Ethylene

Specific investigations: other studies

Administrative data

Endpoint:

specific investigations: other studies

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

no guideline followed

GLP compliance:

yes (incl. QA statement)

Type of method:

in vivo

Endpoint addressed:

other: inflammatory and proliferative changes in rat nasal epithelial cells in vivo

Test material

Test material information

Constituent 1

Reference substance name:

Ethene

IUPAC Name:

Ethene

Constituent 2

Reference substance name:

Ethylene

EC Number:

200-815-3

EC Name:

Ethylene

Cas Number:

74-85-1

Molecular formula:

C2H4

IUPAC Name:

ethene

Test material form:

other: colorless compressed gas

Details on test material:

- Name of test material (as cited in study report): Ethene
- CAS No: 74-85-1
- Molecular formula: C2H14
- Molecular weight: 28.05

Test animals

Species:

rat

Strain:

Fischer 344/DuCrj

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
General: The study design required that animals were obtained at staggered intervals to minimise variability in age between experimental groups-
- Source: Charles River Laboratories, Inc. (Kingston, New York)
- Age at study initiation: 8-9 weeks
- Weight at study initiation: approx 125-150 g
- Fasting period before study: not relevant
- Housing: Animals were housed one per cage in stainless steel cages, with wire mesh floor and non-woven gauze to provide a cushion from the flooring for rodent feet.
- Diet (e.g. ad libitum): Diet consisted of "LabDiet Certified Rodent Diet" in meal form. It was provided ad libitum except during exposure
- Water (e.g. ad libitum): Water was obtained from the municipal water source and provided ad limitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/-1°C
- Humidity (%): 40-70%
- Air changes (per hr): 12-15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hr light/12 hr dark

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

inhalation: gas

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: stainless steel and glass Rochester-style whole-body inhalation exposure chambers
- Method of holding animals in test chamber: rats were housed singly in the chambers
- Source and rate of air: airflow was determined using a differential pressure transducer
- Method of conditioning air:
- System of generating particulates/aerosols: the test material exposure atmospheres were generated by mixing ethylene gas with HEPA-filtered breathing air
- Temperature, humidity, pressure in air chamber: 22 C+/-2C, 40-60%
- Air flow rate: 450 (2m3 chambers) or 900 (4m3 chambers) L per minute
- Air change rate: 12-15 per hr
- Method of particle size determination:
- Treatment of exhaust air:

TEST ATMOSPHERE
- Brief description of analytical method used:
- Samples taken from breathing zone: yes/no

VEHICLE (if applicable)
- HEPA-filtered breathing air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test atmosphere concentrations, sampled from the reference point of the chamber were determined approximately twice per hour with a Miran IA infrared spectrophotometer. The analytical concentration during each exposure was interpolated from a standard curve. The nominal concentration in the chamber was calculated from the amount of test material fed into the inhalation apparatus and the total chamber airflow.

Duration of treatment / exposure:

up to 20 days over a 4 week period

Frequency of treatment:

six hours/day, five consecutive days/week

Post exposure period:

sacrifice was planned after 1, 3, 5, 10, 20 days of exposure (depending on the animal group)

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0
Basis:
nominal conc.
ppm

Doses / concentrations 2

Remarks:

Doses / Concentrations:
10
Basis:
nominal conc.
ppm

Doses / concentrations 3

Remarks:

Doses / Concentrations:
50
Basis:
nominal conc.
ppm

Doses / concentrations 4

Remarks:

Doses / Concentrations:
300
Basis:
nominal conc.
ppm

Doses / concentrations 5

Remarks:

Doses / Concentrations:
10000
Basis:
nominal conc.
ppm

No. of animals per sex per dose:

0 ppm:
Number of rats/experimental group for gene expression analyses = 40
Number of rats/experimental group for light microscopic analyses = 48

10 ppm:
Number of rats/experimental group for gene expression analyses = 5
Number of rats/experimental group for light microscopic analyses = 8

50 ppm:
Number of rats/experimental group for gene expression analyses = 5
Number of rats/experimental group for light microscopic analyses = 8

300 ppm:
Number of rats/experimental group for gene expression analyses = 5
Number of rats/experimental group for light microscopic analyses = 16

