Ethylene

Endpoint summary

Description of key information

In accordance with Section 2 of REACH Annex XI, studies via the oral and dermal routes are not technically feasible as the substance is a gas at room temperature.  The NOAEC for systemic inhalation toxicity in rats is 10,000 ppm (11,473 mg/m3) and the LOEC for local effects (mild rhinitis) is 10 ppm (11.47 mg/m3). 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment.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

Justification for type of information:

N/A

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

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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: 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)

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

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

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.

Positive control:

N/A

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.

Clinical signs:

no effects observed

Description (incidence and severity):

N/A

Mortality:

no mortality observed

Description (incidence):

N/A

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

N/A

Food efficiency:

not examined

Description (incidence and severity):

N/A

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

N/A

Ophthalmological findings:

no effects observed

Description (incidence and severity):

N/A

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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 biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Endocrine findings:

not examined

Description (incidence and severity):

N/A

Urinalysis findings:

no effects observed

Description (incidence and severity):

N/A

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

N/A

Immunological findings:

not examined

Description (incidence and severity):

N/A

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Neuropathological findings:

no effects observed

Description (incidence and severity):

N/A

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Due to the lack of severity associated with the effects described, they were not considered to be adverse.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

N/A

Other effects:

not examined

Description (incidence and severity):

N/A

Details on results:

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

Key result

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)

Key result

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)

Critical effects observed:

not specified

N/A

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 LOEC 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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

11 473 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

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. Details provided are based on the draft report.

Justification for type of information:

N/A

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

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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: 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)

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

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

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.

Positive control:

N/A

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.

Clinical signs:

no effects observed

Description (incidence and severity):

N/A

Mortality:

no mortality observed

Description (incidence):

N/A

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

N/A

Food efficiency:

not examined

Description (incidence and severity):

N/A

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

N/A

Ophthalmological findings:

no effects observed

Description (incidence and severity):

N/A

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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 biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Endocrine findings:

not examined

Description (incidence and severity):

N/A

Urinalysis findings:

no effects observed

Description (incidence and severity):

N/A

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

N/A

Immunological findings:

not examined

Description (incidence and severity):

N/A

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Gross pathological findings:

no effects observed

Description (incidence and severity):

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.

Neuropathological findings:

no effects observed

Description (incidence and severity):

N/A

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Due to the lack of severity associated with the effects described, they were not considered to be adverse.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

N/A

Other effects:

not examined

Description (incidence and severity):

N/A

Details on results:

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

Key result

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)

Key result

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)

Critical effects observed:

not specified

N/A

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 LOEC 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).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

11.47 mg/m³

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Human/Non-human data – oral and dermal

There are no available data following exposures via the oral or dermal routes. Studies are not technically feasible as the substance is a gas at room temperature.

Inhalation

Non-human data

The key study is considered to be a GLP-conducted 13-week inhalation toxicity study in rats at ethylene concentrations of 300, 1000, 3000 and 10,000 ppm (344.2, 1147, 3442 and 11,473 mg/m3) (Dow, 2010). There was no evidence of systemic toxicity based on the lack of toxicologically significant differences in clinical signs (including functional tests, grip performance, and motor activity), body weight, food consumption, body temperature, clinical pathology or organ weights. Evidence of minor local irritation was indicated by histopathological changes in the upper respiratory tract (bilateral, eosinophilic rhinitis with mucous cell hyperplasia/hypertrophy (MCH) and occasional epithelial hyalinosis). The changes, seen at all dose concentrations, were described as very slight or slight, with only minor increase in severity at the highest exposure of 10,000 ppm. Quantitative morphometric analyses substantiated the qualitative histopathologic findings by demonstrating quantifiable increases of both intraepithelial mucosubstances (IM; a quantitative estimate of epithelial remodelling) and eosinophil numeric density (quantitative estimate of nasal inflammation – eosinophilic rhinitis) in the nasal mucosal tissue of ethylene-exposed rats. The NOAEC for systemic toxicity was 10,000ppm (11,473 mg/m3). A NOAEC for local effects was not established, with 300 ppm (344.2 mg/m3) a LOEC for nasal pathology.

 

The local effects described above were investigated further by The Dow Chemical Company (2012). Time- and concentration-dependent changes in histopathology, cellular inflammation, and mucosal gene expression were assessed 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 exposure to ethylene 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 nasal epithelium from F344 rats. An overall LOEC of 10 ppm (11.47 mg/m3) is obtained from this investigation.

A further study assessed whether comparable nasal changes were present in Fischer 344 and Wistar Han rats exposed to target ethylene concentrations of either 0 or 10,000 ppm for 4 or 13 weeks (Hamner Institute 2009, Harkema 2010). There was no mortality, no systemic toxicity and no statistically significant differences in body weights or food consumption during the study. Histopathological findings were described as similar to those seen previously (in Dow 2010). F344 rats had nasal lesions consisting of multifocal subacute rhinitis with accompanying airway epithelial remodelling. The exposure-related inflammatory cell infiltrate consisted mainly of eosinophils and mononuclear leukocytes with lesser numbers of neutrophils. The principal nasal epithelial changes included MCH and eosinophilic hyalinosis. This study included a 4-week exposure period at 10,000 ppm; ethylene-induced inflammatory and epithelial lesions in F344 rats were similar in character, but less severe, to those in F344 rats exposed for 13 weeks. This study also demonstrated a strain-dependent nasal response to ethylene after 13 weeks of exposure, the response being weaker in the Wistar rats.

In two previous studies (Rhudy 1978 and Hamm 1984) groups of rats (strain not specified and Fischer 344) were exposed to ethylene at concentrations of 0, 300, 1000, 3000 and 10,000 ppm for 13 weeks or 0, 300, 1000, or 3000 ppm for 24 months (with groups of animals necropsied and examined after 6, 12 or 18 months). There was no exposure-related mortality and no effects on body weight, weight gain or food consumption. Haematology, clinical chemistry, and urinalyses results also failed to reveal any differences between control and test animals. There were no gross or histopathological alterations attributed to ethylene exposure.

 

Human data

There is no published evidence of adverse effects despite wide industrial use for many years.

 

Summary

Results from recent repeat dose inhalation studies suggest that ethylene may cause mild respiratory tract irritation, with very slight or slight rhinitis observed histologically at exposures of 10 ppm (11.47 mg/m3); only minor increases in severity were apparent at higher exposures (up to 10,000 ppm). The NOAEC for systemic toxicity is 10,000 ppm (11,473 mg/m3).

 

 

 

Justification for classification or non-classification

The health effects of ethylene have been evaluated in a number of repeated-dose toxicity studies conducted in rats and no evidence of systemic toxicity has been seen at any concentration. The only treatment-related effects noted in these studies were changes to the upper respiratory tract following repeated exposures of at least 4 weeks duration, which were considered to be a local adaptive response and showed no dose relationship. The findings are considered not to justify classification under CLP.