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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All genetic toxicity tests listed below had negative results for C14-C20 aliphatic, 2-30% 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 – Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells (OECD TG 479)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

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:

A discussion and report on the read across strategy is given as an attachment in IUCLID Section 13.

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

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 DNA damage and/or repair study

Type of information:

experimental study

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:

The justification for read across is provided as an attachment in IUCLID Section 13.

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.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: According to or equivalent to OECD TG 473. Non-GLP.

Justification for type of information:

The justification for read across is provided as an attachment in IUCLID Section 13.

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:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

primary culture, 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

Vehicle / solvent:

ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

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:

primary culture, other: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

yes 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

Untreated negative controls validity:

not examined

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

Executive summary:

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

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

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:

The justification for read across is provided as an attachment in IUCLID Section 13.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

All genetic toxicity tests listed below had negative results for C14-C20 aliphatic, 2-30% 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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to or similar to guideline study OECD 474.

Justification for type of information:

The justification for read across is provided as an attachment in IUCLID Section 13.

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:

not specified

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 10 ml/kg bw.

Details on exposure:

The animals were randomly divided into 5 dose groups: 4 of the dose groups contained 30 mice (15 males and 15 females). The animals in the first 4 groups were treated with corn oil (the vehicle control) or with 1.0, 2.5, or 5.0 g/kg test material. Doses were administered by oral gavage: dosing volumes were 10 mL/kg. Five male and five female mice from each group were sacrificed 24, 48, or 72 hr after treatment, and the bone marrows were isolated and examined for the presence of micronuclei. The last test group contained 10 mice (5 males and 5 females), which were given 40 mg/kg cyclophosphamide by intraperitoneal injection. All of these mice were sacrificed 24 hr after test material administration.

Duration of treatment / exposure:

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

Frequency of treatment:

One dose was given of either vehicle control or with 1.0, 2.5, or 5.0 g/kg test material.

Post exposure period:

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

Remarks:

Doses / Concentrations:
0, 1.0, 2.5, or 5.0 g/kg
Basis:
actual ingested
oral gavage

No. of animals per sex per dose:

The animals were randomly divided into 5 dose groups: 4 of the dose groups contained 30 mice (15 males and 15 females). The animals in the first 4 groups were treated with corn oil (the vehicle control), or with 1.0, 2.5, or 5.0 g/kg test material. Marrows were isolated and examined for the presence of micronuclei. The last test group contained 10 mice (5 males and 5 females), which were given 40 mg/kg cyclophosphamide.

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. Slides were evaluated at 400x by fluorescent microscopy. A total of 1000 erythrocytes were counted from each animal, and the numbers of polychromatic (PCE) and normochromatic (NCE) erythrocytes were tabulated. To determine micronucleus (MN) frequency, 1000 PCEs were examined and the number of MN per 1000 PCEs was reported.

Statistics:

Statistical analysis included calculation of means and standard deviations as well as a standard one way analysis of variance (ANOVA) at each time period. When the ANOVA was significant, comparisons of vehicle-treated to dosed group means were made according to Duncans Multiple Range test. A standard regression analysis was performed to test for dose-response relationships. Sexes were analyzed separately.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The test material did not produce an increase in micronucleus frequency, regardless of sex or sampling time. Additionally, there was no evidence of bone marrow depression. The positive control (cyclophosphamide) produced a significant increase in micronucleus formation, and the vehicle control values fell within the normal control limits.

Conclusions:

Interpretation of results: negative
These data indicate that kerosenes are not cytotoxic and are not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/kg of body weight. 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:

Kerosenes were tested in the mammalian bone marrow micronucleus assay using CD-1 mice.  The test materials weretested 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 kerosenes are not cytotoxic and are not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 5.0 g/kg of body weight.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 vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Ty

Type of information:

experimental study

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:

The justification for read across is provided as an attachment in IUCLID Section 13.

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.

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1980

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Equivalent or similar to OECD Guideline 483.

Justification for type of information:

The justification for read across is provided as an attachment in IUCLID Section 13.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Assigned to test groups randomly: [yes, under following basis: Litton Bionetics, Inc SOP ]
- Housing: Sanitary cages with bedding
- Diet: ad libitum
- Water: ad libitum

Route of administration:

inhalation

Details on exposure:

Following treatment, the males were sequentially mated to two females per week for 2 weeks. The number of females provided an adequate number of implantations per group per week. After the mating period, the females were removed and housed in other cages until killed.

The males were rested for 2 days at the end of each 5 day exposure period, and two new females were introduced into each cage. Conception took placed in more that 90% of females by the 5th day, and a 2-day rest was beneficial to the males with regard to subsequent weekly matings.

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

5 days per week for eight weeks

Remarks:

Doses / Concentrations:
520 and 2080 mg/m3
Basis:
nominal conc.

No. of animals per sex per dose:

12 males per dose

Control animals:

yes, historical

Positive control(s):

- triethylenemelamine
- Route of administration: acute IP injection
- Doses / concentrations: 0.3 mg/kg

Tissues and cell types examined:

Sperm cells

Details of tissue and slide preparation:

At necropsy, the uteri were examined, and the number of living and dead implantations were counted for each pregnant female.

Evaluation criteria:

Fertility Index, Total number of implantations, dead implantations, proportion of females with one or more dead implantations and the proportion of females with two or more dead implantations.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results show that the test article did not cause significant increases in either pre- or post-implantations loss of embryos when statistically compared with the negative control. It is therefore concluded that the test article did not induce dominant lethal mutations in mice at the two tested dose levels.

Conclusions:

Interpretation of results: negative
These data indicate that the test material did not induce dominant lethal mutations in CD-1 mouse sperm cells at concentrations of 100 and 400 ppm. 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 evaluated for its ability to to induce dominant lethal mutations in sperm cells of CD-1 male mice. The test material was administered at two exposure levels of 520 and 2080 mg/m^3 via inhalation exposure, 6 hours per day, 5 days per week for 8 weeks. The results show that the test article did not cause significant increases in either pre- or post-implantations loss of embryos when statistically compared with the negative control. It is therefore concluded that the test article did not induce dominant lethal mutations in mice at the two tested dose levels. 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

C14-C20 aliphatic, 2-30% aromatics are not mutagenic using in vitro or in vivo genotoxicity assays. In bacterial tests, C16-C20 aliphatic, 2-30% aromatics , and structural analogous materials were not mutagenic in Salmonella strains tested in the presence or absence of metabolic activation. In sister chromatid exchange and in chromosomal aberration studies, structurally analogous test materials, C9-C14 aliphatic, 2-30% aromatics and hydrodesulfurized kerosene were determined to be non-clastogenic. Structurally analogous test materials, hydrodesulfurized kerosene and jet fuel A were also not genotoxic when tested in an in vivo mouse bone marrow micronucleus assay and when tested in mammalian bone marrow chromosome aberration test. All studies were conducted in a manner similar or equivalent to currently established OECD guidelines. C14-C20 aliphatic, 2-30% aromatics are a non-genotoxic agent and 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.

Justification for classification or non-classification

The negative results using in vitro and in vivo genotoxicity assays do not warrant the classification of C14-C20 aliphatic, 2-30% aromatics as genotoxins 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.