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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment. Details provided are based on the draft report.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

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: 22±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
Type of inhalation exposure:
whole body
Vehicle:
other: air
Details on inhalation 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
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Averaged over the entire study the mean chamber concentrations were within 1.5% of the target concentrations.
Duration of treatment / exposure:
13 consecutive weeks (65 days of 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
Control animals:
other: yes, air exposed
Details on study design:
- Dose selection rationale: Based on previous toxicity data and expected saturation kinetics for the metabolic conversion of absorbed ethylene to the reactive intermediate ethylene oxide.
- Additional endpoints were included in the study design to provide data on biomarkers of exposure (HEVal haemoglobin adduct levels) and biomarkers of effect (micronucleus formation in peripheral blood and bone marrow, see section 7.6). The adduct data are not included in this robust summary.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once/day
- Cage side observations included: decreased/increased activity, repetitive behaviour, vocalization, incoordination/limping, injury, neuromuscular function (convulsion, fasciculation, tremor and twitches), altered respiration, blue/pale skin and mucous membranes, severe eye injury (rupture), alterations in faecal consistency and faecal/urinary quantity. In addition, all animals were observed for morbidity, mortality, and the availability of feed and water at least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pre-exposure and once per week throughout the study.

BODY WEIGHT: Yes
- Time schedule: pre-exposure, twice during the first week and at least weekly during the dosing period.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day: Yes
- Time schedule: weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule: pre-exposure and prior to the scheduled necropsy
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters examined: Haematocrit (HCT), Haemoglobin (HGB) concentration, Red blood cell (RBC) count, Total white blood cell (WBC) count, Differential WBC count, Platelet (PLAT) count, Reticulocyte (RET) count, RBC indices: Mean Corpuscular Haemoglobin (MCH), Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin Concentration (MCHC), Prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters examined: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transpeptidase (GGT), Albumin (ALB), Albumin/Globulin Ratio (ALB/GLOB), Cholesterol (CHOL), Creatinine (CREA), Electrolytes (NA, K, PHOS, CL and CA), Globulin (GLOB), Glucose (GLU), Total bilirubin (TBIL), Total protein (TP), Triglycerides (TRIG), blood urea nitrogen (BUN)

URINALYSIS: Yes
- Time schedule for collection of urine: week prior to the scheduled necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters examined: Colour, Appearance, Specific gravity (refractometer), Urine volume, pH, Bilirubin, Glucose, Protein, Ketones, Blood, Urobilinogen, Microscopic examination of microsediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: pre-exposure and during the last week of the exposure period
- Dose groups that were examined: all
- Battery of functions tested: Sensory activity, Grip strength, Motor activity, Rectal temperature

HAEMOGLOBIN ADDUCTS: Yes
Details not included in this robust summary
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals)
ORGAN WEIGHTS: (all animals)
- Tissues/organs: lung, brain, liver, kidneys, heart, adrenals, testes, epididymides, uterus, ovaries, thymus, spleen
HISTOPATHOLOGY: Yes (controls and high dose animals - all tissues listed; relevant gross lesions, lungs, and nasal tissues from all animals in the low and intermediate exposure groups).
- Tissues/organs: adrenals, aorta, bone (including joint), auditory sebaceous gland, bone marrow, brain (cerebrum, brainstem, cerebellum), caecum, cervix, coagulating glands, colon, cranial nerve - optic, duodenum, epididymes, oesophagus, eyes, gross lesions, heart, ileum, jejunum, kidneys, lachrymal/Harderian glands, larynx, liver, lungs, mammary glands (females), mediastinal lymph node, mediastinal tissues, mesenteric lymph node, mesenteric tissues, nasal tissues/pharynx, oral tissues, ovaries, oviducts, pancreas, parathyroid glands, peripheral nerve - tibial, pituitary, prostate, rectum, salivary glands, seminal vesicles, skeletal muscle, skin and subcutis, spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, urinary bladder, uterus, vagina.
Other examinations:
Histochemical and morphometric analyses of the principal inflammatory and epithelial changes in nasal mucosal tissue of F344/DuCrl rats from this study were also conducted. Histochemical processing of tissues required reembedding tissue blocks, and then staining to identify both neutral and acidic mucosubstances stored within the cytoplasm of mucous cells in the nasal respiratory epithelium. To specifically identify infiltrating eosinophils in the nasal mucosa, further sections were stained with a Luna Staining Method which stains cytoplasmic granules of eosinophils a bright red, distinguishing these cells from other cells in the nasal mucosa.
The amount of Intraepithelial Mucosubstances (IM) lining the proximal and distal nasal airways was estimated using computerized image analysis and standard morphometric techniques (Harkema et al., 1989). The volume density (Vs) of AB/PAS-stained IM was morphometrically quantified in the respiratory epithelium lining the selected sites in the nasal airways. The volume of stored mucosubstances per unit of surface area of epithelial basal lamina, or Vs, was calculated using methods described by Harkema et al., 1987) and expressed as nanoliters (nL) of intraepithelial mucosubstances per mm2 of basal lamina.
The numeric density of eosinophils in the nasal mucosa (that includes both epithelium and underlying lamina propria) lining the selected intranasal sites was determined by counting Luna-positively stained cells with distinct eosinophil morphology. The numeric densities of eosinophils within the nasal mucosa at the sites selected for analysis are expressed as the number of eosinophils divided by the length of the underlying basal lamina (numeric cell density; Wagner et al., 2009).
Statistics:
Several different statistical methods were used on study. See other information section.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY: No unscheduled deaths. No treatment-related clinical observations.

