Methane

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

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other: GLP compliant (except for test substance identity, stability and lot number), guideline study, available as unpublished report, no restrictions, fully adequate for assessment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

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

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: other guideline: EPA OPPTS 870.3650 (Combined repeated exposure toxicity study with the Reproduction/Developmental toxicity screening test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Butane

EC Number:

203-448-7

EC Name:

Butane

Cas Number:

106-97-8

Molecular formula:

C4H10

IUPAC Name:

butane

Details on test material:

- Name of test material (as cited in study report): butane
- Physical state: colourless gas
- Analytical purity: 99.5% per supplier
- Analytical purity: 99.23% before study, 99.98% after study
- Lot/batch No.: not available
- Expiration date of the lot/batch: not available
- Stability under test conditions: confirmed
- Storage condition of test material: Ambient
- Other: An archival sample was stored.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, North Carolina 27610
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: males: 241-280 g (mean: 261 g); females 174-229 g (mean 200 g)
- Fasting period before study: none
- Housing: Individually in stainless steel suspended cages with wire mesh floors and fronts except during the mating period. From day 18 of gestation and during lactation, the each female was housed with her litter in plastic cages with bedding
- Diet: Certified Rodent Diet # 5002 (PMI Nutrition Int., St. Louis, Missouri) ad libitum
- Water: ad libitum
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature 20.7-22.4°C
- Humidity: 24.28-77.36%
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 29 September 2003 To: 26 April 2006

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: 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
- Temperature and humidity in chamber: 21-25°C, 42-65%
- Oxygen level: at least 19%
- Air flow rate: minimum flow rate of 200 L/minute (201-221 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, 930.6 ± 28.1, 3022 ± 58 and 9157 ± 269 ppm.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of mating was seen, or for two consecutive weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually in plastic "shoebox" cages with bedding
- After the mating period was over, females without evidence of copulation were removed from the mating cages, housed individually and monitored for visible signs of pregnancy with corresponding bodyweight gain.
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

Males for 2 weeks prior to mating and for an additional 28 days (minimum) after mating.
Females for 2 weeks prior to mating and gestation days 0-19.

Frequency of treatment:

6 hours/day, 7 days/week

Duration of test:

6 weeks

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Details on study design:

Females without evidence of mating that appeared to be pregnant were killed on an estimated gestation day 19.
Females that littered and their offspring were killed on post partum day 4.

Examinations

Maternal examinations:

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Male rats were examined prior to randomisation and once weekly throughout the study. Female rats were examined prior to randomisation and once weekly throughout the premating period and on gestation days 0, 7, 14, 20 and lactation days 0 (except if parturition was not completed on the same day), 1 and 4.

BODY WEIGHT: Yes
- Time schedule for examinations: Male rats were weighed at randomisation and then weekly throughout the study. Females were weighed at randomisation, on the first day of exposure and twice weekly until evidence of copulation was observed, on gestation days 0, 7, 14 and 20, and on lactation days 1 and 4. Females were not fasted prior to recording terminal bodyweights.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Once weekly throughout the premating period. For pregnant or confirmed mated females, food consumption was recorded on gestation days 0-7, 7-14, 14-20 and on lactation days 1-4.

WATER CONSUMPTION: No

- Male animals: All surviving animals (after a minimum of 28 days post-mating).
- Maternal animals: All surviving animals (28 days post-mating).

GROSS EXAMINATION: Yes. Tissues examined: adrenal glands, bone (sternum/femur), bone marrow, brain (medulla/pons, cerebrum and cerebellum), epididymides, heart, kidneys, caecum, colon, rectum, larynx, liver, lungs (with mainstem bronchi), lymph nodes (mesenteric and mediastinal), mammary glands (with adjacent skin), nasopharynx, ovaries (with oviducts), prostate, seminal vesicles, duodenum, ileum, jejunum, spinal cord (cervical, thoracic and lumbar), spleen, stomach, testes, thymus, thyroid with parathyroids, tibial nerve, trachea, urinary bladder, uterus with vagina and all macroscopic lesions and tissue masses.

ORGAN WEIGHTS: Yes. Adrenal glands, brain, epididymides, heart, kidneys, liver, lungs, ovaries, spleen, testes, thymus and uterus with vagina.

