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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
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, 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:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3550
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
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: colourless liquid
- Analytical purity: 99.7% (from Certificate of Analysis)

Test animals

Species:
rat
Strain:
other: Crl:CD® (Sprague-Dawley) IGS BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina, USA
- Age/weight at study initiation: Approximately 12 weeks/342-409 grams (males) and 230-283 grams (females) at initiation of exposures (P) x 15 days. Selected F1 males and females (one male and one female from each litter) were exposed for 7 consecutive days (Postnatal days [PND] 21-27 or 28-34)
- Fasting period before study: None
- Housing: Upon receipt (F0) or selection (F1) and until pairing (F0 only), all F0 and F1 test animals were individually housed in clean, wire-mesh suspended cages. The F0 animals were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire-mesh cages until the scheduled necropsy of the parental generations, and the females were transferred to plastic maternity cages with nesting material. F1 offspring that were weaned on PND 28 for the PND 28-34 exposures were individually housed in suspended wire-mesh cages.
- Diet: Certified Rodent Lab Diet® 5002 (PMI Nutrition International, Inc.) ad libitum except during exposure
- Water: ad libitum ad libitum except during exposure
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.7-22.3°C
- Humidity: 35.4-53.4%
- Air changes: Approximately 10 per hr (animal room); at least 12 to 15 air changes per hour (chamber)
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: Feb 1, 2002 To: Apr 26, 2002

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposures were conducted in 1.0 m3 stainless steel and glass inhalation chambers; operated under dynamic conditions at a slight negative pressure
- Method of holding animals in test chamber: Individually in cages
- One chamber was dedicated for each group for the duration of the study.
- Chamber air supplied by an Inhalation facility HVAC system, conditioned by humidification (if required) and filtration through a HEPA filter and an activated charcoal bed prior to delivery to the chambers.
- System of generating particulates/aerosols: The generation of BTD gas test atmospheres involved delivery of BTD gas directly from the headspace of a single liquefied gas cylinder, followed by distribution and dilution to produce the three required target concentrations.
- Temperature, humidity, pressure in air chamber: 20-26°C, 30-70%, 33 psig
- Air flow rate: 300 mL/minute
- Treatment of exhaust air: Charcoal and HEPA filtration.

TEST ATMOSPHERE
- Brief description of analytical method used: Analyzed exposure concentrations determined approximately every 35 minutes using a gas chromatographic method. Exposure concentrations determined at least 10 times for each chamber during each 6-hour exposure
- Samples taken from breathing zone: yes
- Oxygen content was approximately 19% for all chambers.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: copulatory plug or sperm in vaginal smear, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): individually, in a plastic maternity cage with nesting material
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sample of the test article was collected from each cylinder at the beginning and end of use (during the exposure phase). The samples were analyzed by a validated gas chromatographic method. The area percent purity assessment for 1,3-butadiene and 4-vinyl-cyclohexene, (CAS no. 10-40-3; lot no. 0730CU) as a potential contaminant was determined. No other significant contaminants were observed.
Actual concentrations were 301, 1507 and 6006 ppm
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
7 days/wk
Details on study schedule:
Daily 6-hour exposures, beginning 14 days prior to initiation of the breeding period (15 exposures prior to breeding); F0 males exposed for 83-84 consecutive days; F0 females exposed through gestation day 20 and from lactation day 5 through to the day prior to euthanasia (60-70 total days; F0 females which did not deliver were exposed until one day prior to euthanasia (post-mating day 25). For the lactation exposures, the dams were removed from their litters during each daily six-hour exposure period. Selected F1 males and females (one male and one female from each litter) were exposed for 7 consecutive days (Postnatal days [PND] 21-27 or 28-34).
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 300, 1500 and 6000 ppm
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 301, 1507 and 6006 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
12
Control animals:
yes, sham-exposed
Details on study design:
Exposure levels were selected based on the results of previous studies. The exposure concentrations selected for this screening study were designed to bracket the range of linear and saturable metabolism for 1,3-butadiene (saturation of epoxybutene concentrations in blood occurs at ~1250 ppm). The control group of identical design was exposed to clean, filtered air on a comparable regimen.
Positive control:
none

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- In addition, F0 and F1 animals were observed for appearance, behavior and pharmacotoxic signs prior to exposure, during exposure (for animals visible through the chamber windows) and within one hour after completion of exposure.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weeky for both sexes. Bodyweights were also recorded at the midpoint of study week 1.
- Once evidence of mating was observed, female body weights were recorded on gestation days 0, 7, 14 and 20 and on lactation days 1, 4, 7, 14, 21 and 28.