10000 ppm:
Number of rats/experimental group for gene expression analyses = 40
Number of rats/experimental group for light microscopic analyses = 48

Control animals:

yes, concurrent no treatment

Details on study design:

- Time-dependent Responses to Ethylene
Female F344/DuCrl rats were exposed to 0 or 10,000 ppm ethylene, six hours/day, five consecutive days/week for up to four weeks (up to 20 days of exposure). They were sacrificed after 1, 3, or 5 days

- Concentration-dependent Responses to Ethylene (Exposure-Response):
Female F344/DuCrl rats were exposed to 0, 10, 50, 300, or 10,000 ppm ethylene, six hours/day, five consecutive days/week for four weeks (20 days of exposure) and sacrificed one day (18 hours) following the last (20 th ) day of exposure.

- Persistence of Ethylene-induced Nasal Mucosal Remodeling
Control rats (0ppm) and an additional group of rats were exposed to 0, 300, or 10,000 ppm ethylene for 4 weeks (20 exposure days) was sacrificed after a 13 week post-exposure recovery period.

An overview of the experimental design is shown in the word document attached to "Attached background material"

Examinations

Examinations:

Identification and characterization of early ethylene-induced inflammatory and epithelial cell responses was determined identifying alterations in inflammation, cell proliferation, epithelial morphology, gene expression and serum immunoglobulin levels (IgE, IgG1 and IgG2a).

The effect of exposure concentration on the development of ethylene-induced nasal epithelial lesions following relatively shorter-term repeated exposures, was determined analyzing tissues to identify alterations in inflammation, cell proliferation, epithelial morphology, serum immunoglobulin levels (IgE, IgG1 and IgG2a), and gene expression.

The persistence of ethylene-induced inflammation and nasal epithelial remodeling was investigated analysing nasal tissue samples from these recovery group animals at the end of 4 weeks of exposure of 0, 300, or 10,000 ppm ethylene.

Results and discussion

Details on results:

Mortality: No unscheduled mortality
Clinical Observations: No treatment-related changes
Body weight: No treatment-related effects in the body weights or body weight gains of any treated group compared to the controls
Feed consumption: there were no treatment-related effects in the amount of feed consumed by any treated group when compared to their respective controls.
Serum IgE and IgG Isotype Ratios: No statistically significant difference between the 0 ppm group and the 10,000ppm group on study day 10 and 20.

Any other information on results incl. tables

Anatomic Pathology

Nasal Histopathology: Inflammatory and Epithelial Responses in Nasal Mucosa to Ethylene Exposure

Rats exposed to air only did not present any nasal lesions.

There was no microscopic evidence of nasal epithelial degeneration, cell death (necrosis or apoptosis) or immunohistochemical detection of increased DNA synthesis (BrdU incorporation) in the nasal epithelium of any ethylene-exposed rats.

Morphologic alterations associated with ethylene exposure were observed only on the nasal respiratory epithelium and consisted of:

- Minimal to mild influx of eosinophils and lesser numbers of other inflammatory cells (lymphocytes and neutrophils) in the lamina propria of the nasal mucosa.

- Minimal to mild increase in AB/PAS-stained mucosubstances in the mucous cells of affected nasal respiratory epithelium. 

Histologic examination confirmed no evidence of nasal epithelium degeneration, cell death (apoptosis or necrosis) or immunohistochemical detection of increase DNA synthesis in the nasal epithelium. This demonstrated that cell death and secondary epithelial regeneration were not related to the pathogenesis and inflammatory responses observed in this study.

Time dependency of nasal mucosal changes in rats exposed to 10000 ppm.

An increase in eosinophils was detected as follow:

- 10 Day exposure at 10000 ppm: proximal nasal airways

- 20 Day exposure at 10000 ppm: distal nasal airways

 

An increase in AB/PAS-stained intraepithelial mucosubstances was detected as follow:

- 20 Day exposure at 10000 ppm: respiratory epithelium lining the nasopharyngeal meatus in the distal nasal airways

Persistence of these morphological changes

13 weeks after the rats were exposed to 0, 300, or 10,000 ppm, no eosinophilic inflammation nor high level of AB/PAS-stained mucosubstances was detected in the nasal respiratory epithelium lining the nasopharyngeal meatus.

