Ethylene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Review of a comprehensive database indicates that ethylene is not genotoxic

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: other: clastogenicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Justification for type of information:

N/A

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

not specified

Species:

rat

Strain:

other: F344/DuCrl

Details on species / strain selection:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Inc., Kingston, New York, USA
- Age at study initiation: 8 weeks
- Weight at study initiation: males 157.4-190.7 g, females 112.6-151.8 g
- Housing: 2/cage in stainless steel cages with wire mesh floors (except during exposure when they were singly housed)
- Diet: LabDiet Certified Rodent Diet #5002 in meal form (PMI Nutrition International, St. Louis, Missouri, USA ad libitum except during exposure
- Water: Municipal water ad libitum except during exposure
- Acclimatisation period: at least 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 21±1°C
- Humidity: 40-70% (with occasional transient and minor excursions considered inconsequential to data interpretation)
- Air changes: 12-15 per hr
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: From: 5 November 2006 To: 9 February 2007 (according to study protocol)

Route of administration:

inhalation: gas

Vehicle:

- Vehicle used: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 4m3 stainless steel and glass Rochester-style whole body inhalation chambers (1.5 m x 1.5 m x 1.3 m with a pyramidal top and bottom).
- Method of holding animals in test chamber: individually housed in cages
- Airflow rate: approx 900 L/min
- Air change rate: 12-15 air changes/hour
- System of generation: ethylene gas was mixed with HEPA-filtered breathing air as it entered the exposure chambers. Target chamber concentrations were maintained by adjusting the amount of ethylene gas delivered to each chamber using calibrated mass-flow controllers.
- Temperature, humidity, pressure in air chamber: mean daily chamber temp. 20.7-21.7°C (minimum and maximum recorded hourly values of 18.1 to 23.7°C), mean daily chamber relative humidity 45.9-54.6% (minimum and maximum recorded hourly values of 29.3 and 85.3%), pressure not reported.

TEST ATMOSPHERE
- Brief description of analytical method used: IR spectrophotometry
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

13 consecutive weeks (65 days of exposure)
Micronuclei were evaluated after the first 5 consecutive days of exposure and following 90 days of repeated exposure

Frequency of treatment:

6 hours/day, 5 consecutive days/week

Post exposure period:

N/A

Dose / conc.:

0 ppm (nominal)

Dose / conc.:

300 ppm (nominal)

Remarks:

301.6±6.6 ppm

Dose / conc.:

1 000 ppm (nominal)

Remarks:

1001.4±7.0 ppm

Dose / conc.:

3 000 ppm (nominal)

Remarks:

3024.2±12.5 ppm

Dose / conc.:

10 000 ppm (nominal)

Remarks:

10134.1±35.4 ppm

No. of animals per sex per dose:

10 plus an additional 12 air-exposed males (6/time-point) to serve as positive controls (following cyclophosphamide treatment) for peripheral blood MN assay and an additional 6 air-exposed males to serve as positive controls (following cyclophosamide treatment) for bone marrow MN assay.

Control animals:

other: air exposed

Positive control(s):

cyclophosphamide CAS 6055-19-2
- Route of administration: oral gavage
- Doses / concentrations: 20 mg/kg bw

Tissues and cell types examined:

Micronuclei in peripheral blood reticulocytes and bone marrow (from the femur).

Details of tissue and slide preparation:

Peripheral blood:
Micronuclei in peripheral blood reticulocytes were evaluated in all experimental groups after the first 5 consecutive days of exposure and following 90 days of repeated exposure. Blood samples were obtained via retro-orbital sampling approximately 18 hours after the 5th or the 65th exposure. Micronucleus formation in peripheral blood reticulocytes was determined by flow cytometry. Separate groups of six age-matched, air-exposed rats, treated orally with 20 mg/kg cyclophosphamide two days prior to sacrifice, were included as positive controls at each time point. Whenever possible, up to 20,000 reticulocytes were analyzed per blood sample.

