 23 hour incubation with Brdu), metaphase cells were collected by mitotic shake off

Positive Controls: mitomycin C (MMC, unactivated cultures) and cyclophosphamide (CP, metabolic activation)

Evaluation criteria:

Total sister chromatid exchange (SCE), SCE per chromosome, SCE per cell in the M2 stage of mitosis, percentage of cells in the M1, M1+, or M2 stages of cell cycle.

Statistics:

Evaluation of mutagenic responses was based on statistical comparisons of SCE frequencies in the C9 treated cultures with those in the negative (solvent) control cultures (Bancroft 1957, Hollander and Wolfe 1973).

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cytotoxic at 90.2 ug/mL with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

There were no increases in SCE at any concentration level tested in either the presence or absence of metabolic activation. Cell-cycle delay was not observed at concentration levels below 66.7 ug/mL (w/o activation) or below 50.1 ug/mL (w/ activation). The positive controls produced significant increases in the % SCE as compared to their respective negative controls.

	Dose	Cells	# Chromosomes	# SCE	SCE	SCE/Cell
	ug/mL	Scored			Chromosomes	(Mean +/- SE)
Assay #1: w/o metabolic activation
Negative Control	-	50	1044	443	0.42	8.86 (.36)
Solvent Control	11	50	1038	536	0.52	10.72 (.45)
Positive Control: MMC	0.005	20	420	570	1.36	28.5 (1.13)
C9 Aromatics	2	50	1037	530	0.51	10.6 (.43)
	6.67	50	1038	474	0.46	9.48 (.51)
	20	50	1044	480	0.46	9.6 (.44)
	66.67	50	1038	524	0.5	10.48 (.39)
	200	Toxic	-	-	-	-
Assay #2: w/o metabolic activation
Negative Control	-	50	1038	399	0.38	7.98 (.38)
Solvent Control	11	50	1047	432	0.41	8.64 (.5)
Positive Control: MMC	0.005	20	417	547	1.31	27.35 (1.49)
C9 Aromatics	35	50	1043	428	0.41	8.56 (.49)
	50.1	50	1042	461	0.44	9.22 (.36)
	66.7	50	1041	443	0.43	8.86 (.44)
	90.1	50	Toxic	-	-	-
	Dose	Cells	# Chromosomes	# SCE	SCE	SCE/Cell
	ug/mL	Scored			Chromosomes	(Mean +/- SE)
Assay #1: w/ metabolic activation
Negative Control	-	50	1037	443	0.43	8.86 (.43)
Solvent Control	11	50	1032	430	0.42	8.60 (.49)
Positive Control: MMC	1.5	20	415	379	0.91	18.95 (1.20)
C9 Aromatics	0.667	50	1038	449	0.43	8.98 (.34)
	2	50	1034	484	0.47	9.68 (.43)
	6.67	50	1045	474	0.45	9.48 (.46)
	20	50	1040	441	0.42	8.82 (.45)
	66.67	50	Toxic	-	-	-
Assay #2: w/ metabolic activation
Negative Control	-	50	1048	417	0.4	8.34 (.43)
Solvent Control	11	50	1046	398	0.38	7.96 (.38)
Positive Control: MMC	1.5	20	418	457	1.09	22.85 (.91)
C9 Aromatics	15	50	1043	372	0.36	7.44 (.40)
	20	50	1048	444	0.42	8.88 (0.44)
	35	50	1055	400	0.38	8.00 (.46)
	50.1	50	1047	420	0.4	8.4 (.48)
	66.7	50	Toxic	-	-	-

Conclusions:

Interpretation of results: negative

The test material did not cause sister chromatid exchange in CHO cells, however it is cytotoxic at the highest concentrations tested.