BODY WEIGHT AND WEIGHT GAIN: Statistically significant differences (increases) in mean in-life body weights were identified in the high exposure group. Although there was no time-sex-dose interaction, when the data for male and female rats were combined and analyzed for a time-dose interaction, rats exposed to 10000 ppm had in-life body weights that were statistically identified as different from their respective controls. The magnitude of the effect was slight (≤2.7% higher than control values) and variable over time. Although a treatment-related effect cannot be excluded, the observed difference was considered not to be adverse.

FOOD CONSUMPTION: No treatment-related effects.

OPHTHALMOSCOPIC EXAMINATION: No treatment-related effects.

HAEMATOLOGY: Males and females exposed to 300, 1000 or 3000 ppm ethylene had statistically identified lower mean reticulocyte counts, relative to controls. These control animals had considerably higher reticulocyte counts (203 x 10^9 and 191 x 10^9 reticulocytes/L blood for males and females, respectively) when compared to the laboratory’s historical control database (163-178 x 10^9 and 140-167 x 10^9 reticulocytes/L blood, respectively). Given the lack of dose response and the historical control data, this difference was considered not to be treatment-related. Male and female rats exposed to 300 ppm ethylene had slight, yet statistically identified higher mean prothrombin times when compared to controls. Due to the lack of a dose-response, this difference was considered not to be treatment-related. There were no other statistically significant differences.

CLINICAL CHEMISTRY: Female rats at all exposure concentrations had statistically identified lower mean urea nitrogen values when compared to their respective controls, but all, including controls, (14-17 mg/dL) fell within the range of this laboratory’s historical control values (13-23 mg/dL). Given the lack of dose-response and the overlap with this laboratory’s historical control values, the observed differences in mean blood urea nitrogen values were considered not to be treatment-related. There were no other statistically significant differences.

URINALYSIS: No treatment-related effects.

GROSS PATHOLOGY: There were no treatment-related gross pathological observations. A number of gross pathological observations were noted in control and/or ethylene-exposed rats but were of low incidence and/or commonly observed in this strain of rat and were considered not to be treatment-related.

ORGAN WEIGHTS: Male and female rats exposed to 10000 ppm ethylene had statistically identified lower mean absolute and relative spleen weights. These control animals had considerably higher absolute and relative mean spleen weights when compared to the laboratory’s historical control database, except for a single study in which the absolute spleen weight for females was slightly higher (0.480 g). Given the lack of dose-response and the difference in control values when compared to laboratory historical controls, this difference in mean absolute and relative spleen weights was considered not to be treatment-related.

HISTOPATHOLOGY: Treatment-related histopathological observations were limited to the upper respiratory tract. The morphologic diagnosis was a bilateral, eosinophilic rhinitis with mucous cell hyperplasia/hypertrophy (MCH) and occasional epithelial hyalinosis. Hyaline droplets were not observed in the nasal respiratory epithelium of either male or female control rats, therefore, hyalinosis in the nasal respiratory epithelium was interpreted to be
treatment-related. Exposure-related epithelial and inflammatory responses (MCH and eosinophilic rhinitis, respectively) were greatest in female and male rats exposed to 10000 ppm ethylene, however none of the changes were severe in nature but described as very slight or slight; similar changes of even lesser severity were also noted in both sexes of rats exposed to the lower concentrations of 3000, 1000, and 300 ppm ethylene. Quantitative morphometric analyses substantiated the qualitative histopathological findings by demonstrating that repeated inhalation exposures to ethylene at concentrations as low as 300 ppm caused quantifiable increases of both intraepithelial mucosubstances (IM; a quantitative estimate of epithelial remodelling and MCH) and eosinophil numeric density (quantitative estimate of nasal inflammation – eosinophilic rhinitis) in the nasal mucosal tissue of ethylene-exposed rats.

FUNCTIONAL OBSERVATION BATTERY: There were no treatment-related clinical observations or alterations in functional tests including sensory evaluations, rectal (core) temperature, grip performance, motor activity or body temperature (subcutaneous measurement).

