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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Remarks:
combined repeated dose and carcinogenicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, near guideline study, published in peer reviewed literature, no restrictions, fully adequate for assessment
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Inhalation toxicity studies with 1,3-butadiene. 3. Two year toxicity/carcinogenicity study in rats.
Author:
Owen PE, Glaister JR, Gaunt IF, Pullinger DH
Year:
1987
Bibliographic source:
Am Ind Hyg Assoc J. 48; 407-413.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
Deviations:
not specified
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,3-butadiene
- Physical state: liquid
- The gas supplied to the exposure chambers was analyzed each week for specific impurities: 4-vinyl-1-cyclohexane (dimerization product) and tertiary butyl-catechol (inhibitor preventing polymerization).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: young adults
- Weight on arrival: 40-50 g
- Housing: 5 per sex per cage
- Diet: pelleted SQC rat and mouse No.1 expanded diet (BP Nutrition, Witham, Essex, UK) ad libitum except during exposure
- Water: ad libitum except during exposure
- Acclimation period: yes (duration not reported)

ENVIRONMENTAL CONDITIONS
- Temperature: 20-25°C in the exposure chambers
- Humidity 40-80% in the exposure chambers
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

- stratified body weight randomization procedure used to allocate rats to experimental groups to achieve equal group mean body weights and standard deviations.
- The position of the racks in the chambers and the positions of the cages on the racks were changed according to a pre-determined plan was changed at weekly intervals.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: air
Remarks on MMAD:
MMAD / GSD: No data
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Three 8-m3 exposure chambers of glass and stainless steel. Each chamber was lit by flash-proof lights via two strip glass windows that were inserted into the stainless steel roof.
- Method of holding animals in test chamber: 5 per sex per cage in suspended, stainless steel, wire mesh cages
- Source and rate of air: The chambers were operated at negative pressure, and a separate alarm monitored the differential pressure between the inside and outside of the chambers.
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity in the exposure chambers were within the range of 20°C to 25°C and 40 to 80% on 96% of the occasions that measurements were made.
- Air flow: The main air flow into the chamber was drawn from the room through a filter (efficiency 95% at 1 µm). The flow rate was monitored by means of a pitot tube and inclined manometer.
- Air change rate: The target flow rate was 1000 L/min for all chambers.

TEST ATMOSPHERE
- Generation: To help volatilization, the 1,3-butadiene was passed through a water bath at 30°C. Following volatilization the gas passed through traps to remove the inhibitor, tertiary butylcatechol.
- Brief description of analytical method used: The atmospheres were routinely monitored by an infrared gas analyzer at hourly intervals. The infrared analyzer was calibrated against a gas chromatograph. Even distribution of the test article was established in both exposure chambers before the start of animal exposures and confirmed at regular intervals throughout the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Control chamber, overall mean 0.7 ± 0.8 ppm v/v, < 3.0 ppm v/v on any occasion.
Nominal 1000 ppm, achieved 999.0 ± 30.5 ppm v/v, 94% of all daily mean concentrations were within the range 1000 ± 10% ppm v/v; Coefficients of variation (24 or 27 sample points) 9.19% to 15.69%.
Nominal 8000 ppm, achieved 7886.0 ± 704.3 ppm v/v, 96% of daily mean concentrations within 8000 ± 10% ppm v/v. Coefficients of variation (24 or 27 sample points) 3.68% to 7.63%.
Distribution checked five times and 1,3-butadiene found to evenly distributed throughout each chamber
Duration of treatment / exposure:
105 weeks (females) and 111 weeks (males)
Frequency of treatment:
6 hr/day, 5 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
1000 and 8000 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
110
10 rats/sex were killed after 52 weeks for interim assessment
Control animals:
yes, sham-exposed
Details on study design:
A preliminary 90-day experiment in Sprague-Dawley rats established exposure levels for the long-term study.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily, before and after exposure