Executive summary:

The test material did not cause sister chromatid exchange in CHO cells, however it is cytotoxic at the highest concentrations tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

 

Hydrocarbons, C9 Aromatics:

Genetic Toxicity in vivo - Mammalian Bone Marrow Chromosome Aberration Test (OECD TG 475)

 

Hydrocarbons, C10-C12 Aromatics:

Genetic Toxicity in vivo - Micronucleus Assay in Mouse Bone Marrow (OECD TG 474)

Genetic Toxicity in vivo - Mammalian Bone Marrow Chromosome Aberration Test (OECD TG 475)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to OECD Guideline 474. GLP

Justification for type of information:

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Charles River Breeding Laboratories, Inc.
Sex: Male (65), Female (65)
Age at study initiation: Approximately 9-10 weeks
Weight at study initiation: 23-39g
Housing: Individually
Diet (e.g. ad libitum): Purina Certified Rodent 5002 chow (pellets), ad libitum
Water (e.g. ad libitum): Automatic watering system, ad libitum
Acclimation period: 35d

ENVIRONMENTAL CONDITIONS
Temperature (°F): 68-76
Humidity (%): 40-70%
Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

Corn oil was used. Dose volume did not exceed 1.0 ml/100 g bw.

Details on exposure:

The test material and the carrier were administered by oral gavage as a single dose. The carrier was dosed at a volume equal to the test material dose volume. The individual animal dose volumes did not exceed 1.0 ml/100 g body weight; animals were administered 0.25, 0.5, or 1.0 g test material/ kg body weight. The positive control, cyclophosphamide was administered as a single intraperitoneal injection (40 mg/kg) using water as a carrier.

Duration of treatment / exposure:

Animals were sacrificed 24, 48, and 72 hours after dose administration.

Frequency of treatment:

One dose was given at either 0.25, 0.5, or 1.0 g test material/ kg body weight.

Post exposure period:

Animals were sacrificed 24, 48, and 72 hours after dose administration.

Remarks:

Doses / Concentrations:
0.25, 0.5, 1.0g/kg/bw
Basis:
other: oral gavage

No. of animals per sex per dose:

Male (65), Female (65) ; 5 Males and 5 Females per treatment group

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control, cyclophosphamide was administered as a single intraperitoneal injection (40 mg/kg) using water as a carrier.

Tissues and cell types examined:

Erythrocytes derived from femur bone marrow.

Details of tissue and slide preparation:

Immediately following the sacrifice of the animals, both femurs were removed and the bone marrow was removed and suspended in fetal bovine serum. After the suspension was centrifuged the pellet was resuspended and smears were prepared (two slides per animal).

Evaluation criteria:

Slides were stained using acridine orange; polychromatic erythrocytes (PCE) stained red/orange, nonchromatic erythrocytes (NCE) are unstained (dull green), and micronuclei stain bright yellow. Additional criteria for scoring micronuclei are a circular appearance and a diameter between 1/20 and 1/5 of the cell’s diameter. 1000 PCE from each animal were examined for the presence of micronuclei and the ratio of PCE to NCE was determined for each animal by counting 1000 erythrocytes (PCE and NCE).

Statistics:

Calculation of means and standard deviations of the micronuclei data and a test of equality of group means by a standard one way analysis of variance at each time period (ANOVA). When ANOVA was significant, comparisons of carrier control to dosed group means were made according to Duncan’s Multiple Range Test.

A standard regression analysis was performed to test for a dose response.
Residuals from the ANOVA were analyzed for normality by Wilk’s Criterion. The residuals were normally distributed (values were greater than 0.01 level of significance). Therefore nonparametric analysis was not performed.

Sexes were analyzed separately.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

2(male), 1 (female) animals at 2.5 g/kg; 1 (female) at 1.0 g/kg

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The positive control (cyclophosphamide) induces a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes, indicating that the positive control was clastogenic and was responding in an appropriate manner. Carrier control values for the mean percent of polychromatic erythrocytes and for the mean number of micronucleated polychromatic erythrocytes are within the normal range for the corn oil control.

MRD-90-884 did not induce a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes. A significant regression coefficient (p<0.05) was observed in the mean micronucleated polychromatic erythrocytes for the 48 hour female group. However, the mean values were not significant when compared to the carrier control and were within the normal range of the carrier control. Therefore this statistical response is not believed to be biologically significant. MRD-90-884 was not clastogenic in mouse bone marrow under the conditions of this test.

