Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 July 2003 to 14 August 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline compliant study full reported with no deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
The potential maternal and developmental toxicity of the test substance was evaluated in rats. The test substance was administered by inhalation (whole body exposure) to pregnant rats for approximately 6 hours per day from around the time of implantation to the end of gestation, i.e., days 6-20 of gestation (days 6-20G).
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2,3-Dichloro-1,3-butadiene
- Physical state: Clear to straw-yellow liquid
- Analytical purity: 98.2%
- Impurities (identity and concentrations): 2,4,4-trimethyl-1-pentene or DIB-1 (0.86%); 2,4,4-trimethyl-2-pentene or DIB-2 (0.20%); Toluene (0.58%)
- Lot/batch No.: Haskell number 25953
- Expiration date of the lot/batch:
- Stability under test conditions: The test substance is inherently reactive and may spontaneously polymerize (unless inhibited) or dimerize. Storage conditions were chosen to minimize sample degradation. Based on purity analyses performed prior to and immediately after study conduct, no evidence for instability of the test substance was detected. Analysis of the liquid test substance however showed the concentrations of mixed dichlorobutadiene-based dimers had increased with time but were less than 1,000 ppm, the maximal level considered acceptable for this study
- Storage condition of test material: see stability above

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR rat
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, North Carolina. Crl:CD®(SD)IGS BR rat
- Age at study initiation: 63 days at time of arrival (at GD 1, 2 or 3), treatment began on GD 6
- Weight at study initiation: 201-225g on arrival
- Fasting period before study: not applicable
- Housing: 86 time-mated females were individually housed in suspended wire-mesh stainless steel cages
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 - provided ad libitum except during exposure period
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 3-4 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26°C - target range was 22-24°C
- Humidity (%): 30-70% RH - target range was 40-60% RH
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12 0600 to 1800h


IN-LIFE DATES: From: 27 July 2003 To: 14 August 2003

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: conditioned room air
Details on exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: exposure chambers were constructed of stainless steel and glass with a nominal internal volume of 1.4 m³.
A tangential feed at the chamber inlet promoted uniform chamber distribution of the test atmosphere.
- Method of holding animals in test chamber: stainless steel wire mesh cages individually housing each animal and allowing whole body exposure
- Source and rate of air: The test vapour (saturated nitrogen) was diluted by conditioned, filtered air as it entered the exposure chamber.

- System of generating particulates/aerosols:Chamber atmospheres were generated by passing nitrogen over the liquid test substance.
Nitrogen flows were metered with a Brooks MF Series mass flow controller for the 1 ppm chamber and with a Brooks model 5850E mass flow controller for the 0, 10, and 50 ppm chambers. The test substance was contained within a glass container (one per test chamber) and each glass container was partially immersed in a low-temperature water bath to maintain a constant temperature and to control the evaporation rate. The temperature of the bath was maintained at approximately –8 to –7ºC for the 10 and 50 ppm chambers and approximately –19 to -14ºC for the 1 ppm chamber. The test vapour was diluted by conditioned, filtered air as it entered the exposure chamber.
- Temperature, humidity, pressure in air chamber:
- Air flow rate: . Nitrogen flows ranged from 0.12-0.23, 0.45-0.73, or 2.0-3.3 L/min for the 1, 10, or 50 ppm chambers, respectively.
Nitrogen flow for the control chamber was maintained at 0.8 L/min.
- Air change rate: 10 air changes per hour
- Treatment of exhaust air: Test atmospheres were discharged directly into an exhaust stack


TEST ATMOSPHERE
- Brief description of analytical method used: Purity/stability analysis and dimer quantification of H-25953 were performed by capillary gas chromatography.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance, DCBD, is a reactive liquid and may spontaneously polymerize (unless inhibited) or dimerize. To minimize sample degradation, polymerization inhibitors were added to the test substance prior to shipping, and the test substance was shipped on ice. Upon receipt, the test substance was stored under nitrogen in a freezer maintained at approximately -20°C except when used to generate test atmospheres and when being analyzed.
Purity/stability analysis and dimer quantification of H-25953 were performed by capillary gas chromatography. Mass spectral identification was performed by Gas Chromatography/Mass Spectroscopy (GC/MS) for all peaks > 0.1% normalized area percent.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant
Duration of treatment / exposure:
Each group of rats was exposed 6 hours/day, over a 15-day period.
Chamber atmospheres were generated by passing nitrogen over the liquid test substance. Nitrogen flows were metered with a Brooks MF Series mass flow controller for the 1 ppm chamber and with a Brooks model 5850E mass flow controller for the 0, 10, and 50 ppm chambers. A separate mass flow controller was used for each chamber and a Brooks model 0154/0154E power supply indicator (with digital display) was used to regulate the mass flow controllers. Nitrogen flows ranged from 0.12-0.23, 0.45-0.73, or 2.0-3.3 L/min for the 1, 10, or 50 ppm chambers, respectively. Nitrogen flow for the control chamber was maintained at 0.8 L/min. The test substance was contained within a glass container (one per test chamber) and each glass container was partially immersed in a low-temperature water bath to maintain a constant temperature and to control the evaporation rate. The temperature of the bath was maintained at approximately –8 to –7ºC for the 10 and 50 ppm chambers and approximately –19 to -14ºC for the 1 ppm chamber. The test vapour was diluted by conditioned, filtered air as it entered the exposure chamber. Test atmospheres were generated dynamically using airflows needed to achieve at least 10 air changes per hour in the exposure chamber. Chamber concentrations of test substance were primarily controlled by varying the nitrogen flow over the test substance.
Test atmospheres were discharged directly into an exhaust stack.