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: a detailed observation with palpation of superficial masses was performed at weekly intervals.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly up to week 13, then every 2 weeks to week 52, and monthly thereafter.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 3, 6, 12, and 18 months
- Anaesthetic used for blood collection: Yes (light ether anaesthesia)
- Animals fasted: Yes (Before blood tests, the animals were fasted for 24 hr, but water was available during the last 18 hr of the fast)
- How many animals: 20 preselected animals of each sex from each group.
- Parameters examined: Mean cell volume and haemoglobin concentration, and for counts of erythrocytes, leukocytes (total and differential), platelets and reticulocytes. Packed cell volume, mean cell haemoglobin, and mean corpuscular haemoglobin concentration were also calculated.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 3, 6, 12, and 18 months
- Animals fasted: Yes (Before blood tests, the animals were fasted for 24 hr, but water was available during the last 18 hr of the fast)
- How many animals: 20 preselected animals of each sex from each group (same animals as those used for haematology).
- Parameters examined: Measurements were made of plasma concentrations of glucose (sample taken from caudal vein), blood urea nitrogen, total protein and protein electrophoresis, as well as activities of alkaline phosphatase, glutamic-oxaloacetic transaminase and glutamate-pyruvate transaminase after exposure for 3, 6, and 12 months. At 12 months, leukocyte counts (total and differential) were made in an additional ten animals of each sex from each group, since a possible treatment-related effect had been seen at the 6-month sampling in the high-dose females.

URINALYSIS: Yes
- Time schedule for collection of urine: Individual urine samples were obtained after 3, 6, and 12 months' exposure. Wherever possible, the same 20 males and 20 females were sampled that were used for the blood sampling.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes (4-hr period of food and water deprivation, beginning approximately 2 hr after exposure. During the 2-hr post-exposure period all animals were given access to water).
- Parameters examined: The volume and specific gravity of the urine were measured and a semiquantitative assessment was made for glucose, urobilinogen, ketones, bile pigments, blood, protein, and pH. The insoluble constituents of the urine were examined microscopically after centrifugation.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment and at weeks 1, 4, 15, 26, 52, and 78 of treatment.
- Dose groups that were examined: 40 animals of each sex from each dose group
- Battery of functions tested: The time before falling from a rotating cone was recorded as an index of neuromuscular function.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were examined post mortem. Any that were killed because of poor clinical condition or at scheduled termination were not given food but had water ad libitum overnight before they were anaesthetized with sodium pentobarbitone and exsanguinated.

ORGAN WEIGHTS
- At 52 weeks and at the end of the study: adrenal glands, brain, heart, kidneys, liver, lungs and trachea, spleen and testes.

HISTOPATHOLOGY: Yes
- All tissues from the high-dose and control groups were embedded in wax, sectioned (5 µm), stained with haematoxylin and eosin, and examined microscopically. A less comprehensive selection of tissues were examined from the low-dose group.
- Tissues examined: aorta, adrenals, adipose tissue, liver (caudate, right median, and left lobe), lungs, femur (including bone marrow), brain, caecum, colon, diaphragm, ears (auditory canal and pinna, with adjacent sebaceous glands), epididymides, eyes and optic nerve, gonads, harderian gland, heart, kidneys, larynx, spleen, sternum, stomach, salivary gland (submaxillary), oesophagus (proximal to the tongue), pancreas, parathyroid, pituitary, prostate, sciatic nerve, seminal vesicle, small intestine, spinal cord (high cervical), thymus, thyroid, tongue, trachea, turbinate bones, urinary bladder, uterus, skin (particularly any showing lesions or abnormalities of hair growth) and vagina, all gross lesions lymph nodes (tracheobronchial, cervical and mesenteric).
Other examinations:
Transmission electron micrographs were prepared from the livers for five animals of each sex from the high-dose and control groups at termination.Samples were taken from the left lateral lobe, fixed in osmium, and embedded in epoxy resin. 1 µm sections were cut from the five selected control and high-dose animals of each sex, stained with 1% toluidine blue, and examined with the light microscope. Centrilobular and periportal areas were selected and ultrathin sections were prepared and stained with uranyl acetate and lead citrate. The sections were examined and photographed with a Phillips EM300 operating at 60 KV.
Statistics:
Survival data and palpable subcutaneous masses - Peto and Peto (1972, 1973), and Kaplan and Meier (1956). Lesions/tumour incidences - Peto et al (1980)
Body weights, laboratory investigations, and organ weights - analysis of variance and Student's t-test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
In second year of study statistically significant (p < 0.05 in males; p < 0.01 in females) relationship between increased mortality and the concentration of 1,3-butadiene.
Some rats in the 8000 ppm group, especially females, had wet and ruffled fur and slight limb weakness or incoordination during the first 3 hr of the first exposure day of the week following 2 days without treatment. During the remainder of the week, these signs regressed.