Necropsies of the four animals that died prior to the scheduled sacrifice (2(male), 1 (female) animals at 2.5 g/kg; 1 (female) at 1.0 g/kg) revealed that most common effects were urine stains and a tan mucous like material in the small intestine. The most probable cause of death was due to the toxicity of the test material.

Conclusions:

Interpretation of results: negative

These data indicate that MRD-90-884 is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 1.0 g/Kg of body weight.

Executive summary:

The test material, MRD-90-884 was tested in the mammalian bone marrow micronucleus assay using CD-1 mice.  MRD-90-884 was tested at 24, 48, and 72 hour intervals following exposure and did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes or an increase in the mean number of micronucleated polychromatic erythrocytes. 

Both the positive (cyclophosphamide) and the negative (carrier) controls behaved in an appropriate manner. These data indicate that MRD-90-884 is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 1.0 g/Kg.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable well-documented publication which meets basic scientific principles.

Justification for type of information:

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

inhalation: vapour

Details on exposure:

- Exposure apparatus: 16 m glass and steel chambers.
- Method of holding animals in test chamber: cages
- Source and rate of air: Air was provided by a separate HVAC system.
- Method of conditioning air: Air was filtered for particulates and temperature and humidity controlled.
- System of generating particulates/aerosols: Test atmosphere was generated by heating nitrogen to 200°C by passing it through a 1 L stainless steel cylinder with a 1500 W band heater. The nitrogen then passed through a glass column 7.6 cm diameter and 30 cm long packed with glass beads. Test material was delivered by a metering pump into Teflon tubing, to the bottom of the column. The liquid test substance vaporized as it went up the column with the nitrogen. The vapor then went into the test chambers where dilution with the chamber ventilation air produced the desired concentrations.- Temperature, humidity, pressure in air chamber: Air flow rate, temperature and relative humidity were monitored every half-hour during exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: Measurements made hourly using gas-phase IR.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days

Post exposure period:

6, 24, or 48 hours

Remarks:

Doses / Concentrations:
150 ppm
Basis:
nominal conc.
153 (9.6) ppm measured

Remarks:

Doses / Concentrations:
500 ppm
Basis:
nominal conc.
471 (13.1) ppm measured concentration

Remarks:

Doses / Concentrations:
1500 ppm
Basis:
nominal conc.
1540 (48) ppm measured concentration

No. of animals per sex per dose:

15 male/15 female

Control animals:

yes, concurrent no treatment

Positive control(s):

10 animals of each sex were exposed to cyclophosphamide
- Route of administration: injected intraperitoneally
- Doses / concentrations: 40 mg/kg

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: 4 slides per rat were stained with Giemsa.


METHOD OF ANALYSIS: Slides were scanned for well resolved metaphase spreads. Fifty metaphases per animal were evaluated.

Evaluation criteria:

total number of chromosome aberrations, frequency of aberrations per metaphase, percent metaphases with one or more aberrations, percent metaphases with two or more aberrations

Statistics:

Kruskal-Wallis multiple group comparison test followed by the Mann-Whitney U test

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

reduced body weight gain in 1500 ppm group

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Chromosome Aberrations in Sprague-Dawley Rats

Post-Exposure Interval	Exposure Group	Number	Number of Spreads	Number of Aberrations	% Abrr. Per Metaphase	% Metaphases > 1 Abrr.	% Metaphases > 2 Abrr.
6-hours	Air	8	400	0	0	0	0
	150 ppm	9	450	0	0	0	0
	500 ppm	10	487	0	0	0	0
	1500 ppm	9	450	0	0	0	0
24 hours	Air	9	450	1	0.2	0.2	0
	150 ppm	10	482	0	0	0	0
	500 ppm	10	500	0	0	0	0
	1500 ppm	10	500	1	0.2	0.2	0
	Cyclo-phosphamide	9	453	130	28.7	14.6	8.2
48 hours	Air	4	200	0	0	0	0
	150 ppm	4	200	0	0	0	0
	500 ppm	4	200	0	0	0	0
	1500 ppm	3	150	0	0	0	0

Conclusions:

Interpretation of results: negative

The test substance was not clastogenic at levels up to and including 1500 ppm.

