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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic Toxicity in vitro - Negative - Bacterial reverse mutation assay (OECD TG 471)
Genetic Toxicity in vitro - Negative - In vitro Mammalian Chromosome Aberration Test (OECD TG 473)
Genetic Toxicity in vitro - Negative - In vitro Mammalian Cell Gene Mutation Test (OECD TG 476)
Genetic Toxicity in vitro - Negative - In vitro Sister Chromatid Exchange Assay in Mammalian Cells (OECD TG 479) - Read-Across from Hydrodesulfurized Kerosene

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:
1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Basic data given:comparable to guidelines/standards.
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
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:
other: TA 1535, TA 1537, TA 1538, TA 98 and 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.

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: Basic data given:comparable to guidelines/standards.
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
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 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.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
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

Genetic Toxicity in vivo - Negative - Micronucleus Assay in Mouse Bone Marrow (OECD TG 474) - Read-Across from Jet Fuel A
Genetic Toxicity in vivo - Negative - Mammalian Bone Marrow Chromosome Aberration Test (OECD TG 475)

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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.
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
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 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:
None of the five test materials produced 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.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Conducted in accordance with general scientific principles.
Reason / purpose:
read-across: supporting information
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Balb/c
Sex:
male/female
Details on test animals and environmental conditions:
The animals weighed 30 ± 3 g
Route of administration:
other: i.p. and inhalation
Vehicle:
none
Details on exposure:
The animals were sacrified 30 h after i.p. injection of 0.l, 0.05 or 0.01 ml of white spirit. In addition, male animals were allowed to inhale 50 g/m3 of white spirit, during 5 periods of 5 min spaced by intervals of 5min. According to Carpenter et al. (1975) 50 g/m3 represents about 50 times the limit of toxicity. Animals given 200 mg cyclophosphamide per kg b.w. were used as positive controls.

Carpenter C. P., Kinkead E. R., Geary, Jr. D. L., Sullivan L. J., and King J. M. Petroleum Hydrocarbon Toxicity Studies. Ill. Animal and Human Response to Vapors of Stoddard Solvent. 1975. Toxicology and Applied Pharmacology. 32: 282-297.20 ani
Duration of treatment / exposure:
Experiment #1 - 30 h after i.p. injection of 0.l, 0.05 or 0.01 ml of white spirit
Experiment #2 - male animals were allowed to inhale 50 g/m3 of white spirit, during 5 periods of 5 min spaced by intervals of 5min
Frequency of treatment:
Experiment #1 - once, animals were sacrificed 30 h after i.p. injection
Experiment #2 - 5 periods of 5 min spaced by intervals of 5min
Post exposure period:
none
Remarks:
Doses / Concentrations:
0, 0.l, 0.05, or 0.01 ml
Basis:
other: i.p. injection
Remarks:
Doses / Concentrations:
0, 50 g/m3
Basis:
nominal conc.
inhalation
No. of animals per sex per dose:
20 animals
Control animals:
yes, sham-exposed
Positive control(s):
Animals given 200 mg cyclophosphamide per kg b.w. were used as positive controls.
Tissues and cell types examined:
micronucleus test; 3000 cells examined per dose
Details of tissue and slide preparation:
no data
Evaluation criteria:
micronuclei
Statistics:
Mann Whitney U-test
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
see table

Treatment

Cells with micronuclei (%)
  Male Female

Negative controls

3.8 4.3

Cyclophosphamide (200 mg/kg)

36

31

White Spirit (0.01mL i.p.)

4.4 3.7

White Spirit (0.05mL i.p.)

4.2 4.1

White Spirit (0.1mL i.p.)

4 4

White Spirit (50g/m3 by inhalation)

4.1 N/A
Conclusions:
Interpretation of results: negative
White spirit did not produce chromosomal aberrations in vivo.
Executive summary:

The ability of white spirit to produce chromosomal aberrations in vivo in mammalian somatic cells was studied by examining eight-week-old BALB/c male and female mice for the presence of micronuclei in the polychromatic erythrocytes from bone marrow.

The animals were sacrified 30 h after i.p. injection of 0.l, 0.05 or 0.01 ml of white spirit. In addition, male animals were allowed to inhale 50 g/m3 of white spirit, during 5 periods of 5 min spaced by intervals of 5min. According to Carpenter et al. (1975) 50 g/m3 represents about 50 times the limit of toxicity. Animals given 200 mg cyclophosphamide per kg b.w. were used as positive controls. When compared to control animals, it was concluded that white spirits are not mutagenic or clastogenic.

 

Carpenter C. P., Kinkead E. R., Geary, Jr. D. L., Sullivan L. J., and King J. M. Petroleum Hydrocarbon Toxicity Studies.Animal and Human Response to Vapors of Stoddard Solvent. 1975. Toxicology and Applied Pharmacology. 32: 282-297.

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

Additional information

C9-14 Aliphatics (2-25% Aromatics) are not mutagenic using in vitro or in vivo genotoxicity assays.  Further data derived from read across data from the supporting substances (structural analogue or surrogate), kerosene and jet fuel, also support the conclusion that C9-14 Aliphatics (2-25% Aromatics) are not genotoxic. In bacterial reverse mutation tests, C9-14 Aliphatics (2-25% Aromatics) were not mutagenic in the presence or absence of metabolic activation. Likewise, there were no mutagenic effects reported in an in vitro mammalian gene mutation test (HGPRT forward mutation assay). No in vitro chromosomal effects were reported in a Chinese hamster ovary assay that examined hydrodesufurized kerosene. The test substance, C9-14 Aliphatics (2-25% Aromatics), did not produce chromosomal effects when tested in an in vivo mouse bone marrow micronucleus assay.  These data demonstrate that these substances are not categorizable genotoxins either in vitro or in vivo. Furthermore, no there evidence of hyperplastic responses or pre-neoplastic lesions in sub-chronic and chronic repeat-dose studies in C9-14 Aliphatics (2-25% Aromatics). All studies were conducted in a manner similar or equivalent to currently established OECD guidelines.  C9-14 Aliphatics (2-25% Aromatics) are a non-genotoxic agent and classification is not warranted.

Justification for classification or non-classification

The negative results using in vitro and in vivo genotoxicity assays do not warrant the classification of C9-14 Aliphatics (2-25% Aromatics) fluids 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.