Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, aromatics (2-25%)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All read across genetic toxicity tests listed below had negative results for Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, aromatics (2-25%).

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

Genetic Toxicity in vitro – Mammalian chromosome aberration test (OECD 473)

Genetic Toxicity in vitro – Mammalian cell gene mutation assay (OECD 476)

Genetic Toxicity in vitro – Sister chromatid exchange assay in mammalian cells (OECD 479)

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:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) straddles Hydrocarbons, C9-C14, Aliphatics, (2-25% Aromatics) and Hydrocarbons, C14-C20, Aliphatics (2-30% Aromatics). Read across justification documents have been provided for the same in Section 13 of the dossier. For this substance, a worst case scenario approach has been used for each endpoint.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

only one strain of Salmonella was used

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Not applicable

Species / strain / cell type:

S. typhimurium TA 98

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fractions from Aroclor exposed rats

Test concentrations with justification for top dose:

Tests with and without Metabolic Activation: 0, 1, 5, 10, 25, 50, and 100 uL/plate (2mL test material dissolved in 3mL cyclohexane)
Control Plate: 10uL DMSO

Vehicle / solvent:

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

10uL DMSO Plate

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; benzo(a)pyrene

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 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 50uL/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Positive controls validity:

not applicable

Species / strain:

S. typhimurium TA 100

Metabolic activation:

not applicable

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Positive controls validity:

not applicable

Additional information on results:

Toxicity was noted at doses above 25 uL/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

It is concluded in this study that the test material is not a mutagenic agent. Classification is not warranted 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:

The test material was examined for mutagenic activity in the bacterial reverse mutation test using histidine-requiring Salmonella typhimurium strain TA 98 in the absence and presence of a liver S9 fraction for metabolic activation. The test was performed in triplicate using doses of 0, 1, 5, 10, 50, 100 uL/plate.  Concentrations above 25 uL/plate were found to be cytotoxic. 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. Classification is not warranted 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.

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

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given:comparable to guidelines/standards.

Justification for type of information:

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) straddles Hydrocarbons, C9-C14, Aliphatics, (2-25% Aromatics) and Hydrocarbons, C14-C20, Aliphatics (2-30% Aromatics). Read across justification documents have been provided for the same in Section 13 of the dossier. For this substance, a worst case scenario approach has been used for each endpoint.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Additional strain / cell type characteristics:

other: histidine dependent

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix derived from rat liver

Test concentrations with justification for top dose:

8-5000 μg/plate

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine, N-methyl-N'-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS: 2

Evaluation criteria:

Increases in reversion to prototrophy.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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

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

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

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

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

The test substance is not mutagenic both in the presence and absence of S-9.

Executive summary:

An Ames Salmonella typhimurium assay was performed to assess the mutagenicity of BP 8313. Duplicate testing was performed on the strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100, both in the presence and absence of metabolic activation. Test concentrations were between 8-5000 ug/plate. Positive controls substances were benzo(a)pyrene, 2 -nitrofluorene, 2 -aminoanthracene, 9 -aminoacridine, and N-methyl-N'-nitro-N-nitrosoguanadine. Positive control cultures had significantly increased number of revertant colonies. Test substance cultures exhibited no increase in the number of revertant colonies as compared to negative controls in cultures either with or without metabolic activation. The test substance is not mutagenic.

Reference 3

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:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given:comparable to guidelines/standards.

Justification for type of information:

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) straddles Hydrocarbons, C9-C14, Aliphatics, (2-25% Aromatics) and Hydrocarbons, C14-C20, Aliphatics (2-30% Aromatics). Read across justification documents have been provided for the same in Section 13 of the dossier. For this substance, a worst case scenario approach has been used for each endpoint.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: human peripheral lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S-9 derived from rat livers

Test concentrations with justification for top dose:

1.2, 6.0, 30.0 μg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

DURATION
- Exposure duration: 24 hrs

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphases examined per culture

Evaluation criteria:

Mitotic indexes were calculated for every culture. Gross toxcity was determined by the number of metaphases per 1000 cells scored. 100 metaphases were examined per culture and chromosomal aberrations recorded. Metaphases were analyzed for frequency of cells with aberrations, and aberrations other than gaps.

