Isobutane

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: A 13-week whole-body inhalation toxicity study in rats with neurotoxicity assessments and in vivo genotoxicity assessments

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant (except for test substance identity, stability and lot number), guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day)

Deviations:

yes

Remarks:

The identity and stability was not in compliance with GLP regulations. The lot number was not available.

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Principles of method if other than guideline:

A 13-week whole-body inhalation toxicity study in rats with neurotoxicity assessments. Vaginal smears were taken daily to determine oestrus stage in females for the three weeks prior to and on the day of termination. Testes and ovaries were examined histopathologically. Sperm count, motility and morphology were assessed.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Liquified Petroleum Gas

IUPAC Name:

Liquified Petroleum Gas

Details on test material:

- Name of test material (as cited in study report): liquefied petroleum gas (LPG)
- Substance type: industrial gas
- Supplier: ChevronTexaco Energy Research & Technology Company, 100 Chevron Way, Richmond, CA 94802, USA
- Physical state: colourless gas or liquid in pressurized cylinders
- Analytical purity: 100% LPG
- Lot/batch No.: 120701-01
- Composition of test material, percentage of components (weight %): methane 0.012%, ethane 1.809%, propane & propylene 93.513%, n-butane 2.854%, n-pentane 0.064%, isobutane 1.246%, neopentane 0.003%, isopentane 0.404%, 2,3-dimethylbutane 0.006%, cyclopentane <0.001%, isobutene 0.038%, 1,3-butadiene <0.001%, t-2-butene 0.009%, c-2-butene 0.007%, 3-methyl-1-butene 0.004%, 1-pentene 0.014%, 2-methyl-1-butene 0.005%, t-2-pentene 0.003%, c-2-pentene 0.003%, 2-methyl-2-butene 0.005%, benzene <0.001%
- Expiration date of the lot/batch: 31 December 2007
- Storage condition of test material: Ambient conditions in an outside solvent shed except when in use in the inhalation laboratory.

Test animals

Species:

rat

Strain:

other: Sprague-Dawley CD

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, Kingston, New York 12484, USA
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: Males mean 280 g (range 243-308 g); females mean 209.1 g (range 187-231 g)
- Fasting period before study: None
- Housing: Individually in stainless steel suspended cages with wire mesh floors and fronts.
- 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: 17-25°C
- Humidity: 22-99%
- Air changes (per hr): Not reported
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 20 April 2005 To: 27 July 2005

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure (if applicable):

whole body

Vehicle:

other: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Stainless steel and glass whole body exposure chambers with a volume of approximately 1000 L
- Method of holding animals in test chamber: Individually housed in stainless steel, wire mesh cages within the exposure chamber, with the placement of animals in chambers rotated weekly to ensure uniform exposure.
- Generation: Pre-study trials had evaluated the optimal set of conditions and equipment that generated a stable and uniform atmosphere at the target exposure levels. Test substance flowed from cylinder into a copper tubing coil, maintained in a warm water bath. From the coil the test substance flowed through a metering valve to a mass flowmeter and then via tubing to the turret of the exposure chamber, where it was mixed with room air.
- Temperature, humidity in exposure chamber: 19-28°C, 22-61%
- Air flow rate: Operated at a minimum rate of 203 L/min. Final airflow set to provide at least one air change /5 mins (12/hour) and a T99 equilibrium time of at most 23 mins.
- Oxygen level: at least 19%
- Animal loading factor: below 5%
- Method of particle size determination: yes. Samples drawn for 20 secs at a flow rate of 5 L/min. MMAD, GSM and total mass concentration were calculated.
- Treatment of exhaust air: coarse filter, a HEPA filter and activated charcoal

TEST ATMOSPHERE
- Brief description of analytical method used: Infrared spectrophotometer (IR) 4 times per chamber per day. Gas chromatography (GC) used to characterise at least 5 major components (comprising at least 90% by weight of test substance) once/week/chamber to show test substance stability and comparison between neat test substance and test atmospheres.
- Samples taken from breathing zone: yes

