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Genetic toxicity in vivo

Description of key information
Whilst no data exist for most of the streams in the Other Petroleum Gases category, mutagenicity data exist for the main components of the streams. A review of an extensive database indicates that Other Petroleum Gases and their main components are not genotoxic. However, benzene and 1,3-butadiene may present in some streams and have been shown to be mutagenic.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant (except for minor deviations listed below), guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
The identity and stability of test material was not in compliance with GLP regulations. The lot number was not available. The identity, strength, purity, composition and purity of positive control was not performed at HLS.
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Principles of method if other than guideline:
A 13-week whole-body inhalation toxicity study in rats with neurotoxicity assessments and in vivo genotoxicity assessments
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
other: Sprague-Dawley CD
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Species: Albino rats (Outbred) VAF/Plus®, Sprague-Dawley derived (CD®), Crl:CD®(SD)IGS BR
- Source: Charles River Laboratories, Kingston, New York 12484, USA
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: Males mean 280 g (range 243-308 g); females mean 209.1 g (range 187-231 g)
- Fasting period before study: None
- Housing: Individually in stainless steel suspended cages with wire mesh floors and fronts.
- Diet: Certified Rodent diet No 5002 (PMI Nutrition International, St Louis, Missouri, USA) ad libitum
- Water: Municipal water ad libitum
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 17-25°C
- Humidity: 22-99%
- Air changes (per hr): Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 20 April 2005 To: 27 July 2005
Route of administration:
inhalation: gas
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel and glass whole body exposure chambers with a volume of approximately 1000 L
- Method of holding animals in test chamber: Individually housed in stainless steel, wire mesh cages within the exposure chamber, with the placement of animals in chambers rotated weekly to ensure uniform exposure.
- Generation: Pre-study trials had evaluated the optimal set of conditions and equipment that generated a stable and uniform atmosphere at the target exposure levels. Test substance flowed from cylinder into a copper tubing coil, maintained in a warm water bath. From the coil the test substance flowed through a metering valve to a mass flowmeter and then via tubing to the turret of the exposure chamber, where it was mixed with room air.
- Temperature, humidity in exposure chamber: 19-28°C, 22-61%
- Air flow rate: Operated at a minimum rate of 203 L/min. Final airflow set to provide at least one air change /5 mins (12/hour) and a T99 equilibrium time of at most 23 mins.
- Oxygen level: at least 19%
- Animal loading factor: below 5%
- Method of particle size determination: yes. Samples drawn for 20 secs at a flow rate of 5 L/min. MMAD, GSM and total mass concentration were calculated.
- Treatment of exhaust air: coarse filter, a HEPA filter and activated charcoal

TEST ATMOSPHERE
- Brief description of analytical method used: Infrared spectrophotometer (IR) 4 times per chamber per day. Gas chromatography (GC) used to characterise at least 5 major components (comprising at least 90% by weight of test substance) once/week/chamber to show test substance stability and comparison between neat test substance and test atmospheres.
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day, 5 days/week
Post exposure period:
Approximately 18-24 hours
Remarks:
Doses / Concentrations:
0, 1000, 5000, 10000 ppm
Basis:
other: target conc.
Remarks:
Doses / Concentrations:
0.0 ± 0.0, 1019 ± 58, 5009 ± 174, 9996 ± 261 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
5/sex/group of the main study animals were used for micronucleus assessment together with 5/sex/group as positive controls
Control animals:
yes, sham-exposed
Positive control(s):
cyclophosphamide monohydrate (CP)
- Route of administration: intraperitonael injection
- Doses / concentrations: 40 mg/kg bw
- supplied by: Sigma Aldrich, Inc., 3050 Spruce St., St Louis, MO 63103, USA
Tissues and cell types examined:
Bone marrow from the right femur
Details of tissue and slide preparation:
5 rats/sex/group, were exposed to liquified petroleum gas by inhalation at exposure levels of 0, 1000, 5000 or 10000 ppm for a 13 week (5 days per week) exposure period. A group of non-exposed (5/sex) positive control animals (40 mg/kg cyclophosphamide, injected ip with a 4.0 mg/mL solution @ 10 mg/kg, within 24 hours prior to sacrifice) were also dosed. The test animals were sacrificed under carbon dioxide anaesthesia. The time
between last exposure and tissue harvest was approximately 18 to 24 hours. The right femurs were removed and sampled. Unstained slides were prepared and stained (Acridine orange) and evaluated using a fluorescent microscope for determination of micronucleus response.
Evaluation criteria:
Incidences of micronucleated immature erythrocytes were calculated.
Statistics:
Results obtained for each treatment group were compared with control results using non-parametric statistical methods with sexes combined. For incidences of micronucleated immature erythrocytes, exact one-sided p-values were calculated by permutation. Comparison of several dose levels was made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance is detected. For inter-group comparisons a straightforward permutation test was used. For assessment of effects on the proportion of immature erythrocytes, equivalent permutation tests based on rank scores were used i.e. exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
After 13 weeks of exposure, there were no treatment-related differences in micronucleus incidence.
Conclusions:
Interpretation of results (migrated information): negative
After 13 weeks of exposure of rats to liquified petroleum gas, there were no treatment-related differences in micronucleus incidence at concentrations up to 10000 ppm, compared to the air control animals. The no observed adverse effect concentration (NOAEC) was 10000 ppm.
Executive summary:

This study was designed to assess the potential inhalation toxicity of liquified petroleum gas (LPG) when administered via whole-body exposures to rats for 13 weeks. The assessment included routine toxicology parameters as well as detailed evaluations of genotoxicity parameters.

Sprague-Dawley CD® rats were exposed for six hours per day to 0 (air control), 1000, 5000 or 10000 ppm of LPG for 5 days per week for 13 consecutive weeks (highest exposure concentration was selected for safety reasons and approximated 50% of the lower explosive limit).

At the end of the treatment period, all animals were euthanized and necropsied and the incidences of micronucleated immature erythrocytes were calculated in 5 males & females per dose concentration.

After 13 weeks of exposure of rats to liquified petroleum gas, there were no treatment-related differences in micronucleus incidence at concentrations up to 10000 ppm, compared to the air control animals. The no observed adverse effect concentration (NOAEC) was 10000 ppm.

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

Additional information

Additional information from genetic toxicity in vivo:

Methane CAS Number 74-82-8

In vitro data

The key study is considered to be a bacterial mutation assay (NTP, 1993), a recognised core assay type for investigating mutation in vitro.

Methane was tested in a standard Ames test but using gas chambers to allow appropriate examination of a gaseous material. Salmonella typhimurium (TA1535, TA97, TA98 and TA100) was treated with methane both with and without auxiliary metabolic activation (S9). A range of doses of methane was used, and the S9 was prepared from both rat and hamster livers and added at two levels.

Methane was not mutagenic in this test system.

In vivo data

No in vivo genotoxicity data are available for methane.

Human information

There is no information indicating any adverse effects of methane.

 

Summary

Methane has been examined for mutagenicity in vitro in the Ames test using a test system that is suitable for examining gaseous materials. It was tested both in the absence and presence of different levels of S9 fractions from both rat and hamster. It was non-mutagenic in the assay. This result is what would be expected from the simple chemical structure of methane. There are no functional groups in the molecule, and it would not be expected to undergo significant metabolism to any reactive species. It carries no alerts for possible genotoxic activity based on established Structure Activity Relationship (SAR) principles (Tennant RW and Ashby J (1991). Classification according to chemical structure, mutagenicity to Salmonella and level of carcinogenicity of a further 39 chemicals by the US National Toxicology Program.  Mutat Res 257 (3) 209-227). 

 

Ethane CAS Number 74-84-0

No data were identified. There are no functional groups in the ethane molecule, and it would not be expected to undergo significant metabolism to any reactive species. It carries no alerts for possible genotoxic activity based on established Structure Activity Relationship (SAR) principles (Tennant and Ashby, 1981). 

 

Propane CAS Number 74-98-6

In vitro data

The key study is considered to be a bacterial mutation assay (Kirwin et al, 1980), a recognised core assay type for investigating mutation in vitro.

Propane was tested in a standard Ames test but using gas chambers to allow appropriate examination of a gaseous material. Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) was treated with propane both with and without auxiliary metabolic activation from rat liver (S9). A range of doses of propane was used, up to 50% atmosphere. Propane was not mutagenic in this test system.

In vivo data

No in vivo genotoxicity data are available for propane.

Human information

There is no information indicating any adverse effects of propane.

 

Summary

Propane has been examined for mutagenicity in vitro in the Ames test using a test system that is suitable for examining gaseous materials. It was tested both in the absence and presence of different levels of S9 fractions from both rat and hamster. It was non-mutagenic in the assay. This result is what would be expected from the simple chemical structure of propane. There are no functional groups in the molecule, and it would not be expected to undergo significant metabolism to any reactive species. It carries no alerts for possible genotoxic activity based on established Structure Activity Relationship (SAR) principles (Tennant and Ashby, 1981). 