Frequency of treatment:
Daily
Duration of test:
15 days
Doses / concentrations
Remarks:
Doses / Concentrations:
1, 10 and 50 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
22 pregnant females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
An inhalation pilot developmental study with DCBD was conducted in time-mated pregnant female rats at targeted exposure concentrations of 0, 10, 25, and 50 ppm for 6 hours per day during gestation days 6-20. (1) Initial exposure levels on gestation day 6 were 0, 50, 100, and 150 ppm.
However, due to clinical signs of toxicity during exposure (lethargy, partially closed eyes, salivation) and weight loss at every exposure concentration on gestation day 6, the exposure concentrations for the remaining exposures in the 100 and 150 ppm groups were adjusted to 10 and 25 ppm for the groups formerly exposed to 100 and 150 ppm, respectively, beginning on gestation day 7. No clinical signs of toxicity were observed subsequently. Mean daily body weight gain and food consumption for maternal rats over the interval of gestation days 6-21 were significantly reduced at all exposure concentrations. Although some evidence of recovery was observed in the animals exposed to 10 and 25 ppm, their body weight and body weight gain values continued to be less than the control values by gestation day 21. No mortality or test substance-related gross post-mortem changes were observed in the maternal rats for any exposure concentration. Fetal body weight values for the 10, 25, and 50 ppm groups were significantly reduced (13%, 7%, and 7% respectively). There were no test substance-related effects on resorptions or live fetuses, nor were there any test substance-related external alterations observed in fetuses from any exposure concentration. Based on these data, 50 ppm was selected as the high concentration for this main study since maternal and fetal toxicity were expected to be observed. The intermediate exposure concentration selected for the main study was 10 ppm. Although effects observed at 10 ppm in the pilot study may have been related to the first exposure day when they were exposed to 100 ppm, it was expected that an exposure concentration of 10 ppm for the main study would produce minimal or no maternal and/or fetal toxicity. The low exposure concentration selected for the main study was 1 ppm, since it was likely that maternal or fetal toxicity would not occur at this concentration.

- Rationale for animal assignment (if not random): Upon arrival, dams were assigned to lots according to their gestation day. Rats were ranked within their respective lots on the basis of GD0 body weights and assigned to control and experimental groups by random sampling from the ranked list.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily from arrival to end of study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily observations from GD 6 to GD 20. Animals were assessed three times during exposure and immediately post exposure

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from arrival to termination of study

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Measured on gestation days 4, 6, 8, 10, 12, 14, 16, 18, 20 and 21

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21 - surviving females were examined at post-mortem
- Organs examined: Intact and empty uterus, corpora lutea counts, implantation sites
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: relative positions of different types of implants were recorded
Fetal examinations:
- External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [ half per litter ]
- Skeletal examinations: Yes: [all live foetuses per litter ]
- Head examinations: Yes: [half per litter ]
Statistics:
Descriptive statistics including mean, standard deviation, and standard error of the mean were used to summarise exposure concentration, for all of the parameters listed below.
For litter parameters, the proportion of affected foetuses per litter or the litter mean was used as the experimental unit for statistical evaluation. For each parameter analysed with a trend test, the test was applied to the data sequentially. If a significant dose-response was detected, data from the top dose group were excluded and the test repeated until no further trend was detected. The level of significance selected was p < 0.05.
Levene's Test for homogeneity and Shipiro-Wilks test for normality; Dunnett's Test; Kruskall-Walis test and Dunn's test:
Maternal Weight, Maternal Weight Change, Maternal Food Consumption, Live Foetuses, Dead Foetuses, Resorptions, Implantations.