Statistics:

Statistical analysis was done on the frequencies of aberrant metaphases, both with and without gap type aberrations. Since there was no significant difference between cultures with and without metabolic acitivation, the data was pooled.

Key result

Species / strain:

other: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

140 μg/ml caused a marked reduction in cell growth, 28 μg/ml caused a 58% reduction in mitotic index, 30 μg/ml caused a slight reduction in mitotic index

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative

The test substance caused no significant chromosomal damage to human peripheral lymphocytes, therefore the test substance in non-clastogenic.

Executive summary:

An in vitro cytogenic assay was performed on human peripheral lymphocytes to evaluate the clastogenicity of BP 8313. The cells were exposed to 1.2, 6.0, or 30.0 µg/ml of the test substance for 24 hrs, both with and without metabolic activation. Cyclophosphamide was used as a positive control. 100 metaphases were examined from each culture for chromosomal aberrations, and the mitotic index calculated. Since there was little difference in results for cultures with and without metabolic activation, the data were pooled for the statistical analysis. The positive control substance induced significant increases in chromosomal aberrations. The test substance did not increase the number of aberrations in human peripheral lymphocytes as compared to negative controls. The test substance is not clastogenic either in the presence or absence of S-9.

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

Study period:

1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Equivalent or similar to OECD Guideline 476.

Justification for type of information:

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) straddles Hydrocarbons, C9-C14, Aliphatics, (2-25% Aromatics) and Hydrocarbons, C14-C20, Aliphatics (2-30% Aromatics). Read across justification documents have been provided for the same in Section 13 of the dossier. For this substance, a worst case scenario approach has been used for each endpoint.

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)

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

na

Metabolic activation:

with and without

Metabolic activation system:

S9 homogenate

Test concentrations with justification for top dose:

Without activation: 6.25 nl/ml to 37.5 nl/ml
With activation: 3.91 nl/ml to 62.5 nl/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [ethanol]
- Justification for choice of solvent/vehicle:The test material was miscible with ethanol.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 24 hours

SELECTION AGENT (mutation assays): BrdU

NUMBER OF REPLICATIONS: Variable with or without activation

NUMBER OF CELLS EVALUATED: 3x10^6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

None of the assayed treatments induced a mutant frequency that exceeded the minimum criterion of 40.8 x 10^-6

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test material induced a good range of toxicities for evaluation of the test material (percent relative growths, 65.3% to 2.8%). The toxicities did show some variability between replicate samples. In the presence of metabolic activation, no indication of mutagenic activity was observed. The average cloning efficencies for the solvent and untreated negative controls varied from 119.1% without activation to 82.7% with activation which demonstrated very good cloning conditions for the assays. The negative control mutant frequencies were all in the normal range and the positive compounds yielded normal mutant frequencies that were greatly in excess of the background.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

It is concluded in this study that the test material is not a mutagenic agent with or without activation. Classification is not warranted 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:

The test material was examined for mutagenic activity in the mouse lymphoma forward mutation assay in the absence and presence of a liver S9 fraction for metabolic activation. The test material did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. Treatments up to 37.5 nl/ml without activation and 62.5 nl/ml with activation were assayed and high toxicities were induced without inducing significant increases in the mutant frequency. It is concluded in this study that the test material is not a mutagenic agent with or without activation. Classification is not warranted 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.

Reference 5

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

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

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Equivalent or similar to OECD TG 479.

Justification for type of information:

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) straddles Hydrocarbons, C9-C14, Aliphatics, (2-25% Aromatics) and Hydrocarbons, C14-C20, Aliphatics (2-30% Aromatics). Read across justification documents have been provided for the same in Section 13 of the dossier. For this substance, a worst case scenario approach has been used for each endpoint.