OTHER FINDINGS: N-terminal hydroxyethylvaline (HEVal) adducts of haemoglobin: details not included in this robust summary

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
10 000 ppm
Sex:
male/female
Basis for effect level:
other: no evidence of toxicity at highest dose concentration tested (equivalent to 11473 mg/m3)
Dose descriptor:
LOEC
Effect level:
300 ppm
Sex:
male/female
Basis for effect level:
other: treatment-related mucous cell hyperplasia/hypertrophy (MCH) in the nasal airways at lowest concentration tested (344.2 mg/m3 equivalent)

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the presence of treatment-related histopathological alterations in the nasal airways of male and female rats repeatedly exposed to ethylene at all concentrations tested, a no observed-adverse effect concentration (NOAEC) was not identified. The LOAEC was determined to be 300 ppm (equivalent to 344.2 mg/m3).
Executive summary:

A GLP-compliant, guideline subchronic (13-wk) inhalation toxicity study of ethylene was conducted in F344/DuCrl rats, with target exposure concentrations (300, 1000, 3000 and 10000 ppm) based on expected saturation kinetics for the metabolic conversion of absorbed ethylene to the reactive intermediate ethylene oxide. Additional endpoints were included in the study design to provide data on biomarkers of exposure (HEVal haemoglobin adduct levels) and biomarkers of effect (micronucleus formation in bone marrow and in peripheral blood).

 

There were no unscheduled deaths, no treatment-related clinical observations or alterations in 1) functional tests including sensory evaluations, rectal (core) temperature, grip performance, or motor activity, 2) feed consumption, or 3) body temperature (subcutaneous measurement). Statistically significant differences were identified in the following endpoints: reticulocyte count, coagulation, urea nitrogen, mean absolute and relative spleen weight and mean relative testes weight. In each case, based on historical control data or lack of dose-response, the observed effects were interpreted not to be treatment-related. Statistically significant increases in mean in-life body weights were identified in the high exposure group. Although there was no time-sex-dose interaction, when the data for male and female rats were combined and analyzed for a time-dose interaction, rats exposed to 10000 ppm had in-life body weights that were statistically identified as different from their respective controls. The magnitude of the effect was slight (≤2.7% higher than control values) and variable over time. Although a treatment-related effect cannot be excluded, the observed difference was not considered to be adverse.

Treatment-related histopathological observations were limited to the upper respiratory tract. The morphologic diagnosis was a bilateral, eosinophilic rhinitis with mucous cell hyperplasia/hypertrophy (MCH) and occasional epithelial hyalinosis. Hyaline droplets were not observed in the nasal respiratory epithelium of either male or female control rats, therefore, hyalinosis in the nasal respiratory epithelium was interpreted to be treatment-related. Exposure-related epithelial and inflammatory responses (MCH and eosinophilic rhinitis, respectively) were generally most severe (reported as slight or mild alterations) in female and male rats exposed to 10000 ppm ethylene, but similar changes of lesser severity (very slight or minimal) were also noted in both sexes of rats exposed to the lower concentrations of 3000, 1000, and 300 ppm ethylene. Quantitative morphometric analyses substantiated the qualitative histopathological findings by demonstrating that repeated inhalation exposures to ethylene at concentrations as low as 300 ppm caused quantifiable increases of both intraepithelial mucosubstances (IM; a quantitative estimate of epithelial remodeling and MCH) and eosinophil numeric density (quantitative estimate of nasal inflammation – eosinophilic rhinitis) in the nasal mucosal tissue of ethylene exposed rats.

A benchmark dose (BMD) analysis of the morphometric data enumerating the exposure related influx of eosinophils into nasal respiratory mucosa was conducted in order to establish BMD and BMDL (lower limit of the 95% confidence interval on the BMD) values. This inflammatory response (eosinophilic rhinitis), specifically the data from the mid-septum of the proximal nasal cavity (T2 section; male and female rats), was selected for BMD analysis because of the consistency and the magnitude of the ethylene-induced inflammatory and epithelial changes in this location. BMD analysis indicated exponential models gave the best fit to the T2 eosinophil data. Female rats appeared to be marginally more sensitive to exposure-related eosinophilic rhinitis in the proximal mid-septum T2 region, resulting in lower BMD and BMDL values, compared to values calculated for similarly exposed male rats. The lowest BMD/BMDL values come from the female exponential 4 model with a BMD of 34.71 ppm ethylene and a corresponding BMDL of 21.3 ppm ethylene. Comparable values for males are 69.1 ppm and 44.4 ppm, respectively.

Based on the presence of treatment-related histopathological alterations in the nasal airways of male and female rats repeatedly exposed to ethylene at all concentrations tested, a no observed-adverse effect concentration (NOAEC) was not identified. Based on the observation of ethylene-induced eosinophilic rhinitis, a benchmark dose (BMD) of 34.71 ppm ethylene and a corresponding BMDL (lower limit of the 95% confidence interval on the BMD) of 21.3 ppm ethylene were calculated. Under the conditions of this study, the lowest-observed-effect concentration (LOEC) for male and female F344/DuCrl rats repeatedly exposed to ethylene for a total of 13 weeks was 300 ppm (344.2 mg/m3).