 

Isobutane CAS Number 75-28-5

In vitro data

The key study is considered to be a bacterial mutation assay (Kirwin et al, 1980), a recognised core assay type for investigating mutation in vitro.

Isobutane was tested in a standard Ames test but using gas chambers to allow appropriate examination of a gaseous material. Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) was treated with isobutane both with and without auxiliary metabolic activation from rat liver (S9). A range of doses of isobutane was used, up to 50% atmosphere. Isobutane was not mutagenic in this test system.

In vivo data

No in vivo genotoxicity data are available for isobutane.

Human information

There is no information indicating any adverse effects of isobutane.

 

Summary

Isobutane has been examined for mutagenicity in vitro in the Ames test using a test system that is suitable for examining gaseous materials. It was tested both in the absence and presence of different levels of S9 fractions from both rat and hamster. It was non-mutagenic in the assay. This result is what would be expected from the simple chemical structure of propane. There are no functional groups in the molecule, and it would not be expected to undergo significant metabolism to any reactive species. It carries no alerts for possible genotoxic activity based on established Structure Activity Relationship (SAR) principles (Tennant and Ashby, 1981). 

 

Butane CAS Number 106-97-8

In vitro data

The key studies are considered to be bacterial mutation assays (Kirwin et al 1980, NTP, 2005), and an in vitro cytogenetic assay (Safepharm, 2008). These are recognised core assay types for investigating mutation in vitro.

Butane was tested in a standard Ames test but using gas chambers to allow appropriate examination of a gaseous material (Kirwin et al 1980). Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA100) was treated with butane both with and without auxiliary metabolic activation from rat liver (S9). A range of doses of butane was used up to a dose of 50% atmosphere.  In a second Ames test, Salmonella typhimurium (TA1535, TA97, TA98 and TA100) was treated with butane both with and without auxiliary metabolic activation (S9). In this study, a range of doses of propane was again used, and the S9 was prepared from both rat and hamster livers and added at two levels. Butane was not mutagenic in either of these studies.

Butane was examined in an in vitro cytogenetic assay in human lymphocytes in both the absence and presence of auxiliary metabolic activation. It was tested using contained exposures to allow appropriate examination of a gaseous material. Butane was not mutagenic in this assay.

In vivo data

No in vivo genotoxicity data from mammalian systems are available for butane. Butane has been examined in the Sex Linked Recessive Lethal assay in drosophila, at a dose level of 35% atmosphere (NTP, 2005). The material was reported to give a negative response.

Human information

There is no information indicating any adverse effects of butane.

 

Summary

Butane has been examined for mutagenicity in vitro in the Ames test using a test system that is suitable for examining gaseous materials. It was tested both in the absence and presence of different levels of S9 fractions from both rat and hamster. It was non-mutagenic in the assay.   Furthermore, butane has been examined in an in vitro cytogenetic assay in human lymphocytes to current OECD guideline standards, again under exposure conditions appropriate for a gaseous material. It was negative in this assay. Butane has therefore been examined, and found to be negative, for both gene mutation and cytogenetic endpoints in vitro. This result is what would be expected from the simple chemical structure of butane. There are no functional groups in the molecule, and it would not be expected to undergo significant metabolism to any reactive species. It carries no alerts for possible genotoxic activity based on established Structure Activity Relationship (SAR) principles (Tennant and Ashby, 1981).

 

Propene CAS No 115-07-1

In vitro

Propene has been found to be without significant genotoxic activity across endpoints including bacterial gene mutation (Inveresk, 2003), mammalian cell gene mutation and mammalian cell cytogenetics (McGregor et al., 1991). 

In vivo

A bone marrow micronucleus assay exposed rats to doses of propene up to 10000ppm for 6h/day and 5 days/week for a total of 20 exposures, and gave a negative result (Pottenger et al., 2007). An examination of HPRT mutation in rats exposed to the same regime, using splenic T-lymphocytes similarly gave a negative result (Walker et al., 2004). These studies support the negative outcome of chronic (2-year) inhalational exposure of rats and mice to concentrations up to 10000ppm (half the lower explosion limit) of propene. Taken together these studies underpin the conclusion that propene is not a genotoxic carcinogen.

