Registration Dossier

Diss Factsheets

Toxicological information

Repeated dose toxicity: inhalation

Currently viewing:

Administrative data

short-term repeated dose toxicity: inhalation
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Guideline study
GLP compliance:
yes (incl. QA statement)
Limit test:

Test material

Constituent 1
Reference substance name:
Hydrocarbons, C4, ethylene-manuf.-by-product
EC Number:
EC Name:
Hydrocarbons, C4, ethylene-manuf.-by-product
Cas Number:
Hydrocarbons, C4, ethylene-manuf.-by-product
Constituent 2
Reference substance name:
C4 Crude Butadiene (low 1,3-Butadiene Content)
C4 Crude Butadiene (low 1,3-Butadiene Content)
Constituent 3
Reference substance name:
25167-67-3, 64742-83-2, 68187-60-0, 68476-44-8, 68955- 28-2, 68956-54-7
25167-67-3, 64742-83-2, 68187-60-0, 68476-44-8, 68955- 28-2, 68956-54-7
Details on test material:
- Name of test material (as cited in study report): C4 Crude Butadiene (low 1,3-Butadiene Content)
- Supplier: Texas Petrochemicals, LP
- Substance type: Chemical stream
- Physical state: vapour
- Composition of test material, percentage of components: approx. composition: 10% 1,3-butadiene, 4% isobutane, 4% n-butane, 29% trans-2-butene, 29% 1-butene, 11% isobutylene, 12% cis-2-butene
- Lot/batch No.: lot # 268193
- Storage condition of test material: Supplied in cylinders and stored at approximately 4°C in an enclosed storage building, and kept at approximately 16°C during use

Test animals

other: Crl:CD® (Sprague-Dawley) IGS BR
Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: Approximately 200-300 g (males) and 155-225 g (females)
- Housing: one per cage in stainless steel cages
- Diet: LabDiet® Certified Rodent Diet #5002 (PMI Nutrition International, St. Louis, Missouri, USA) in meal form ad libitum (except during exposures)
- Water: Municipal water ad libitum (except during exposures)
- Acclimation period: At least 1 week

- Temperature: 22 ± 3 °C
- Humidity: 40-70%
- Air changes: 12-15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 24 October 2000 To: 29 November 2000

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
other: air
Remarks on MMAD:
MMAD / GSD: no data
Details on inhalation exposure:
- Exposure apparatus: 2 m3 stainless steel and glass Rochester-style exposure chambers (1.3m tall x 1.2m wide x 1.2m deep with a pyramidal top and bottom).
- Method of holding animals in test chamber: in stainless steel cages within the exposure chamber.
- Temperature, humidity in air chamber: 21.20 ± 1.67, 21.42 ± 0.46, 20.59 ± 0.47, 22.06 ± 0.49°C for 0, 2, 10 and 20 mg/L exposure groups respectively / 46.01 ± 3.36, 53.41 ± 6.93, 51.25 ± 7.86, 39.94 ± 7.32% for 0, 2, 10 and 20 mg/L exposure groups respectively.
- Air flow rate: approximately 450 L/minute
- Air change rate: 12-15 calculated air changes per hour.

- Brief description of analytical method used: Gas chromatography
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The distribution of total hydrocarbons within the five sample locations in each chamber ranged from 91.35 to 104.94% of the mean reference value. The analytical concentrations for each chamber were 0.0, 2.17, 9.81 and 19.1 mg/L for the targeted concentrations of 0, 2, 10, and 20 mg/L, respectively.
Duration of treatment / exposure:
36-37 days
Frequency of treatment:
6 hours/day, 7 days/wk
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 2, 10, 20 mg/L
other: target concentration
Doses / Concentrations:
0.0, 2.17, 9.81, 19.1 mg/L
analytical conc.
No. of animals per sex per dose:
Control animals:
yes, sham-exposed
Details on study design:
Groups of 12 male and 12 female CD rats were exposed to vapours of the test material daily by inhalation for approximately six hours/day at exposure levels of 0, 2, 10, or 20 mg/L. The main study (repeated-exposure general toxicity and neurotoxicity endpoints) males and females were exposed for 36 and 37 days, respectively. Effects on general toxicity, neurobehavioral activity, clinical chemistry, and haematology were evaluated. In addition, a gross necropsy with extensive histopathological examination of tissues was conducted. The study also contained reproductive and developmental toxicity satellite groups (summarized separately, see "Toxicity to reproduction", section 7.8.1 and "Developmental toxicity", section 7.8.2.).
Positive control:


Observations and examinations performed and frequency:
- Time schedule: twice/day
- Cage side observations included: skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), animal behaviour, moribundity, mortality, and the availability of feed and water.

- Time schedule: At least once pre-exposure and weekly thereafter. The examination included cage-side, hand-held and open field observations that were recorded categorically or using explicitly defined scales.

FUNCTIONAL TESTS (sensory evaluation, rectal temperature, grip performance, and motor activity): Yes
- Time schedule: Pre-exposure and during final week of exposure.