HISTOPATHOLOGY: Yes. ORGAN WEIGHTS: Yes. Adrenal glands, brain, epididymides, heart, kidneys, liver, lungs, ovaries, spleen, testes, thymus and uterus with vagina.

HISTOPATHOLOGY: Yes (male and female main study 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), protate, seminal vesicles, smnall 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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes satellite females
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes

Fetal examinations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Number of live and dead pups, pup abnormalities and sex of pups. Pups were observed twice daily (morning and afternoon).
Each pup was examined on lactation day 0 and 4.
Individual pup weights and sex were recorded on lactation days 1 and 4

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.

Macroscopic postmortem examinations (external only) were performed on all surviving F1 pups on lactation day 4.

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.

Indices:

Male and female mating indices, pregnancy rates, male and female fertility indices, gestation indices and the incidence of dams with no viable pups, were analysed statistically. Live birth index, litter survival and mean pup survival indices (days 0 and 4) were analysed statistically.

Historical control data:

None required

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no effects on mating, fertility, gestational indices or reproductive performance in either male or female rats exposed to concentrations up to 9000 ppm butane for up to 6 weeks prior to, during, and after mating.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEC

Effect level:

9 000 ppm

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEC

Effect level:

21 394 mg/m³ air

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no effects on numbers of live or dead pups, pup abnormalities, sex or weight.

Effect levels (fetuses)

1

Dose descriptor:

NOAEC

Effect level:

9 000 ppm

Sex:

male/female

Basis for effect level:

other: Developmental Toxicity

2

Dose descriptor:

NOAEC

Effect level:

21 394 mg/m³ air

Sex:

male/female

Basis for effect level:

other: Developmental Toxicity

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

Conclusions:

There were no effects on gestation duration, number of live and dead pups, pup abnormalities or pup sex and weights. The NOAEC for butane for developmental toxicity was 9000 ppm. Equivalent to 21394 mg/m3

Executive summary:

The study assessed the potential toxicity, including neurotoxicity and reproductive performance in male and female rats following butane exposure at900, 3000 and9000 ppm. It also was designed to investigate effects inboth sexes on mating behavior and on gonadal function, as well aseffects on conception, development, parturition andpup survival to lactation day 4. Male and female rats were exposedfor 6 hours/day, 7 days/week for 2 weeks prior to mating initiation.Main study females were evaluated for subchronic effects and were exposed once daily (6 hours/day), seven days/week for 4 weeks (28 days). A satellite group of females was evaluated for reproductive effects only - exposed once daily (6 hours/day), seven days/week for at least two weeks prior to mating initiation, then once daily during mating and gestation (days 0-19). For satellite female rats without evidence of mating that appeared to be pregnant, exposure was terminated on the estimated gestation day 19.Main study male rats were exposed during the mating and post-mating periods until euthanized for a minimum exposure of 28 days. There was no effect on survival. There were no exposure-related clinical effects or effects on body weight, food consumption, FOB or motor activity parameters for either sex. Furthermore there were no exposure-related differences in haematology, clinical chemistry and no macroscopic or microscopic changes at post-mortem. All mated female animals were found pregnant and delivered live pups. Mating indices for the butane male rats treated were comparable to control. Mating, fertility and gestation indices for the female rats were comparable to control. Most of the females in each group mated at the first opportunity. There were also no treatment-related differences in the other reproductive parameters up to the time of parturition including the percent of females completing delivery and the duration of gestation. There were no treatment-related differences in all parturition parameters including the total number of pups delivered, the number of pups dying, the viability (4 day survival) index, the number of implantation sites and corpora lutea per dam, the pup sex ratio and the number of live pups/litter. The pups were unremarkable during the lactation period. There were no exposure-related differences in body weights or weight gains in the pups feeding from test substance exposed animals compared to the pups feeding from air control animals. Furthermore there were no macroscopic changes at post-mortem. Exposure of male and female rats to target concentrations of 900, 3000 and 9000 ppm of butane resulted in no general systemic effects, no effects on fertility and reproductive performance and no effects on pup survival and development to postnatal Day4.                A no observed adverse effect concentration(NOAEC) of 9000 ppm was determined for this study's systemic, reproductive, and neonatal endpoints.Equivalent to 21394 mg/m3 (MW 58.12g/mol).