Ethane

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP compliant (except for minor exception listed below), guideline study, available as unpublished report, no restrictions, fully adequate for assessment.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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

no lot number available of test material

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 002

Cas Number:

74-84-0

Molecular formula:

C2H6

Test material form:

gas

Details on test material:

- Name of test material (as cited in study report): ethane
- Supplier: MG Industries, 3 Great Valley Parkway, Malvern, Pennsylvania 19355, USA
- Substance type: Industrial gas
- Physical state: colourless gas
- Analytical purity: 99.0% per supplier
- Lot/batch No.: Not available
- Stability under test conditions: 99.86% before study, 99.88% after study
- Storage condition of test material: Ambient

Specific details on test material used for the study:

- Name of test material (as cited in study report): ethane
- Supplier: MG Industries, 3 Great Valley Parkway, Malvern, Pennsylvania 19355, USA
- Substance type: Industrial gas
- Physical state: colourless gas
- Analytical purity: 99.0% per supplier
- Lot/batch No.: Not available
- Stability under test conditions: 99.86% before study, 99.88% after study
- Storage condition of test material: Ambient

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

According to OECD 422 test guideline

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species: Albino rats (Outbred) VAF/Plus®, Sprague-Dawley derived (CD®), Crl:CD®(SD)IGS BR
- Source: Charles River Laboratories, Raleigh, North Carolina 27610, USA
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: Males mean 256 g (range 230-284 G); females mean 197 g (range 167-217 g)
- Fasting period before study: None
- Housing: Individually in stainless steel suspended cages with wire mesh floors and fronts (except for mating period when 1 male and 1 female were housed together)
- Diet: Certified Rodent diet No 5002 (PMI Nutrition International, St Louis, Missouri, USA) ad libitum
- Water: Municipal water ad libitum
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature: 20.7 - 23.2°C
- Humidity: 19.96-74.37%
- Air changes (per hr): Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 24 November 2003 To: 17 January 2004

Administration / exposure

Route of administration:

inhalation: gas

Type of inhalation exposure:

whole body

Vehicle:

other: air

Mass median aerodynamic diameter (MMAD):

>= 1.428 - <= 1.643 µm

Remarks on MMAD:

MMAD / GSD: MMAD: 1.607, 1.477, 1.643, 1.428 µm; GSD: 2.045, 2.385, 2.005, 1.897; total mass concentration: 3.04 x 10E-3, 3.24 x10E-3, 3.61xE10-3, 2.69x10E-3 mg/m3 for target exposure concentrations of 0, 1600, 5000 and 16000 ppm respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 L glass and stainless steel whole-body exposure chamber
- Method of holding animals in test chamber: housed individually, the placement of animals in the chamber was rotated daily to ensure uniform exposure
- System of generating particulates/aerosols: the test substance was delivered from a single cylinder, through a regulator and two backpressure gauges via a flowmeter into the exposure chambers
- Time to T99: 23 minutes maximum
- Airflow rate: 204 Lpm
- Temperature and humidity in chamber: 20-24°C, 28-59%
- Oxygen level: at least 19%
- Air flow rate: minimum flow rate of 200 L/minute
- Air change rate: final airflow set to provide at least one air change in 5 mins (12 air changes/hour)
- Method of particle size determination: determined weekly using a TSI Aerodynamic Particle Sizer
- Treatment of exhaust air: filtered through a system which consisted of a coarse filter, a HEPA filter and an activated charcoal bed

TEST ATMOSPHERE
- Brief description of analytical method used: Infrared spectrophotometer (IR) 4 times per chamber per day
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Exposure levels were determined using an infrared spectrophotometer 4 times/chamber/day. The test substance was evenly distributed within each chamber. The mean (± SD) analytical concentrations were 0.0 ± 0.0, 1599 ± 59, 5186 ± 285 and 16380 ± 626 ppm.

Duration of treatment / exposure:

Males: 2 weeks prior to mating and a minimum of 28 days after mating.
Females: 2 weeks prior to mating and post-mating for an additional 4 weeks.