- Time schedule: At least once pre-exposure, on day of exposure, weekly thereafter and at termination.

- Time schedule: Weekly throughout the study for females. In the males, feed consumption was determined on test days 1-7, 7-14, and 28-36. Determination of feed consumption for the males was discontinued during the two-week mating period due to co-housing.




- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Prothrombin time (PT), Haematocrit (Hct), Haemoglobin (HgB) concentration, Erythrocyte count (RBC), Total leukocyte count (WBC), Platelet (PLAT) count, Differential WBC leukocyte count, RBC indices (MCH, MCV, and MCHC).

- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: Yes (carbon dioxide)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Alkaline phosphatase (AP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Albumin (ALB), Creatinine (CREAT), Electrolytes (Na, K, PO4, Cl and Ca), Glucose (GLU), Total bilirubin (TBIL), Total protein (TP), Urea nitrogen (UN).

Sacrifice and pathology:
GROSS PATHOLOGY: All males and main study females were fasted overnight and necropsied after the end of the mating period (males on test day 37 and females on test day 38).

ORGAN WEIGHTS: testes, epididymides, ovaries, liver, kidneys, lungs, adrenals, thymus, spleen, brain, and heart.

HISTOPATHOLOGY: All gross lesions (from any group). The following tissues were examined from control and high dose groups: Adrenals, kidneys, prostate, rectum, larynx, liver, seminal vesicles, bone marrow, lungs, brain (cerebrum, brainstem, cerebellum), caecum, mediastinal lymph node, spinal cord (cervical, thoracic, lumbar), cervix, spleen, coagulating glands, mesenteric lymph node, stomach, colon, testes, nasal tissues, thymus, duodenum, thyroid gland, epididymides, ovaries, oviducts, trachea, urinary bladder, gross lesions, uterus, heart, peripheral nerve –tibial, vagina, ileum (with peyer’s patch), jejunum.
Adult body weights, body weight gains, feed consumption, organ weights, clinical chemistry data and appropriate haematological data were evaluated by ANOVA. Detailed clinical observation incidence scores for ranked observations and sensory evaluation scores were statistically analyzed by a z-test of proportions. Rectal temperature and grip performance were analyzed by an analysis of covariance with dose as the factor and time as the covariate. Motor activity was analyzed by a repeated-measure design with treatment as a between subjects factor and the repeated factor of time.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: There were no deaths or treatment-related clinical observations noted

BODY WEIGHT AND WEIGHT GAIN: No significant differences in body weights were observed for the males or females at any dose level tested throughout the duration of the study

FOOD CONSUMPTION: No significant differences in feed consumption were observed for the males or females at any dose level tested throughout the duration of the study

HAEMATOLOGY AND CLINICAL CHEMISTRY: There were no treatment-related changes for males and females at any dose level for prothrombin time, haematology values or clinical chemistry measures. Females exposed to 2 mg/L had a statistically identified increase in haematocrit value, and a statistically identified decrease in serum total protein. Given the lack of dose response, effects on related parameters, and similar effects in males, these were considered incidental findings that were toxicologically insignificant.

NEUROBEHAVIOUR: Sensory evaluation and fore/hindlimb grip performance data revealed no treatment-related findings. Treatment did not affect motor activity total counts (treatment-by-time interaction, p =0.0930). However, the treatment-by-time-by-epoch interaction was significant (p = 0.0098). Examination of the data suggested that this effect could be reasonably attributed to the significant time-by epoch interaction (p = 0.0001) rather than to a true treatment effect. This was confirmed following calculation of linear contrasts to determine which group(s), if any, were different from the control group. These analyses revealed that none of the three treatment groups were significantly different from control (alpha > 0.02) when the time-by-epoch-by-treatment interaction was considered.

RECTAL TEMPERATURE: There were no effects of exposure on rectal temperature

ORGAN WEIGHTS: There were no effects of exposure on organ weights

GROSS PATHOLOGY: There were no effects of exposure on gross pathology

HISTOPATHOLOGY: There were no effects of exposure on histopathology

Effect levels

Dose descriptor:
Effect level:
20 other: mg/L air (20,000 mg/m3)
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables


Applicant's summary and conclusion

Repeated inhalation exposure of C4 Crude Butadiene, to male and female rats at up to 20 mg/L produced no evidence of any adverse effects. The NOAEC for repeated dose toxicity was 20 mg/L (20,000 mg/m3), the highest concentration tested.
Executive summary:

Male and female Sprague Dawley rats were exposed to C4 Crude Butadiene at levels of 0, 2, 10, or 20 mg/L for 6 h/day, 7 days/week for up to 37 days by inhalation exposure. There was no evidence of any adverse effects on clinical observations, organ weights, gross or histopathological assessment of organs, neurobehavioural activity, clinical chemistry or haematology endpoints. Based on these data, the no-observable-adverse effect concentration (NOAEC) for repeated dose toxicity was 20 mg/L (20,000 mg/m3), the highest concentration tested.