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:

not specified

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:

S-9

Test concentrations with justification for top dose:

0, 0.007, 0.013, 0.025, 0.05 uL/mL (without activation)
0, 0.05, 0.1, 0.2, 0.4 uL/mL (with activation)

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

yes

Positive controls:

yes

Positive control substance:

triethylenemelamine

Details on test system and experimental conditions:

For the SCE assay CHO cells were seeded in duplicate for each treatment condition and were incubated at 37°C in a humidified atmosphere for 16 to 24 hours. Treatment was carried out by refeeding two complete sets of flasks with complete medium for the non activation study or with S-9 reaction mixture for the activated study to which was added 50 μl of dosing solution of test control or article in solvent or solvent alone. In the non activation study the cells were exposed for about 25 hours. At the end of the treatment period, the treatment medium was removed, the cells rinsed and then exposed to 0.01mM BrdUrd and colcemid (0.1 μg/ml) for a further 2 hours. In the activation study exposure was for 2 hours. After the exposure period, the treatment medium was removed, the cells were washed re-fed with medium containing BrdUrd and then incubated for a further 26 hours. Colcemid was added for the last 2 hours of incubation.For activated and non activated assays metaphase cells were harvested 2 hours after addition of colcemid. Cells were collected and fixed and stored until slides were prepared.

Evaluation criteria:

Slides were coded and scored without regard to treatment group. Only cells with 20 ±2 centromeres were selected for evaluation of SCEs. A total of 4 doses were scored including the highest test article dose where sufficient second-division metaphase cells wee available. SCEs were scored in 25 cells from each duplicate culture to make up a total of 50 cells per treatment. The percentage of cells in first (M1), second (M2) or third division (M3) metaphase was also recorded for a total of 100 metaphase cells scored. TEM was used as positive control in the non activated assay at a concentration of 0.025 μg/ml. CP was used in the activation assay at a concentration of 2.5 μg/ml.

Statistics:

The test material was considered positive if it induced a doubling in SCE frequency over the solvent control at a minimum of three consecutive dose levels or if a dose responsive and statistically significant increase was observed over a minimum of three dose levels.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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

Additional information on results:

The test material was soluble at all concentrations tested. The study in both the presence and absence of S9 was repeated since there was a poor metaphase cell yield. The responses to the positive and negative control materials fulfilled the requirements for the assays. The test material did not cause an increase in SCEs in the absence of exogenous activation. The test material did cause a increase in SCEs at two non adjacent doses (0.05 and 0.4 uL/mL) in the activation assay. However, the increased activity was only seen in one of two treatment flasks. These increases appeared to be random and of no biological significance. It was concluded that the test material was negative in the SCE assay.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results: negative

It was concluded that the test material was negative in the SCE assay.

Executive summary:

It was concluded that the test material was negative in the SCE assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Read across genetic toxicity tests listed below had negative results for Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, aromatics (2-25%).

Genetic Toxicity in vivo – chromosome aberration assay (OECD 475)

Link to relevant study records

Reference

Endpoint:

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

Type of information:

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

Adequacy of study:

key study

Study period:

1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to or similar to guideline study OECD 475: GLP.

Justification for type of information:

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) straddles Hydrocarbons, C9-C14, Aliphatics, (2-25% Aromatics) and Hydrocarbons, C14-C20, Aliphatics (2-30% Aromatics). Read across justification documents have been provided for the same in Section 13 of the dossier. For this substance, a worst case scenario approach has been used for each endpoint.

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:

intraperitoneal

Details on exposure:

A pilot study was carried out in 4 male and 4 female young adult Sprague Dawley rats. These animals were given a single intraperitoneal (i.p.) dose (3 g/kg) of API 81-07. During the following 48 hours observation, no animals died. The doses selected for the cytogenetics study were therefore 0.3, 1 and 3 g/kg. Three groups of 15 male and 15 female rats were given a single i.p. dose of either 0.3, 1 or 3 g API 81-07/kg. At six, 24 and 48 hours after dosing 5 males and 5 females were killed at each dose level. An additional 15 males and 15 females were untreated and served as negative controls. These animals were otherwise treated the same as the test animals. A positive control group of 5 males and 5 females was administered 0.8 mg/kg Triethylenemelamine (TEM) as a single i.p. dose. These positive control animals were killed 24 hours after administration of the positive control substance. Three hours prior to being killed with CO2, animals were injected i.p. with 4 mg/kg of colchicine. After the animal was killed, the adhering soft tissue and epiphyses of both tibiae were removed and the marrow was flushed from the bone and transferred to Hank's balanced salt solution. The marrow button was collected by centrifugation and was then re suspended in 0.075M KCl. The centrifugation was repeated and the pellet resuspended in fixative (methanol:acetic acid, 3:1). The fixative was changed once and left overnight. Cells in fixative were dropped onto glass slides which were then air dried and stained with Giemsa. Slides were coded and scored for chromosomal aberrations. 50 spreads were read for each animal where feasible. A mitotic index based on at least 500 counted cells was also recorded. The index was calculated by scoring the number of cells in mitosis per 500 cells on each read slide.

Duration of treatment / exposure:

Three groups of 15 male and 15 female rats were given a single i.p. dose of either 0.3, 1 or 3 g API 81-07/kg. At six, 24 and 48 hours after dosing 5 males and 5 females were killed at each dose level. An additional 15 males and 15 females were untreated and served as negative controls.

Frequency of treatment:

Single i.p. dose of either 0.3, 1 or 3 g API 81-07/kg

Remarks:

Doses / Concentrations:
0, 0.3, 1 or 3 g/kg.
Basis:
analytical conc.
i.p.

No. of animals per sex per dose:

15 male and 15 female rats

Control animals:

yes, concurrent no treatment

Positive control(s):

These animals were otherwise treated the same as the test animals. A positive control group of 5 males and 5 females was administered 0.8 mg/kg Triethylenemelamine (TEM) as a single i.p. dose. These positive control animals were killed 24 hours after administration of the positive control substance.

Details of tissue and slide preparation:

Three hours prior to being killed with CO 2 , animals were injected i.p. with 4 mg/kg of colchicine. After the animal was killed, the adhering soft tissue and epiphyses of both tibiae were removed and the marrow was flushed from the bone and transferred to Hank's balanced salt solution. The marrow button was collected by centrifugation and was then resuspended in 0.075M KCl. The centrifugation was repeated and the pellet re suspended in fixative (methanol:acetic acid, 3:1). The fixative was changed once and left overnight. Cells in fixative were dropped onto glass slides which were then air dried and stained with Giemsa. Slides were coded and scored for chromosomal aberrations. 50 spreads were read for each animal where feasible. A mitotic index based on at least 500 counted cells was also recorded. The index was calculated by scoring the number of cells in mitosis per 500 cells on each read slide.

Evaluation criteria:

Data interpretation and evaluation Gaps were not counted as significant aberrations. Open breaks were considered as indicators of genetic damage as were configurations resulting from the repair of breaks. The latter included translocations, multiradials, rings, multicentrics, etc. Reunion figures such as these were weighed slightly higher than breaks since they usually resulted from more than one break. Cells with more than one aberration were considered to indicate more genetic damage than those with evidence of single events. Consistent variations from the euploid number were also considered in the evaluation of mutagenic potential.

The type of aberration, its frequency and its correlation to dose in a given time was considered in evaluating the test material as being positive or negative.

Statistics:

Statistical evaluation Performed by Student's t-tests on four parameters:
1. Number of structural aberrations per animal
2. Number of numerical aberrations per animal
3. % cells with one or more structural aberrations per animal
4. % cells with 2 or more structural aberrations per animal.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The data are given in the report for males, females and as male and female pooled data. When the results for males were compared with those for controls and the females were compared to controls, no statistically significant differences were found. The data summarized below, are the pooled data for males and females. The structural aberration frequency did not differ significantly from the negative control at any tested dose. The percentage of cells showing one or more structural aberrations or 2 or more structural aberrations were also similar to the negative controls. A concurrent positive control group induced significant increases in aberrations.

Conclusions:

Interpretation of results: negative
The test material did not cause chromosome aberration in the test model.