  

Nasal Morphometry: Quantitative Estimate of Inflammatory and Epithelial Responses to Ethylene Exposure

Eosinophil Numeric Density

After 20 days treatment (treatment week 4), an exposure-related, minimal to mild influx of eosinophils was present in the proximal nasal airways of all rats exposed to 10, 50, 300 and 10,000 ppm ethylene, but there was little evidence of a dose-response.

 

Minimal eosinophilic inflammation was also evident in the respiratory airway mucosa bordering the nasopharyngeal meatus in the distal nasal airways from rats exposed to 10,000 ppm ethylene (lower treatment levels not affected).

 

Volume Density of Intraepithelial Mucosubstances

No significant increase in volume densities was detected in the proximal midseptum of rats exposed to 10, 50, 300, or 10,000 ppm compared to the controls.

In contrast, after 20-days of ethylene exposure the volume densities of intraepithelial mucosubstances in the distal nasopharyngeal meatus was found to be much greater than in the controls.

A statistically significant increase in the volume density of intraepithelial mucosubstances in the nasopharyngeal meatus was found in rats exposed for 5, 10 and 20 days to 10,000 ppm ethylene.

 

 

Gene Expression Changes in the Nasal Mucosa after Ethylene Exposures

Proximal Nasal Septum

- Gob5, IL-13, IL-5, and YM1 gene expressions: increased after 20-day exposures to 10,000 ppm

- IL-4 gene expression: significant increase at the end of exposure Day 5

- IL-13 was expression: significant increase 18 hours after exposure Day 3 and in the 300 ppm group exposure after 20 days exposure.

No significant mucosal gene expression changes were found after 20 days exposure to 5 ppm

 

Distal Nasopharyngeal meatus

-Gob5, Muc5AC, IL-5, and IL-13 expression: significant increase in the mucosal tissues lining the nasopharyngeal meatus after 20 days of exposure to 10,000 ppm.- No significant increases in mucosal target gene expression in this region after 20day exposures to 10, 50, or 300 ppm (except for a slight statistically significant increase in the endogenous house-keeping gene GAPD in rats exposed to 10 ppm).

Applicant's summary and conclusion

Conclusions:

Repeated exposure to ethylene was associated with a generally concentration and time dependent increase in minimal to mild inflammatory changes in the rat nasal respiratory mucosa, but was without effect on cell death (apoptosis or necrosis) or epithelial cell regeneration. The changes were generally site specific, co-located and limited to the upper respiratory tract, and resolved following a 13 week non-exposure period.

Executive summary:

Time- and concentration-dependent changes in histopathology, cellular inflammation, and mucosal gene expression were investigated in nasal tissue from female F344 rats exposed to 0, 10, 50, 300 or 10,000 ppm ethylene vapour for up to 4 weeks. Additional recovery groups (exposed to 300 or 10,000 ppm ethylene for 4 weeks) were maintained for a further 13 weeks to establish the reversibility of any effects seen.

 

Repeated ethylene exposure was associated with a generally concentration and time dependent increase in minimal to mild nasal inflammation and increased storage of mucosubstances in the rat nasal respiratory mucosa, but was without effect on cell death (apoptosis or necrosis) or epithelial cell regeneration (BrdU incorporation). Systemic IgE or IgG isotypes were unaffected by exposure. Where present, the nasal changes were site specific, generally co-located and limited to the upper respiratory tract i.e the eosinophilic inflammatory response was greater in nasal respiratory mucosa of the proximal nasal airways rather than in the distal airways, and increases in intraepithelial mucosusbstances were more prominent in the respiratory mucosa lining the distal nasal airways than in the proximal airways.Expression of Th2 cytokines (e.g., IL-5, IL-13) and YM1/2 gene expression in the nasal mucosa was much greater than that of Th1 cytokines after exposure to ethylene. Lesions observed after 4 weeks exposure to 300 or 10,000 ppm ethylene were not present following a 13 week recovery period.

 

The results indicate that sub-acute exposure to ethylene vapour at concentrations up to 10,000 ppm is associated with reversible inflammatory changes in rat nasal epithelium.