Executive summary:

A rat bone marrow cytogenicity study was performed to determine the clastogenicity of high flash aromatic naphtha. 15 male and 15 female rats were exposed via inhalation to 150, 500, or 1500 ppm of high flash aromatic naphtha for 6 hrs/day for 5 days. Rats were sacrificed at 6, 24, or 48 hrs after end of exposure, and the bone marrow then examined for chromosome and chromatid aberrations.

There was no increase in the number of aberrations as compared to negative controls in any of the exposure groups. A significant increase in the number of aberrations was seen in the positive control group, therefore the test is valid.

In conclusion, the test substance is not clastogenic.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Source of data is from secondary literature. Acceptable well-documented study report which meets basic scientific principles: GLP.

Justification for type of information:

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

inhalation: vapour

Details on exposure:

- Exposure apparatus: 16 m glass and steel chambers.
- Method of holding animals in test chamber: cages
- Source and rate of air: Air was provided by a seperate HVAC system.
- Method of conditioning air: Air was filtered for particulates and temperature and humidity controlled.
- System of generating particulates/aerosols: Test atmosphere was generated by heating nitrogen to 200 degree C by passing it through a 1 L stainless steel cylinder with a 1500 W band heater. The nitrogen then passed through a glass column 7.6 cm diameter and 30 cm long packed with glass beads. Test material was delivered by a metering pump into Teflon tubing, to the bottom of the column. The liquid test substance vaporized as it went up the column with the nitrogen. The vapor then went into the test chambers where dilution with the chamber ventilation air produced the desired concentrations.- Temperature, humidity, pressure in air chamber: Air flow rate, temperature and relative humidity were monitored every half-hour during exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: Measurements made hourly using gas-phase IR.
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days

Post exposure period:

6, 24, or 48 hours

Remarks:

Doses / Concentrations:
0, 150, 500, 1500 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 153 (9.6), 471 (13.1), 1540 (48) ppm
Basis:
analytical conc.

No. of animals per sex per dose:

15 male/15 female

Control animals:

yes, concurrent no treatment

Positive control(s):

10 animals of each sex were exposed to cyclophosphamide
- Route of administration: injected intraperitoneally
- Doses / concentrations: 40 mg/kg

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: 4 slides per rat were stained with Giemsa.
METHOD OF ANALYSIS: Slides were scanned for well resolved metaphase spreads. Fifty metaphases per animal were evaluated.

Evaluation criteria:

total number of chromosome aberrations, frequency of aberrations per metaphase, percent metaphases with one or more aberrations, percent metaphases with two or more aberrations

Statistics:

Kruskal-Wallis multiple group comparison test followed by the Mann-Whitney U test

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

reduced body weight gain in 1500 ppm group

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Chromosome Aberrations in Sprague-Dawley Rats Post-Exposure Interval Exposure Group Number Number of Spreads Number of Aberrations % Abrr. Per Metaphase % Metaphases > 1 Abrr. % Metaphases > 2 Abrr. 6-hours Air 8 400 0 0 0 0   150 ppm 9 450 0 0 0 0   500 ppm 10 487 0 0 0 0   1500 ppm 9 450 0 0 0 0 24 hours Air 9 450 1 0.2 0.2 0   150 ppm 10 482 0 0 0 0   500 ppm 10 500 0 0 0 0   1500 ppm 10 500 1 0.2 0.2 0   Cyclo-phosphamide 9 453 130 28.7 14.6 8.2 48 hours Air 4 200 0 0 0 0   150 ppm 4 200 0 0 0 0   500 ppm 4 200 0 0 0 0   1500 ppm 3 150 0 0 0 0

Conclusions:

Interpretation of results: negative

The test substance was not clastogenic at levels up to and including 1500 ppm.