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Female food consumption was recorded on gestation days 0, 7, 14 and 20 and lactation days 1, 4, 7, 14, 21 and 28. Food consumption was calculated and reported as g/animal/day and g/kg/day for the corresponding body weight change intervals. Food efficiency (body weight gained as a percentage of food consumed) was also calculated.
Oestrous cyclicity (parental animals):
No data
Sperm parameters (parental animals):
Parameters examined in all F0 males:
- epididymis weight, sperm motility, sperm morphology
- The left testis and epididymis from all F0 males from all exposure groups were stored frozen, homogenized and evaluated for determination of homogenization-resistant spermatid count and sperm production rate, using the method described by Blazak et al and the Hamilton-Thorne CASA system.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were weighed, killed and discarded.

PARAMETERS EXAMINED
- The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.
- Detailed physical examinations and body weights were recorded for each pup on PND 1, 4, 7, 14, 21 and 28; food consumption was not recorded for F1 pups.

GROSS EXAMINATION OF DEAD PUPS: yes
- Pups dying from PND 0-4: necropsied using a fresh dissection technique described by Stuckhardt and Poppe (1984).
- Pups that died after PND 4 but prior to weaning were given an external examination and discarded.
Postmortem examinations (parental animals):
SACRIFICE
- All surviving F0 animals killed as soon as possible after the F1 litters were weaned.

GROSS NECROPSY
- Gross necropsy consisted of examination of the external surface, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, lymph nodes, thyroid glands, mammary glands, and the thoracic, abdominal and pelvic cavities including viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
- the following F0 parental tissues and organs were collected and placed in 10% neutral buffered formalin (except as noted): Coagulating gland, testes with epididymides and vas deferens, prostate, seminal vesicles, lungs, mammary glands, uterus with oviducts and cervix, ovaries, vagina, pituitary and all gross lesions.
- The following organs from all F0 parental animals euthanized at scheduled termination and for all F0 females that failed to deliver offspring were weighed: brain, prostate, epididymides (total and cauda), seminal vesicles with coagulating glands (with accessory fluids), pituitary, testes, ovaries, uterus with oviducts and cervix.

OTHER
- Microscopic examination of the following tissues was conducted for all F0 parental animals from the control and high-exposure groups and from all parental animals dying spontaneously or euthanized in extremis or that failed to breed, conceive, or deliver offspring, if applicable: epididymides (right; caput, corpus, and cauda), ovaries, testes (right).
In addition, due to decreases in absolute and relative seminal vesicle weights observed in the 1500 and 6000 ppm groups, seminal vesicles from all males in all dose groups were examined microscopically.
Postmortem examinations (offspring):
SACRIFICE
- Pups exposed to the test article on PND 21-27 were euthanized on PND 28. Pups exposed to the test article on PND 28-34 were euthanized on PND 35.
- Pups that were stillborn or those that died between birth and PND 4, any pups considered moribund and euthanized in extremis during the lactation period were subjected to macroscopic pathological evaluation.
- All remaining pups were euthanized on PND 28. Pups were examined externally, euthanized by carbon dioxide inhalation, and discarded without macroscopic internal evaluation.
Statistics:
Parametric one-way analysis of variance (ANOVA) – bodyweight, body weight gain, food consumption, gestation length, precoital interval, number of pups born, live litter size, pup weights, organ weights (absolute and relative to final body weight), epididymal and testicular sperm numbers and sperm production rate; Chi-square test with Yates correction factor– mating and fertility indices; Kruskal Wallis with Mann-Whitney U test – sex ratios, postnatal survival, percentage of motile sperm with normal morphology.
Reproductive indices:
Reproductive performance
Offspring viability indices:
Clinical observation, body weight

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
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
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Clinical observations indicative of chromodacryorrhea, chromorhinorrhea, and salivation were observed in F0 males and females at 6000 ppm. Similar, but less severe observations were reported at 300 and 1500 ppm. The observations were not considered adverse at these lower levels because the signs were always transient and only reported during the one-hour post-exposure observations.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Persistent reductions in body weight parameters were seen in F0 and F1 males and females (see below)..

FOOD CONSUMPTION (PARENTAL ANIMALS)
Transient reductions in food consumption (week 0-1) for F0 males and females were observed.

REPRODUCTIVE PERFORMANCE
Male and female mating indices were 100% for all groups on study. Male and female fertility indices were 83.3%, 100%, 100%, and 91.7%, for the control, 300, 1500, and 6000 ppm groups, respectively.

GESTATION LENGTH
The mean gestation lengths for the control, 300, 1500, and 6000 ppm groups were 21.7, 21.8, 21.6, and 22 days, respectively.

