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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethylene
EC Number:
200-815-3
EC Name:
Ethylene
Cas Number:
74-85-1
Molecular formula:
C2H4
IUPAC Name:
ethylene
Constituent 2
Reference substance name:
acetene, ethene, bicarburretted hydrogen, elayl, olefiant gas
IUPAC Name:
acetene, ethene, bicarburretted hydrogen, elayl, olefiant gas
Details on test material:
- Name of test material (as cited in study report): ethylene
- Supplier: Air Liquide America L.P., LaPorte, Texas, USA
- Physical state: colourless compressed gas
- Analytical purity: 99.4% with 0.12% oxygen and 0.47% nitrogen
- Lot/batch No.: A25106-ET

Test animals

Species:
rat
Strain:
other: F344/DuCrl
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)

Administration / exposure

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
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 300, 1000, 3000, and 10000 ppm
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
0, 301.6±6.6, 1001.4±7.0, 3024.2±12.5 or 10134.1±35.4 ppm (study mean ± standard deviation)
Basis:
analytical conc.
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

Examinations

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.

Results and discussion

Test results
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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): 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.