The number of normochromatic erythrocytes (NCE), MN-NCE, RET and MN-RET were recorded for each sample and the frequency of MN-RET calculated to provide an indication of genotoxic potential. The frequency of reticulocytes relative to total erythrocytes was determined to provide an indication of stem cell toxicity. For each of the treatment groups, a mean and standard deviation were calculated to describe the frequency of RET, MN-NCE, and MN-RET observed.

Bone marrow:
At the time of terminal kill the bone marrow from one femur of each rat was aspirated into foetal calf serum, centrifuged, and the cell pellet resuspended in a drop of serum which was used to prepare a wedge film on a microscope slide. The slides were allowed to air dry and fixed with methanol prior to staining with Acridine Orange.

Two thousand polychromatic erythrocytes (PCE) were examined from each animal in all experimental groups selected for analysis and the number of micronucleated polychromatic erythrocytes (MN-PCE) was recorded. In order to determine the proportion of PCE among erythrocytes in the bone marrow, approximately 200 erythrocytes (PCE + NCE) from each animal were examined and expressed as percentages: (PCE x 100/PCE + NCE).

Evaluation criteria:

A test material was considered positive in this assay if the following criterion was met: Statistically significant increase in MN-PCE/MN-RET frequency that was equal to or greater than 2-fold at one or more dose levels accompanied by a dose-response.
A test material was considered negative in this assay if the following criterion was met: No statistically or biologically significant dose-related increase in MN-PCE/MN-RET as compared to the negative control.

Statistics:

A two-fold or greater change in average response at one or more exposure levels was needed in MNRET or MN-PCE before statistical analysis was initiated. The data was first tested for equality of variance using Bartlett's test and if significant, then the data were subjected to a transformation to obtain equality of the variances. The raw data on the counts of MNPCE in bone marrow were transformed by first adding one to the count and then taking the natural log of the adjusted number. MNPCE, percent PCE and MN-RET were evaluated using a two-way ANOVA. Results for MN-PCE, percent PCE, MN-RET were analyzed using a one-way ANOVA comparing the positive control treatment to the concurrent negative controls.

Key result

Sex:

male/female

Genotoxicity:

negative

Remarks:

peripheral blood and bone marrow

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

N/A

Peripheral Blood:

There was no significant difference in frequencies of MN-RET in ethylene-exposed rats when compared to control rats. Since there was not a two-fold or greater change in the average response at one or more exposure level, statistical analysis was not initiated. The adequacy of the experimental conditions for the detection of induced micronuclei was demonstrated by the observation of a significant increase in the frequencies of MN-RET in the positive control group. In addition, the percent RET values of the positive control animals were found to be significantly lower than those of the negative control animals.

 

Based upon these results, it was concluded that repeated exposure (5 or 90 days) to up to 10000 ppm ethylene did not induce any increase in the frequencies of micronucleated peripheral blood reticulocytes.

 

Bone Marrow:

There was no difference between frequencies of MN-PCE in bone marrow from ethylene-exposed rats when compared to control rats. Since there was not a two-fold or greater change in the average response at one or more exposure level, statistical analysis was not initiated. The adequacy of the experimental conditions for the detection of induced micronuclei was demonstrated by the observation of a significant increase in the frequencies of micronucleated polychromatic erythrocytes in the positive control group. The percent PCE values of the positive control animals were found to be significantly lower than those of the negative control animals.

 

Based upon these results, it was concluded that repeated exposure (up to 90 days) to up to 10000 ppm ethylene did not induce any increase in the frequencies of micronucleated bone marrow polychromatic erythrocytes.

Body temperature:

Relative body temperature measurements of all animals collected three times during each exposure day during the first, second, and 13th weeks of exposure and one exposure day per week for the remaining 10 weeks revealed no treatment-related effects.