Executive summary:

The test substance was not clastogenic at levels up to and including 1500 ppm.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Hydrocarbons, C9-C10, aromatics, >1% Naphthalene are a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Read across data is available for Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics and the worst case scenario for each end point has been presented.

In Vitro

In Vitro Gene Mutation in Bacterial Cells

Hydrocarbons, C9, aromatics

 

A key bacterial reverse mutation assay (Shell, 1998) 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.

 

Hydrocarbons, C10-C13, aromatics, >1% naphthalene

 

In a key bacterial reverse mutation assay (Petrochem Carless, 2001), the test material (Hydrocarbons, C10-C13, aromatics, >1% naphthalene) was examined for mutagenic activity using histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 98, and 100, and the E. coli strain WP2 uvr A in the absence and presence of a liver S9 fraction for metabolic activation. Concentrations above 500 µg/plate were found to be cytotoxic and so the test was performed in triplicate using doses of 0, 32, 63, 125, 250, 500 µg/plate. In all cases, the test material did not induce any significant changes in the number of revertant colonies. It was concluded in this study that the test material is not a mutagenic agent and is not classified under the new Regulation (EC) 1272/2008 on classification, labelling, and packaging of substances and mixtures (CLP).  

In a second key study (ExxonMobil, 1991) Hydrocarbons, C10-C13, aromatics, >1% naphthalene was examined for mutagenic activity in the bacterial reverse mutation test using histidine-requiring Salmonella typhimurium strains TA 1535, 1537, 1538, 98, and 100 in the absence and presence of a liver S9 fraction for metabolic activation. Concentrations above 320 µg/plate were found to be cytotoxic and so the test was performed in triplicate using doses of 0, 3.2, 10, 32, 100, 320 µg/plate. In all cases, the test material did not induce any significant changes in the number of revertant colonies. It was concluded in this study that Hydrocarbons, C10-C13, aromatics, >1% naphthalene is not a mutagenic agent and is not classified under the new Regulation (EC) 1272/2008 on classification, labelling, and packaging of substances and mixtures (CLP).

In Vitro Mammalian Chromosome Aberration in Mammalian Cells

Hydrocarbons, C9, aromatics

 

A key study assessed the potential of high flash aromatic naphtha to cause chromosome aberrations in Chinese hamster Ovary (CHO) cells (Schreiner et al., 1989). Cells were exposed to concentrations of 15.0, 30.1, 45.0, 60.0, 75.0, 90.0 micrograms/mL without metabolic activation and 20.0, 25.0, 37.5, 40.1, 50.0, 60.1, 70.0, 80.2 micrograms/mL with metabolic activation. Cells were than evaluated for chromosomal aberration frequency, the percentage of cells with aberrations, the percentage of cells with more than one aberration, the presence or absence of a dose-response, and the complexity of the aberrations. There were no increases in chromosomal aberrations at any concentration level tested in either the presence or absence of metabolic activation. The test material was not clastogenic, however, it was cytotoxic at concentrations of 80.2 micrograms/mL or above.

 

In Vitro Gene Mutation in Mammalian Cells 

Hydrocarbons, C9, aromatics

 

A mammalian cell gene mutation assay was performed on Chinese hamster ovary cells to assess the mutagenicity of high flash aromatic naphtha to mammalian cells (Schreiner et al., 1989). The test was performed both with (0.02, 0.04, 0.06, 0.08, 0.1, 0.13, 0.16, 0.2 microliters/mL) and without (0.01, 0.02, 0.04, 0.06, 0.07, 0.08, 0.1, 0.13 microliters/mL) metabolic activation. Cells were then examined for mutation frequency.

 

Only positive controls showed a significant increase in mutation frequency. No increase in mutation frequency was seen at any concentration either with or without metabolic activation. An analysis of relative population growth showed a reduction in population growth to 35.2% or greater of negative controls at exposures to concentrations or 0.07 microliters/mL or above. The test substance was therefore not mutagenic, however, it was cytotoxic at concentrations of 0.07 µL/mL or above.