Frequency of treatment:

6 hours/day, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm

Remarks:

Dose Group 1
Analytical concentration:
0.0 ± 0.0 ppm

Doses / concentrations 2

Dose / conc.:

1 600 ppm

Remarks:

Dose Group 2
Analytical concentration:
1599 ± 59 ppm

Doses / concentrations 3

Dose / conc.:

5 000 ppm

Remarks:

Dose Group 3
Analytical concentration:
5186 ± 285 ppm

Doses / concentrations 4

Dose / conc.:

16 000 ppm

Remarks:

Dose Group 4
Analytical concentration:
16380 ± 626 ppm

No. of animals per sex per dose:

12/sex/group

Control animals:

yes, sham-exposed

Details on study design:

Dose selection rationale:
Based on results of a 2-week range-finding study (HLS Study No. 03-6144) which showed no toxicity at 160, 1600 and 16000 ppm. The high level was established at 16000 ppm since it is 50% of the lower explosion limit for the test substance.

Positive control:

n/a

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (mortality and clinical condition)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: At randomisation, first day of exposure and weekly thereafter.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Pre-test and weekly thereafter.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Study termination
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes (overnight)
- How many animals: 12/sex/group
- Parameters examined: haemoglobin concentration, haematocrit, erythrocyte count, platelet count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, total leukocyte count, reticulocyte count, differential leukocyte count, erythrocyte and platelet morphology (from peripheral blood smear), prothrombin time, activated partial thromboplastin time..

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Study termination
- Anaesthetic used for blood collection: Yes (CO2/O2)
- Animals fasted: Yes (overnight)
- How many animals: 12/sex/group
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, blood urea nitrogen, creatinine, glucose, cholesterol, total protein, triglycerides, albumin, total bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorus, gamma-glutamyl transpeptidase, globulin, albumin/globulin ratio

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During last week of exposure
- Dose groups that were examined: All
- Battery of functions tested: sensory observations (startle response to auditory stimuli, tail pinch response), neuromuscular observations (grip strength - hindlimb and forelimb), physical observations (rectal temperature) and motor activity assessments

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (all animals)
- examined: external surfaces, all orifices, cranial cavity, nasal cavity, neck and its associated tissues and organs, thoracic, abdominal and pelvic cavities and their associated tissues and organs and external surfaces of the brain.
ORGAN WEIGHTS: Yes (all animals)
- organs weighed: adrenal glands, brain, epididymes, heart, kidneys, liver, lungs (with mainstem bronchi), ovaries (with oviducts), spleen, testes, thymus, uterus with vagina.
HISTOPATHOLOGY: Yes (control and high dose only).
- tissues examined: adrenal glands, bone (sternum/femur), brain (cerebellum, cerebrum and cerebellum), epididymes, heart, kidneys, large intestine (caecum, colon and rectum), liver, lungs (with mainstream bronchi), lymph node (mesenteric), lymph node (mediastinal), mammary glands (with adjacent skin), ovaries (with oviducts), prostate, seminal vesicles, small intestine (duodenum, ileum and jejunum), spinal cord (cervical, thoracic and lumbar), spleen, stomach, testes, thymus, thyroids with parathyroids, tibial nerve, trachea, urinary bladder, uterus with vagina, all macroscopic lesions and tissue masses.

Other examinations:

n/a

Statistics:

Group mean values of parameters for all the exposure groups were compared to the control group mean values at each time interval, using appropriate statistical methods.

Evaluation of equality of group means was by made appropriate statistical method, followed by multiple comparison test if needed. Bartlett's test was used to determine if groups had equal variances. For all functional obeservation battery, clinical pathology, pre- and post-implantation loss parameters, if the variances were equal, parametric procedures were used, if not, non-parametric procedures were used. All other data was analysed only by parametric methods.

The parametric method was a standard one-way analysis of variance (ANOVA) using the F ratio to assess significance. If significant differences among the means were identified, additional tests were used to determine which means were significantly different from the control: Dunnett's, Williams or Cochran and Cox's modified t-test. The non-parametric method was a Kruskal-Wallis test and if differences were indicated, Shirley's test, Steel's test or a Pairwise Comparison was used to determine which means differed from controls. Bartlett's test for equality of variance was conducted at 1% significance level while all other statistical tests were conducted at 5% and 1% significance levels.

Statistical evalutaions were not performed when the standard deviation for the control group was 0. When 75% of the values for a clinical pathology parameter were the same, Fisher's Exact Test was performed followed by Mantel's tets.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was a low incidence of transient red nasal discharge or red/brown staining between the 7th and 28th days of exposure in all groups including the control group. These findings were slightly more prevalent among the test substance exposed animals (Table 1).

Mortality:

no mortality observed

Description (incidence):

n/a

Body weight and weight changes:

no effects observed

Description (incidence and severity):

n/a

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Marginally lower food consumption at 16000 ppm compared to controls was seen during 1st week of exposure, but differences from control were less than 5% and were transient and, therefore, were considered not to be adverse (Table 2).

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:

not examined

Description (incidence and severity):

n/a

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease (up to 15% in the high level group) in reticulocytes in the 5000 ppm and 16000 ppm level group females was observed in an exposure-related pattern but there was no accompanying change in erythrocyte numbers or other haematology parameters (Table 3). No similar changes were observed in males. In isolation, this minor inter-group difference was considered to be a test-item related and was within the normal range of historical values.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase in sodium concentration in the 16000 ppm exposed males was observed but the absolute difference was only 2% and there was no similar difference in the females (Table 4). In isolation, this minor inter-group difference was considered to be a test-item related and was within the normal range of historical values.

Endocrine findings:

not examined

Description (incidence and severity):

n/a

Urinalysis findings:

not examined

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):

Females in the 5000 ppm level group showed an increase in uterus weight (relative to body weight) but no similar difference was observed in the 16000 ppm level group (Table 5) In isolation, this minor inter-group difference was considered to be a test-item related and was within the normal range of historical values.

Gross pathological findings:

no effects observed

Description (incidence and severity):

n/a

Neuropathological findings:

not examined

Description (incidence and severity):

n/a

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

n/a

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

n/a

Other effects:

not examined

Description (incidence and severity):

n/a

Details on results:

No systemic toxicity (no effects on survival, haematological or clinical chemistry parameters, food consumption, body weight, organ weight, and histopathology) or neurological effects (as measured by clinical observations, functional observational battery, and motor activity) were observed.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

16 000 ppm

Sex:

male/female

Basis for effect level:

other: highest concentration tested

2

Key result

Dose descriptor:

NOAEC

Remarks:

systemic toxicity

Effect level:

19 678 mg/m³ air

Sex:

male/female

Basis for effect level:

other: highest concentration tested

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1 Summary of Clinical Observations

		Day of the study
Males	Group	-7	0	7	14	21	28	30	32	Total
No. of animals examined	I	12	12	12	12	12	12	6	6
	II	12	12	12	12	12	12	6	6
	II	12	12	12	12	12	12	6	6
	IV	12	12	12	12	12	12	6	6
Nasal discharge - red	I	0	0	0	0	0	0	0	0	0
	II	0	0	0	0	1	1	0	0	1
	II	0	0	0	0	1	1	0	0	1
	IV	0	0	0	0	0	0	0	0	0
Males	Group	-7	0	7	14	21	28	30	32	Total
No. of animals examined	I	12	12	12	12	12	12	6	6
	II	12	12	12	12	12	12	6	6
	II	12	12	12	12	12	12	6	6
	IV	12	12	12	12	12	12	6	6
Red/brown stains - snout	I	0	0	0	0	1	0	0	0	1
	II	0	0	0	0	2	1	0	0	2
	II	0	0	0	0	4	0	0	0	4
	IV	0	0	0	0	5	0	0	0	5

Table 2 Summary of Mean Feed Consumption (g/kg/day)