Details on mating procedure:

Animals not mated

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. Particle size distribution measurements were also made once weekly using a TSI Aerodynamic Particle Sizer. The mean (± standard deviation) analytical exposure concentrations of LPG were
determined to be 0.0 ± 0.0, 1019 ± 58, 5009 ± 174 and 9996 ± 261 ppm for the air control and the exposure groups, respectively. Particle sizing results indicated that the atmospheres were essentially gas/vapour only, as expected, since there was no substantial difference in particle concentration between the test substance chambers and the air control chamber. Analysis of the major components in the neat test substance and the test atmospheres showed an acceptably close comparison between the neat test substance and the vaporized test substance. The data were consistent pretest and during the study indicating stability of the test substance and the atmosphere generation techniques.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours per day, 5 days per week

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 1000, 5000, 10000 ppm
Basis:
other: target concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.0 ± 0.0, 1019 ± 58, 5009 ± 174, 9996 ± 261 ppm
Basis:
analytical conc.

No. of animals per sex per dose:

15/sex/group

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: Based on results of a range-finding study (HLS Study number 03-6140) which showed no effects at 100, 1000 or 10000 ppm and no more than 50% of the lower explosion limit (LEL = 21000 ppm)

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (mortality and severe toxic effects)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During exposure, animals were observed at least once as a group for behavioural effects, signs of toxicity, and mortality. 10/sex/group were examined twice pretesting and weekly during the study period for clinical signs of toxicity

BODY WEIGHT: Yes
- Time schedule for examinations: On assignment to treatment group, on day of treatment began, and weekly thereafter

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-test and at study termination (all animals)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (60% carbon dioxide/40% oxygen)
- Animals fasted: Yes (overnight)
- How many animals: 10 sex/group
- Parameters examined: haemoglobin concentration, haematocrit, erythrocyte count, platelet count, mean platelet volume, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, red cell distribution width, total leukocyte count, reticulocyte count, differential leukocyte count, erythrocyte and platelet morphology, prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (60% carbon dioxide/40% oxygen)
- Animals fasted: Yes (overnight)
- How many animals: 10 sex/group
- Parameters examined: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, lactate dehydrogenase, blood urea nitrogen, creatinine, glucose, creatinine kinase, cholesterol, total protein, albumin, total bilirubin, direct bilirubin, sodium, potassium, chloride, calcium, inorganic phosphorus, gamma-glutamyl transferase, triglycerides, globulin, albumin/globulin ratio, indirect bilirubin

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes (10/sex/group)
- Time schedule for examinations: Pre-test and during 2nd, 4th, 8th and 13th week of exposure. Tested on a non-exposure day at least 16 hours after exposure.
- Dose groups that were examined: All
- Battery of functions tested: sensory observations, neuromuscular observations, physical observations and motor activity assessments

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily to determine oestrus stage in females for the three weeks prior to and on the day of termination.

Sperm parameters (parental animals):

Testes were examined histopathologically. Sperm count, motility and morphology were assessed.

Litter observations:

Not applicable

Postmortem examinations (parental animals):

SACRIFICE: Fasted overnight prior to exsanguination following carbon dioxide inhalation.

GROSS PATHOLOGY: Yes (all animals) - included external examinations as well as examination of the brain, spinal cord, organs and tissues of the cranial, thoracic, abdominal, and pelvic cavities and neck.

ORGAN WEIGHTS: Yes (all animals). Adrenal gland, brain, epididymes, heart, kidneys, liver, lungs (with mainstem bronchi), ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroids (with parathyroids), Zymbals gland.

