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Genetic toxicity in vitro

Description of key information

The genetic toxicity test listed below had a negative result for Hydrocarbons, C16-C20, n-alkanes, isoalkanes, cyclics, aromatics (2-30%).

 

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

 

The read across genetic toxicity tests listed below had negative results for Hydrocarbons, C16-C20, n-alkanes, isoalkanes, cyclics, aromatics (2-30%).

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

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

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

Genetic Toxicity in vitro – Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells (OECD TG 479)

Link to relevant study records

Referenceopen allclose all

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.
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 examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Species / strain:
S. typhimurium TA 97
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Species / strain:
E. coli WP2
Metabolic activation:
not applicable
Genotoxicity:
not determined
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not examined
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
Toxicity was noted at doses above 25 uL/plate.
Remarks on result:
other: all strains/cell types tested
Remarks:
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.

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:
A discussion and report on the read across strategy is given 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 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:
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.

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:
A discussion and report on the read across strategy is given 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:
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.

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:
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:
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:
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The read across genetic toxicity tests listed below had negative results for Hydrocarbons, C16-C20, n-alkanes, isoalkanes, cyclics, aromatics (2-30%).

 

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

Genetic Toxicity in vivo - Mammalian Spermatogonial Chromosome Aberration Test (OECD TG 483)

Link to relevant study records

Referenceopen allclose all

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:
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.

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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:
A discussion and report on the read across strategy is given 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

AMES data is available for Hydrocarbons, C16-C20, n-alkanes, isoalkanes, cyclics, aromatics (2-30%). Additional in vitro genetic toxicity data is available for structural analogues, Hydrocarbons, C11-C14, n-alkanes, isoalkanes, cyclics, aromatics (2-25%) and Kerosene. In vivo data is available for structural analogues, Kerosene and Jet Fuel A. Petroleum substances of similar carbon number and aromatic content, principally kerosene and jet fuel, are typically in the range of C9-C16. These substances also contain similar types of molecules in similar proportions to those in C14-C20 aliphatic [2-30% Aromatics] Hydrocarbon solvents. In general, hydrocarbon solvents are more highly refined than petroleum substances. Accordingly, the petroleum substances typically represent a “worse case” with respect to hydrocarbon solvents and can be used for read across on that basis. 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

Kerosene

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

Kerosene

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

Jet Fuel A

In a key study (API, 1980a) the test material (Jet Fuel A) 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/m3 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).

Justification for classification or non-classification

The negative results in in vitro and in vivo genotoxicity assays from Hydrocarbons, C16-C20, n-alkanes, isoalkanes, cyclics, aromatics (2-30%) and structural analogues do not warrant the classification of Hydrocabons, C16-C20, n-alkanes, isoalkanes, cyclics, aromatics (2-30%) as genotoxic under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).