 

Hydrocarbons, C10, aromatics

 

A second mammalian cell gene mutation assay was performed on Chinese hamster ovary cells to assess the mutagenicity of Hydrocarbons, C10, aromatics to mammalian cells (Schreiner et al., 1989). The test material was not mutagenic, however, it was cytotoxic at concentrations of 0.1 μL/mL or above.

 

In Vitro Sister Chromatid Exchange in Mammalian Cells

 

Hydrocarbons, C9, aromatics

 

The study examined the potential of high flash aromatic naphtha to cause sister chromatid exchange in Chinese hamster ovary (CHO) cells (Schreiner et al., 1989). CHO cells were exposed to test substance concentrations of 2.0, 6.67, 20.0, 35.0, 50.1, 66.7 micrograms/mL without metabolic activation, and 0.667, 2.0, 6.67, 15.0, 20.0, 35.0, 50.1 micrograms/mL with metabolic activation. The sister chromatid exchange frequency of cells in the treatment groups was then compared to the frequency in the control group. There were no increases in SCE at any concentration level tested in either the presence or absence of metabolic activation. The test material did not cause sister chromatid exchange in CHO cells, however it was cytotoxic at the highest concentrations tested.

 

Hydrocarbons, C10, aromatics

 

The test material did not cause sister chromatid exchange in CHO cells, however it was cytotoxic at the highest concentrations tested (Schreiner et al., 1989).

In Vivo

Hydrocarbons, C9, aromatics

 

A rat bone marrow cytogenicity study was performed to determine the clastogenicity of high flash aromatic naphtha (Schreiner et al., 1989). 15 male and 15 female rats were exposed via inhalation to 150, 500, or 1500 ppm of high flash aromatic naphtha for 6 hrs/day for 5 days. Rats were sacrificed at 6, 24, or 48 hrs after end of exposure, and the bone marrow then examined for chromosome and chromatid aberrations. There was no increase in the number of aberrations as compared to negative controls in any of the exposure groups. A significant increase in the number of aberrations was seen in the positive control group, therefore the test is valid. In conclusion, the test substance was not clastogenic.

 

Hydrocarbons, C10, aromatics

 

The test substance was not clastogenic at levels up to and including 1500 ppm (Schreiner et al., 1989).

 

Hydrocarbons, C10-C13, aromatics, >1% naphthalene

 

The test material, Hydrocarbons, C10-C13, aromatics, >1% naphthalene was tested in the mammalian bone marrow micronucleus assay using CD-1 mice (ExxonMobil, 1991). The test material was tested at 24, 48, and 72 hour intervals following exposure and did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes or an increase in the mean number of micronucleated polychromatic erythrocytes. Both the positive (cyclophosphamide) and the negative (carrier) controls behaved in an appropriate manner. These data indicate that Hydrocarbons, C10-C13, aromatics, >1% naphthalene is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 1.0 g/kg.

Furthermore, no there evidence of hyperplastic responses or pre-neoplastic lesions in sub-chronic and chronic repeat-dose studies in either the Hydrocarbons, C9 aromatics or the C10 -C12 Aromatics. All studies were conducted in a manner similar or equivalent to currently established OECD guidelines. Hydrocarbons, C9 aromatics and C10 -C12 Aromatics are non-genotoxic agent sand classification is not warranted.

Justification for classification or non-classification

There is no data available for Hydrocarbons, C9-C10, aromatics, >1% Naphthalene. Hydrocarbons, C9-C10, aromatics, >1% Naphthalene is a combination of Hydrocarbons, C9 Aromatics and Hydrocarbons, C10-C12 Aromatics. Based on the negative results from read across in vitro and in vivo genotoxicity assays, Hydrocarbons, C9-C10, aromatics, >1% Naphthalene does not warrant classification as genotoxins under Regulation (EC) 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP).