Gases (petroleum), light steam-cracked, butadiene conc.

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

other: reproduction and developmental screening assay

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

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other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment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-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

guideline study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Hydrocarbons, C4, ethylene-manuf.-by-product

EC Number:

270-691-3

EC Name:

Hydrocarbons, C4, ethylene-manuf.-by-product

Cas Number:

68476-52-8

IUPAC Name:

Hydrocarbons, C4, ethylene-manuf.-by-product

Constituent 2

Reference substance name:

C4 Crude Butadiene (low 1,3-Butadiene Content)

IUPAC Name:

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

IUPAC Name:

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

Species:

rat

Strain:

other: Crl:CD® (Sprague-Dawley) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- 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

ENVIRONMENTAL CONDITIONS
- 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 (if applicable):

whole body

Vehicle:

other: air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- 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.

TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatography
- Samples taken from breathing zone: yes

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: until pregnancy occurred or 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal lavage referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

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:

Two weeks prior to breeding, during breeding (up to two weeks), and continuing through day 19 of gestation. The dams were then allowed to deliver their litters, which were retained until postnatal day 4. The males were exposed for 36-37 days.

Frequency of treatment:

6 hours/day, 7 days/wk

Details on study schedule:

as above

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 2, 10, 20 mg/L
Basis:
other: target concentration

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.0, 2.17, 9.81, 19.1 mg/L
Basis:
analytical conc.
over 37 exposure days

No. of animals per sex per dose:

12

Control animals:

yes, sham-exposed

Details on study design:

none

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- 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 (males), clinical examinations were conducted on all females pre-exposure and weekly throughout the pre-breeding and breeding periods. Mated (sperm positive or plug positive) females received clinical examinations on gestation day (gd) 0, 7, 14, and 21. Females that delivered litters were subsequently evaluated on lactation day (ld) 0, 1, and 4. Females that failed to mate or failed to deliver litters were examined weekly. This examination included a careful, hand-held evaluation of the skin, fur, mucous membranes, respiration, nervous system function (including tremors and convulsions), swelling, masses, and animal behaviour.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule: at least once during the pre-exposure period and on the first day of exposure. Male body weights continued to be recorded weekly throughout the study and at termination. Females were weighed weekly during the pre-mating and mating periods. During gestation, females were weighed on days 0, 7, 14, and 20. Females that delivered litters were weighed on days 1 and 4 of lactation. Females that failed to mate or deliver a litter were not weighed during the gestation or lactation phases. Body weight gains were determined for the females on gestation days 0-7, 7-14, 14-20, 0-20, and lactation days 1-4.

FOOD CONSUMPTION: Yes
- Time schedule: Days 1-7, 7-14, and 28-36 for males (not measured during the two-week mating period due to co-housing). For females, weekly during the pre-breeding period, not measured during the mating period, following mating, gestation days 0-7, 7-14, 14-20, and on lactation days 1-4. Feed consumption was not recorded for females that failed to mate or deliver a litter.

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

A cross section through the approximate centre of both testes of control and high dose males was embedded in paraffin, sectioned at 5 μm and stained with modified periodic acid-Schiffs-hematoxylin. The presence and integrity of the 14 stages of spermatogenesis was qualitatively evaluated. Microscopic evaluation included a qualitative assessment of the relationships between spermatogonia, spermatocytes, spermatids and spermatozoa seen in cross sections of the seminiferous tubules. In addition, sections of both testes were examined for the presence of degenerative changes e.g., vacuolation of the germinal epithelium, multinucleated giant cells, a decrease in the thickness of the germinal epithelium, a preponderance of Sertoli cells, sperm stasis, inflammatory changes, mineralization, and fibrosis.

Litter observations:

Females examined for signs of parturition including signs of difficulty or unusual duration. All litters were examined as soon as possible after delivery. The lungs of any pups found dead during the initial examination were subjected to a flotation test to determine viability status at birth. The following information was recorded on each litter: the date of parturition, litter size on the day of parturition (day 0), the number of live and dead pups on postnatal days 0, 1, and 4, and the sex and the weight of each pup on postnatal days 1 and 4. Any visible physical abnormalities or behaviour changes in the neonates was recorded during the lactation period.

Postmortem examinations (parental animals):

see Repeated Dose Toxicity Section 7.5.3

Postmortem examinations (offspring):

gestation and postnatal survival indices, percent pre- and post-implantation loss

Statistics:

Adult body weights and feed consumption, maternal body weight gains, and pup body weights were analyzed by ANOVA. Gestation length, average time to mating (precoital interval) and litter size were analyzed using a nonparametric ANOVA. Pregnancy rates and mating, conception, fertility and gestation indices were analyzed by the Fisher exact probability test. Evaluation of the neonatal sex ratio was performed by the binomial distribution test. Post-implantation loss, pup survival indices, and other incidence data among neonates were analyzed using the litter as the experimental unit by a censored Wilcoxon test.

Reproductive indices:

mating, conception and fertility, time to mating, gestation length, litter size

Offspring viability indices:

as above

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

Reproductive function / performance (P0)

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

See Repeated Dose Toxicity Section 7.5.3

Effect levels (P0)

Dose descriptor:

NOAEC

Remarks:

reproductive toxicity

Effect level:

20 other: mg/L air (nominal), (20,000 mg/m3)

Basis for effect level:

other: no evidence of adverse effects on any measures of reproductive function at the highest dose tested (20 mg/L)

Remarks on result:

other: Generation not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Effect levels (F1)

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

20 other: mg/L air (nominal) (20,000 mg/m3)

Sex:

male/female

Basis for effect level:

other: Reproductive toxicity

Remarks on result:

other: Generation not specified

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

There were no deaths or treatment-related clinical observations noted. No significant differences in parental body weights, body weight gains or feed consumption were observed at any dose level tested throughout the duration of the study. The only exception to this was a statistically identified increase in feed consumption noted for the 10 mg/L satellite females during the premating period (days 7-14). However, this increase was considered spurious, as feed consumption increases were not noted during subsequent gestation and lactation periods and similar changes in feed consumption were not observed at the highest exposure level of 20 mg/L. There were no treatment-related effects at any dose level on any of the reproductive parameters evaluated in this study. These included measures of reproductive performance (mating, conception and fertility, time to mating, gestation length, litter size), offspring survival (gestation and postnatal survival indices, percent pre- and post-implantation loss), pup body weight and pup sex ratio. The only statistically identified change in any of these parameters was an increase in post-implantation loss occurring only at the low-dose. This was considered a spurious finding, given the lack of a dose response. Of the 12 females mated in each group, the number of viable litters produced was 11, 11, 11, and 12 for the 0, 2, 10 and 20 mg/L exposure level groups, respectively. External morphological alterations observed in the pups were limited to a hernia observed in a single pup from the high dose group. Given the low incidence of this finding, it was considered spurious and unrelated to exposure.

Applicant's summary and conclusion

Conclusions:

Repeated inhalation exposure of C4 Crude Butadiene, to male and female rats at levels up to 20 mg/L produced no evidence of adverse effects on any measures of reproductive function.

Executive summary:

Male and female Sprague Dawley rats were exposed to C4 Crude Butadiene by inhalation at levels of 0, 2, 10, or 20 mg/L (6h/day, 7 d/week) for two weeks prior to breeding, during breeding (up to two weeks), and continuing through day 19 of gestation. After birth pups were maintained until postnatal day 4. There were no treatment-related adverse effects on any measures of reproductive function. Based on these data, the NOAEC for reproductive toxicity was 20 mg/L (20,000 mg/m3), the highest concentration tested.