SPERMATOGENIC ENDPOINTS
There were no test article-related effects on spermatogenesis at any exposure level tested. Testicular and epididymal sperm count data were comparable across all study groups. Mean sperm motility was 86.3%, 82.8%, 83.3%, and 81.1% for the control, 300, 1500, and 6000 ppm groups, respectively.

ORGAN WEIGHTS (PARENTAL ANIMALS)
At 6000 ppm there were increased F0 male brain weight relative to final body weight (p<0.05) and reduced F0 male seminal vesicle/coagulating gland weight relative to brain weight (p<0.05). At 1500 ppm there was reduced F0 male seminal vesicle/coagulating gland weight relative to brain weight (p<0.05). Although the reductions occurred in a dose-related manner, there were no microscopic correlates when seminal vesicles were examined and there were no functional deficits in reproductive outcome in this group. Therefore, the reductions in mean absolute and relative (to final body and to brain weight) seminal vesicle weights were attributed to biological variation.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Remarks:
Systemic toxicity
Effect level:
300 other: ppm (663 mg/m3)
Sex:
male/female
Basis for effect level:
other: Effects on body weight parameters in F0 males.
Dose descriptor:
NOAEC
Remarks:
Reproductive
Effect level:
6 000 other: ppm (13,276 mg/m3)
Sex:
male/female
Basis for effect level:
other: Highest dose level tested

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

See below

Effect levels (F1)

Dose descriptor:
NOAEC
Remarks:
Systemic toxicity
Generation:
F1
Effect level:
300 other: ppm (663 mg/m3)
Sex:
male/female
Basis for effect level:
other: Post-weaning exposure during PND 21-27 or PND 28-34

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Occasional occurrences of dried red material (perioral and perinasal regions) were observed in four 6000 ppm exposed F1 offspring (three males and one female).

Treatment- related decreases in body weights and body weight gains were observed in F0 males at 1500 and 6000 ppm. Body weight losses were observed during the first week and body weight gains were reduced over the next few weeks in a dose-related fashion. As a result of the treatment-related effects on weight changes, at 1500 ppm, mean male body weights were 1-7% lower than controls and at 6000 ppm, mean body weights were 5-8% lower than controls over the course of the study. Differences from the control group were statistically significant at 6000 ppm at study week 1 and from study week 3 to study week 8. Mean final body weights were 5% lower than controls at 6000 ppm.

There were no treatment-related effects on body weight parameters in F0 females at any dose levels or F0 males at 300ppm.

Treatment- related decreases in body weights and body weight gains were observed in F1 males and females at 1500 and 6000 ppm during the pnd 21-27 period. The reduced body weights of the males were not statistically significant but those of the females were. Body weight gains were statistically significantly reduced in both sexes. Reduced body weight gains were also evident for males and females at 1500 and 6000 ppm during the pnd 28-34 period

There were no treatment-related effects on body weight parameters in F1 males or females at 300ppm.

Applicant's summary and conclusion

Conclusions:
Exposure to 1,3-butadiene at concentrations of up to 6000 ppm (13,276 mg/m3) for two weeks prior to mating, during mating and through gestation and lactation resulted in adverse clinical signs and reductions in body weight. NOAELs for F0 parental toxicity and F1 offspring toxicity were both 300 ppm (663 mg/m3), whilst the NOAEL for reproduction was 6000 ppm (13,276 mg/m3, the highest concentration tested).
Executive summary:

Male and female rats were exposed to 1,3-butadiene by inhalation at target concentrations of 300, 1500 or 6000 ppm (663, 3319 or 13,276 mg/m3) for two weeks prior to mating, during mating and through gestation and lactation. F1 males and females were exposed for 7 days post weanling (pnd 21-27 or 28-34). At 6000 ppm adverse clinical signs were noted. At 1500 and 6000 ppm there were persistent reductions in body weight parameters in F0 and F1 males and females and transient reductions in food consumption (week 0-1) for F0 males and females. There were no effects on gonadal function, mating behavior, conception, gestation, parturition, lactation of the F0 generation, and the development of F1 offspring from conception through weaning. 1,3 -Butadiene at 300 ppm had no effects on F0 or F1 animals. The NOAEL for F0 parental systemic toxicity was 300 ppm (663 mg/m3). The NOAEL for effects on gonadal function, mating behaviour, conception, gestation, parturition, lactation of the F0 generation, and the development of F1 offspring from conception through weaning was 6000 ppm. (13276 mg/m3) The NOAEL for systemic toxicity for F1 animals following post-weaning exposure was 300 ppm (663 mg/m3).