BODY WEIGHT AND WEIGHT GAIN
Initially body weight gains of both sexes at 8000 ppm and of males at 1000 ppm were slightly but significantly lower than the controls during the first 12 weeks of the study. By the end of the first year the mean body weights of the control and treated groups were similar.

ORGAN WEIGHTS
Higher relative liver weights in both sexes at 1000 and 8000 ppm, except high-dose females at week 52. Absolute kidney weight and the relative weight were increased in male rats exposed to 8000 ppm at 2 years,.
Higher kidney weights related to increased severity of nephrosis compared with control. Higher relative heart weight in the 8000 ppm group may have been related to blood pressure changes resulting from the developing kidney changes
There were higher relative lung and spleen weights in 8000 ppm males at 2 years.

HAEMATOLOGY
White cell counts were increased in 8000 ppm females at 52 weeks but not at 78 weeks or in males and were therefore considered not to be toxicologically significant.

EFFECTS ON THE KIDNEY
Blood urea nitrogen was increased in treated males at 13 and 26 weeks but not in females or after 52 weeks. The higher incidence of nephrosis in male rats exposed to 8000 ppm for 2 years was associated with evidence of proteinuria.

OTHER NON-NEOPLASTIC CHANGES
There was a higher incidence of focal epithelialization in the lungs of high dose males exposed for 2 years (5/45 for controls compared with 10/31 for high dose males killed at week 111).

HISTOPATHOLOGY: NEOPLASTIC
See IUCLID Section 7.7 Carcinogenicity

Effect levels

Dose descriptor:
NOAEC
Effect level:
1 000 other: ppm (2212 mg/m3)
Sex:
male/female
Basis for effect level:
other: no adverse effects on systemic toxicity at 1000 ppm (2212 mg/m3), some toxic effects (increased heart weight and kidney nephrosis) were seen at 8000 ppm (17701 mg/m3)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

% increase in relative organ weight compared to concurrent control

 

Males

Females

 Organ

1 year

2 year

1 year

2 year

1000 ppm

8000 ppm

1000 ppm

8000 ppm

1000 ppm

8000 ppm

1000 ppm

8000 ppm

No of rats

10

10

50

32

10

10

32

24

Kidneys

-

-

-

24**

16

-

-

-

Liver

5*

24**

11**

25**

-

-

18**

21**

Heart

-

-

-

16*

-

-

-

-

Lung

-

-

-

14*

-

-

-

-

Spleen

-

-

-

21*

-

-

-

(33)

- not significantly different from control

* statistically significantly different from control p≤0.05

** statistically significantly different from control p≤0.01

() value higher but difference from control not statistically significant

 

Incidence of nephropathy in male rats

Severity of nephropathy in Males

Incidence decedent/terminal

0 ppm

1000 ppm v/v

8000 ppm v/v

Examined

55/45

50/50

69/31

Normal

9/4

18/7

9/0

Minimal

17/12

12/20

10/1

Slight

17/19

11/16

25/17

Moderate

3/7

3/4

9/2

Marked

1/2

1/2

5/9

severe

6/1

5/1

11/2

A statistically significant dose related trend (p≤0.05) for "fatal" nephropathy was established.

"Fatal" was defined for the statistical treatment by the Peto method of analysis.

Applicant's summary and conclusion

Conclusions:
Male and female rats were exposed to 1,3-butadiene at 0, 1000, or 8000 ppm (2212 and 17701 mg/m3) for up to 2 years. Non-neoplastic findings were limited to increased weights of liver, kidney, heart, lung and spleen, nephrosis of the kidney and focal metaplasia in lung. A NOAEC of 1000 ppm (2212 mg/m3) for systemic toxicity was established with some toxic effects (increased heart weight and kidney nephrosis) occurring at 8000 ppm.
Executive summary:

A 2-year inhalation study was conducted in Sprague-Dawley rats with 1,3-butadiene. Groups of 110 male and 110 female rats inhaled 1,3-butadiene at 0, 1000, or 8000 ppm for 6 hr/day, 5 days/week. Interim clinical pathology, neuromuscular, and histopathology investigations were carried out. The study terminated at 20 to 25% survival (105 weeks for females, 111 weeks for males). Following exposure to 1,3-butadiene there were no effects on haematology, blood chemistry, urine analysis or neuromuscular function that definitely could be associated with treatment. Treatment-related non-neoplastic findings were associated with changes in clinical condition, suppression of body weight gain, reduced survival, and increases in certain organ weights.