 

Petroleum gases, liquefied (main components propane and propene)

In vitro/vivo data

HLS (2009) assessed the potential inhalation toxicity of liquified petroleum gas when administered via whole-body exposures to rats for 13 weeks. The assessment included evaluations of genotoxicity parameters.

Rats were exposed for six hours per day to 0 (air control), 1000, 5000 or 10000 ppm of LPG for 5 days per week for 13 consecutive weeks (highest exposure concentration was selected for safety reasons and approximated 50% of the lower explosive limit). At the end of the treatment period, all animals were euthanized and necropsied and the incidences of micronucleated immature erythrocytes were calculated in 5 males & females per dose concentration. After 13 weeks of exposure, there were no treatment-related differences in micronucleus incidence at concentrations up to 10000 ppm, compared to the air control animals. The no observed adverse effect concentration (NOAEC) was 10000 ppm.

 

Summary for the category

In summary, simple short chain alkanes (i.e methane, ethane, propane, butane, isobutane) can be considered in a similar manner, and data are available for methane, propane, butane and isobutane in the Ames test, testing under exposure conditions appropriate for gaseous materials, that similarly show them to be non-mutagenic. Furthermore, butane (the C4 analogue alkane) has been examined in an in vitro cytogenetic assay in human lymphocytes to current OECD guideline standards, again under exposure conditions appropriate for a gaseous material. It was negative in this assay. The data are further supported by in vivo data on liquefied petroleum gas. After 13 weeks of exposure of rats, there were no treatment-related differences in micronucleus incidence at concentrations up to 10000 ppm, compared to the air control animals.

The available data for the short-chain alkanes indicates no genotoxic activity across endpoints of bacterial gene mutation, in vitro clastogenicity and in vivo. A consideration of the data available for the alkene propene similarly supports a conclusion of no genotoxic activity, including data from in vivo cytogenetic and in vivo gene mutation endpoints. These data support the conclusion that Other Petroleum Gases are unlikely to express any significant genotoxic activity in vitro or in vivo. However, specific components which have been identified as present in some streams and shown to be mutagenic in vivo are: benzene and 1,3-butadiene:

Benzene

(Classification: CLP - Category 1B, H340)

Benzene has been extensively examined for mutagenicity both in vitro and in vivo in a range of recognised core assay types. It has shown mixed results for mutagenicity in vitro although in mammalian cells there is overall evidence for potential mutagenic activity (EU RAR 2008). Benzene has been shown to be mutagenic in vivo in both somatic cells and germ cells (Ciranni et al, 1991).

Benzene could be present in the Other Petroleum Gases category at levels up to 1%.

1,3-Butadiene

(Classification: CLP - Category 1B, H340)

In vitro, positive results were obtained in bacterial mutation assays (Araki et al 1994, and a mammalian cell cytogenetic assay (Asakura et al 2008). In vivo, somatic cell cytogenetic and gene mutation studies in the mouse and dominant lethal studies in the mouse were positive (Adler et al 1994). Similar studies in the rat were negative.There are many studies on human in occupational settings. The available data on several groups of 1,3-butadiene-exposed workers, both in 1,3-butadiene monomer production and in the polymerization of 1,3-butadiene, did not show any association between 1,3-butadiene exposure and increased gene mutations, primarily HPRT mutations (Albertini et al, 2001, 2003 & 2007, Wickliffe et al 2009). One group of investigators have shown a relationship in workers exposed to 1,3-butadiene but a different method was used by these investigators to measure the HPRT mutation than in the other studies, and there are questions on whether co-exposures were adequately accounted for. Nevertheless, a recent study from these investigators has shown that reduced exposures to all potential genotoxic agents in these facilities have resulted in negative findings (Wickliffe et al, 2009). No 1,3-butadiene-related chromosome aberrations have been demonstrated in humans (Albertini et al 2001, 2003, 2007).

 


Justification for selection of genetic toxicity endpoint
The available data indicates no genotoxic potential across endpoints of bacterial gene mutation, in vitro clastogenicity and in vivo. 

Justification for classification or non-classification

There is no evidence that the main components of the Other Petroleum Gases category are genotoxic therefore no classification is warranted.

Category streams containing <0.1% benzene and/or <0.1% 1,3 -butadiene are considered unlikely to be mutagenic and no labelling will be required under CLP, however streams containing ≥0.1% benzene or ≥0.1% 1,3-butadiene are classified Cat 1B, H340.

Some streamsare already classified as Muta 1B:H340 under CLP (see Section 3).