HISTOPATHOLOGY: Yes.
- tissues examined: (control and high dose only) adrenal glands, aorta (thoracic), bone (sternum/femur), brain (medulla/pons, cerebrum and cerebellum), epididymes, oesophagus, eye, heart, kidneys, large intestine (caecum, colon and rectum), lachrymal gland, larynx, liver, lungs (with mainstream bronchi), lymph node (mesenteric), lymph node (mediastinal), mammary glands, muscle (biceps femoris), nasopharngeal tissue, nerve (sciatic), optic nerve, ovaries, pancreas, prostate, salivary gland with submandibular lymph node, seminal vesicles, small intestine (duodenum, ileum and jejunum), spinal cord (cervical, thoracic and lumbar), spleen, stomach, testes, thymic region, thyroids with parathyroids, trachea, urinary bladder, uterus (body/horns with cervix), Zymbals glands, all macroscopic lesions and tissue masses.

EXAMINATION OF TESTES: To identify potential treatment-related effects such as retained spermatids, missing germ cell layers or types, multinucleated giant cells, or sloughing of spermatogenic cells into the lumen. A longitudinal section of the intact epididymis was examined to assess caput, corpus and cauda regions to identify such lesions as sperm granulomas, leukocytic infiltration (inflammation), aberrant cell types within the lumen, or the absence of clear cells in the cauda epididymal epithelium. As an increased incidence of "mid-tail blob" was seen in high dose sperm, the testes from all groups were examined.

EXAMINATION OF OVARIES: Histopathological examination of the ovary included evaluation of five sections taken at least 100 pm apart from the inner third of each ovary. These examinations could detect depletion of the primordial follicle population and enumerate the total number of primordial follicles for comparison with the ovaries from control animals. These examinations could also confirm the presence or absence of growing follicles and corpora lutea, in comparison to the control ovaries.

Postmortem examinations (offspring):

Not applicable

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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, treatment-related

Details on results (P0)

There was a slight increase in the incidence of "mid-tail blob" (cytoplasmic droplet) in the 10000 ppm group only when compared to control, but it was not statistically significant. This may reflect relative immaturity of the affected sperm, and the increased incidence (1.8 to 6.8%) affected 4 out of 10 males examined. Usual incidence of this finding in historical controls is 0-2% in each animal although occasional control animals have shown 6-8% incidence. There were no correlative findings in animals showing these higher incidences, in terms of histological findings or general condition and, therefore, it was considered that the difference from control was incidental and not a treatment-related effect.

There was no treatment-related effects on oestrous cycle in females at any exposure concentration.

No treatment-related effect on survival, clinical observations, ophthalmology, terminal body weight, food consumption, functional observational battery, motor activity parameters, haematological parameters, clinical chemistry values, macroscopic or microscopic evaluations, or organ weights was observed at any exposure concentration.

Effect levels (P0)

Dose descriptor:

NOAEC

Remarks:

(reproductive effects)

Effect level:

10 000 ppm

Sex:

male/female

Basis for effect level:

other: no treatment-related effects on oestrous cycle in females or sperm count, motility or morphology in males at the highest concentration tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

No treatment-related effects on oestrous cycle in females or sperm count, motility or morphology in males at any exposure concentration.

The no observed adverse effect concentration (NOAEC) for liquified petroleum gas for reproductive effects was 10000 ppm in this study.

Executive summary:

This study was designed to assess the potential inhalation toxicity of liquified petroleum gas (LPG) when administered via whole-body exposures to rats, six hours per day to 0 (air control), 1000, 5000 or 10000 ppm of LPG for 5 days per week for 13 consecutive weeks (highest exposure concentration was selected for safety reasons and approximated 50% of the lower explosive limit).

At the end of the treatment period, all animals were euthanized and necropsied. The following parameters were evaluated: viability, clinical observations, body weights, feed consumption, functional observation battery and motor activity, oestrus cycles, sperm assessments, micronucleus assessment, clinical pathology, organ weights, macroscopic and microscopic observations.

No treatment-related effects on oestrous cycle in females or sperm count, motility or morphology in males at any exposure concentration.

The 10000 ppm exposure level was considered a no observed adverse effect level concentration.