Males	Dose Group	1	2	3	4
Day 0	Mean	109	110	110	106
	S.D.	6.2	10.0	4.3	7.7
	N	12	12	11	12
Day 7	Mean	90	89	88	87
	S.D.	5.5	5.8	4.7	5.1
	N	12	12	11	12
Day 14	Mean	76	75	76	73
	S.D.	3.7	4.3	3.8	3.1
	N	12	12	12	12
Females	Dose Group	1	2	3	4
Day 0	Mean	97	94	6	98
	S.D.	4.6	6.9	5.7	5.6
	N	11	12	11	10
Day 7	Mean	86	84	85	82
	S.D.	4.0	3.7	4.7	5.6
	N	12	10	10	9
Day 14	Mean	82	83	82	82
	S.D.	4.6	5.5	4.6	3.5
	N	12	12	11	10
Day 21	Mean	79	79	80	82
	S.D.	2.6	5.6	4.4	4.1
	N	12	12	12	12
Day 28	Mean	75	73	75	75
	S.D.	3.1	5.6	3.9	2.6
	N	12	12	12	12

Table 3 Summary of Haematological Findings

Females		Reticulocytes
Group		x10^9/L
1	Mean	204.9
	S.D.	32.89
	N	12
2	Mean	191.3
	S.D.	35.19
	N	12
3	Mean	180.7*
	S.D.	21.83
	N	12
4	Mean	174.3
	S.D.	19.09
	N	12

*Significantly different from control mean, p<0.05

Table 4 Summary of Clinical Biochemistry Findings

Males		Na+
Group		mEq/L
1	Mean	147
	S.D.	2.2
	N	12
2	Mean	147
	S.D.	1.9
	N	12
3	Mean	149
	S.D.	2.0
	N	12
4	Mean	150**
	S.D.	1.6
	N	12

**Significantly different from control mean, p<0.01

Table 5 Summary of % Organ to Body Weight (Female)

Group		Terminal body weight (g)	Uterus w/ vagina
1	Mean	240	0.3793
	S.D.	19.3	0.0820
	N	12	12
2	Mean	237.2	0.4083
	S.D.	18.3	0.0950
	N	12	12
3	Mean	239.8	0.4835*
	S.D.	19.6	0.1386
	N	12	12
4	Mean	236.9	0.3693
	S.D.	18.1	0.0557
	N	12	12

*Significantly differen from control, p=0.05 with modified T test

Applicant's summary and conclusion

Conclusions:

The no observed adverse effect concentration (NOAEC) of ethane was 16000 ppm in this study. Equivalent to 19678 mg/m3.

Executive summary:

This OECD 422 test guideline study, the potential toxicity, including neurotoxicity and reproductive performance, in male and female rats following ethane exposure at 1600, 5000and 16000 ppm (highest exposure level was 50% of the lower explosive limit) was assessed. It also was designed to investigate effects in both sexes on mating behaviour and on gonadal function, as well as effects on conception, development, parturition and pup survival to lactation day 4. Male and female rats were exposed for 6 hours/day, 7 days/week for 2 weeks prior to mating initiation. Main study males and females were then evaluated for subchronic effects and were exposed once daily (6 hours/day), seven days/week for 4 weeks (28 days).

There was no effect on survival. There were no exposure-related clinical effects or effects on body weight, food consumption, functional obeservation battery or motor activity parameters for either sex (except the16000 ppm exposed animals showed marginally lower food consumption during the first week of exposures).There were no exposure-related differences in haematology, clinical chemistry and no macroscopic or microscopic changes at post-mortem.

Exposure of male and female rats to target concentrations of 1600, 5000 or 16000 ppm of ethane by whole-body inhalation for 4-6 weeks resulted in no general systemic or neurotoxic effects apart from a very marginal reduction of food consumption during the first week of exposure at 16000 ppm and this transient difference was not considered adverse. Therefore, a no-observed-adverse effect concentration (NOAEC) of 16000 ppm (equivalent to 19678 mg/m3 (mw 30.07g/mol)) was determined for all endpoints.