Executive summary:

The test material did not cause chromosome aberration in the test model.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) straddles Hydrocarbons, C9-C14, Aliphatics, (2-25% Aromatics) and Hydrocarbons, C14-C20, Aliphatics (2-30% Aromatics). Read across justification documents have been provided for the same in Section 13 of the dossier. For this substance, a worst case scenario approach has been used for each endpoint.

There is no in vitro or in vivo data available for Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, aromatics (2-25%). However, data is available for structural analogues, Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, aromatics (2-25%); Hydrocarbons, C16-C20, n-alkanes, isoalkanes, cyclics, aromatics (2-30%); and Hydrodesulfurized kerosene. This data is read across to based on analogue read across and a discussion and report on the read across strategy is provided as an attachment in IUCLID Section 13.

In Vitro

In vitro gene mutation study in bacteria

Hydrocarbons, C16-C20, n-alkanes, isoalkanes, cyclics, aromatics (2-30%)

In an Ames assay, the test material (Hydrocarbons, C16-C20, n-alkanes, isoalkanes, cyclics, aromatics (2-30%)) was examined for mutagenic activity in the bacterial reverse mutation test using histidine-requiring Salmonella typhimurium strain TA 98 in the absence and presence of a liver S9 fraction for metabolic activation (Dow, 1987). The test was performed in triplicate using doses of 0, 1, 5, 10, 50, 100 uL/plate. Concentrations above 25 uL/plate were found to be cytotoxic. 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. Classification is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, aromatics (2-25%)

An Ames Salmonella typhimurium assay was performed to assess the mutagenicity of hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) (DHC, 1984a). Duplicate testing was performed on the strains TA 1535, TA 1537, TA 1538, TA 98, and TA 100, both in the presence and absence of metabolic activation. Test concentrations were between 8-5000 ug/plate. Positive controls substances were benzo(a)pyrene, 2 -nitrofluorene, 2 -aminoanthracene, 9 -aminoacridine, and N-methyl-N'-nitro-N-nitrosoguanadine. Positive control cultures had significantly increased number of revertant colonies. Test substance cultures exhibited no increase in the number of revertant colonies as compared to negative controls in cultures either with or without metabolic activation. The test substance was not mutagenic.

In Vitro Chromosome Aberration in Mammalian Cells

Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, aromatics (2-25%)

An in vitro cytogenic assay was performed on human peripheral lymphocytes to evaluate the clastogenicity of hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) (DHC, 1984a). The cells were exposed to 1.2, 6.0, or 30.0 µg/ml of the test substance for 24 hrs, both with and without metabolic activation. Cyclophosphamide was used as a positive control. 100 metaphases were examined from each culture for chromosomal aberrations, and the mitotic index calculated. Since there was little difference in results for cultures with and without metabolic activation, the data were pooled for the statistical analysis. The positive control substance induced significant increases in chromosomal aberrations. The test substance did not increase the number of aberrations in human peripheral lymphocytes as compared to negative controls. The test substance was not clastogenic either in the presence or absence of S-9.

In vitro Gene Mutation study in Mammalian Cells

Hydrodesulfurized kerosine

In a key mammalian cell gene mutation assay (API, 1984), the test material (Hydrodesulfurized kerosine) was examined for mutagenic activity in the mouse lymphoma forward mutation assay in the absence and presence of a liver S9 fraction for metabolic activation. The test material did not induce significant increases in the mutant frequency at the TK locus in L5178Y mouse lymphoma cells. Treatments up to 37.5 nl/ml without activation and 62.5 nl/ml with activation were assayed and high toxicities were induced without inducing significant increases in the mutant frequency. It is concluded in this study that the test material is not a mutagenic agent with or without activation. Classification is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

 

It was also concluded that Hydrodesulfurized kerosine was negative in the SCE assay (API, 1987).

In Vivo

Hydrodesulfurized kerosine

Hydrodesulfurized kerosene was not genotoxic when tested in an in vivo mammalian bone marrow chromosome aberration test (API, 1984).

Justification for classification or non-classification

The negative results observed in read across in vitro and in vivo genotoxicity assays do not warrant the classification of Hydrocarbons, C12-C16, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).