Conclusions:

Interpretation of results: negative
Repeated exposure (5 or 90 days) to up to 10000 ppm ethylene did not induce any increase in the frequencies of micronucleated peripheral blood reticulocytes, or any increase in the frequencies of micronucleated bone marrow polychromatic erythrocytes.

Executive summary:

GLP-compliant, guideline subchronic (13-wk) inhalation toxicity study of ethylene was conducted in rats, with target exposure concentrations (300, 1000, 3000 and 10000 ppm). Additional endpoints were included in the study design to provide data on biomarkers of effect (micronucleus formation in peripheral blood and bone marrow).

Repeated exposure (5 or 90 days) to up to 10000 ppm ethylene did not induce any increase in the frequencies of micronucleated peripheral blood reticulocytes. Equivalent to 11473 mg/m3.

Repeated exposure (up to 90 days) to up to 10000 ppm ethylene did not induce any increase in the frequencies of micronucleated bone marrow polychromatic erythrocytes. Equivalent to 11473 mg/m3.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Non-human information

 

In vitro data

The key studies are considered to be a bacterial mutation assay (Victorin & Stahlberg, 1988) and a mammalian cell cytogenetic assay (Riley, 1996).  These are two recognised core assay types for investigating mutation in vitro.

Ethylene was tested in an Ames plate assay modified to allow exposure to a gaseous material (Victorin & Stahlberg, 1988). Salmonella typhimurium strain TA100 was selected as a strain sensitive to base pair substitution mutations since these were considered the most likely to occur from a simple alkene rather than possible frameshift mutations. Exposures were carried out both with and without auxiliary metabolic activation (S9). A range of doses was used from 0.5 to 20% atmospheres. Ethylene was negative in this assay.

Ethylene was tested for cytogenetic activity in CHO cells both in the presence and absence of S9. A range of doses up to approximately 275 µg/ml (approximately 10mM) was used, with exposure for 3 hours, under modified conditions appropriate for a gaseous material. Ethylene was negative in this assay.

There are additional reports of mutation data using further strains of Salmonella and E coli that support the above conclusion of no mutagenic activity (Agrochemical Draft Assessment Report (DAR), fourth stage review under Council Directive 91/414/EEC, 2008).

 

In vivo data

The key studies are considered to be bone marrow micronucleus studies in the mouse and rat (Vergnes et al, 1994; Dow 2010). This is a recognised core assay type for investigating mutation in vivo.

Ethylene was studied in a rodent bone marrow micronucleus assay in male B6C3F1 mice and Fischer 344 rats exposed by inhalation to atmospheres of 0, 39, 966, 2995 ppm for 6 hours/day, 5 days/week for 4 weeks.  Samples of bone marrow cells were taken for cytogenetic analysis at 24 hours after the final exposure. No significant increases in micronucleated polychromatic erythrocytes were found (Vergnes et al 1994). 

In another bone marrow micronucleus assay, Dow (2010) exposed male and female F344/DuCrl rats to ethylene at 0, 302, 1001, 3024, or 10,134 ppm for 6 hours/day, 5 days/week for 13 weeks. Blood samples were taken and examined 18 hours after exposure at day 5 and day 90 of the study. Bone marrow samples were taken after 90 days of the study. No significant increases in micronucleated erythrocytes were found. Ethylene gave a negative result in these two cytogenetic studies.

Ethylene was also negative in an assay examining for HPRT gene mutation in splenic T cells in male F344 rats and B6C3F1 mice exposed by inhalation to 0, 39, 966 and 2992 ppm ethylene for 6 hours/day, 5 days/week for 4 weeks (Walker et al 2000).

 

Human information

There is no information indicating any adverse effects of ethylene. 

 

Summary

Ethylene has been examined for mutagenicity both in vitro and in vivo in a range of recognised core assay types. It has shown negative results for mutagenicity both in vitro and in vivo. It is concluded that the available data indicate that ethylene has no significant genotoxicity.

Justification for classification or non-classification

Ethylene does not warrant classification for genotoxicity under CLP.