Cochran-Armitage Test:
Incidence of Pregnancy, Clinical Observations, Maternal Mortality, Females with Total Resorptions, Early Deliveries.
Exact Mann-Whitney with a Bonferroni-Holm adjustment - Incidence of foetal alterations
Analysis ofCovariance; Dunnett-Hsu: Fetal weight (Covariates: litter size, sex ratio); Sex ratio (Covariate: litter size)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
There were no maternal mortalities at any of the test concentrations. Clinical signs of toxicity were evident at the high dose, 50 ppm, after the first and third exposure but gasping respiration was not evident following exposure or after later exposures.
Effects were noted in the high dose group -reduced body weight and lower weight gains over GD 7-12, weights at termination, corrected for gravid uterus, were 11.5% lower than controls. Large weight losses were recorded in the high dose group in the first few days of treatment and recovery of weight gain was slow over the remaining exposure period. No adverse effects on bodyweight were recorded in the 10 or 1 ppm groups.
There were no treatment related effects on food consumption in the 10 or 1 ppm groups but in the 50 ppm group, reduced food consumption was consistent with lower weight gains in the early stages of gestation. There were no macroscopic abnormalities resulting from treatment noted during necropsy of the dams.
Reproductive effects included a significant reduction in mean foetal weight in the high dose group but no effects in the 10 or 1 ppm groups. The number of resorptions per litter, litter size, and foetal sex ratio were unaffected by exposure to 50, 10 or 1 ppm DCBD.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEC
Effect level:
>= 10 ppm
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
>= 10 ppm
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No test substance related foetal malformations were apparent at any dose level. Malformations noted in the control group included a malformed paw (clubbed and digital soft tissue fusion) for one foetus and a bent pelvis for a second control rat). Malformations observed in the 10 ppm and 50 ppm groups (Three foetuses in the 10 ppm group had malformations including filamentous tail, cleft lip, micrognathia, imperforate anus, and microphthalmia. One foetus in the 50 ppm group had absent vertebra) were not attributed to an effect of treatment since there was no apparent dose relationship.
No treatment related foetal variations were observed in any the dose groups.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Analytical measurements of purity, stability and dimer concentrations confirmed the suitability of the test material for use in inhalation exposure. The analysed chamber concentrations were virtually indistinguishable from nominal values. The analytically determined overall mean concentrations ± S.E.M. of DCBD in the exposure chambers targeted to 1, 10, or 50 ppm were 1.0 ± 0.0072, 10 ± 0.050, or 50 ± 0.070, respectively. The daily mean chamber concentrations for 6 hours of exposure were consistent throughout the study.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, maternal toxicity occurred at an exposure concentration of 50 ppm based on decreased maternal body weight, weight gain, food consumption, and clinical signs of toxicity during exposure. Developmental toxicity occurred at 50 ppm, evident as decreased foetal weight. Therefore, the maternal and foetal no-observed-adverse-effect levels (NOAEL) were both considered to be 10 ppm.
Executive summary:

Groups of 22 time-mated pregnant female Crl:CD®(SD)IGS BR rats were exposed (whole body) to the test substance at atmospheric concentrations of 1, 10, or 50 ppm (whole body exposure) for 6 hours per day during gestation days 6-20 (days 6-20G). Control group females were exposed to conditioned air alone according to the same exposure regimen. During the in-life portion of the study, maternal body weights, food consumption, and clinical signs data were collected. On day 21G, dams were euthanized and subjected to a gross external and internal examination. Uterine contents were described; all fetuses were removed and individually weighed, sexed, and examined for external alterations. Approximately one-half of the fetuses were subjected to visceral and head evaluations; all fetuses were examined for skeletal alterations. There was no test substance-related maternal mortality, nor were there any test substance-related maternal gross post-mortem findings. Maternal toxicity was observed at 50 ppm which was characterized by statistically significant, test substance-related reductions in maternal body weight, weight gain, and food consumption. Gasping was observed during the first exposure period in 50 ppm females, and laboured breathing was observed during the third exposure period for 50 ppm females. Subsequently, there were no test substance-related clinical signs of toxicity either during exposure or following the daily exposure periods. Fetuses in the 50 ppm group had a test substance-related reduction (6.2% lower than the control mean) in mean fetal weight. There were no test substance-related malformations or variations observed at any exposure concentration. Fetal viability, resorptions, sex ratio, and litter size were comparable across all groups. Under the conditions of the study, maternal toxicity occurred at an exposure concentration of 50 ppm based on decreased maternal body weight, weight gain, food consumption, and clinical signs of toxicity during exposure. Developmental toxicity occurred at 50 ppm, evident as decreased fetal weight. Therefore, the maternal and fetal no-observed-effect levels (NOEL)a were both considered to be 10 ppm.