Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
other: Draft review paper evaluatiing various reproduction/developmental studies
Adequacy of study:
weight of evidence
Study period:
Not stated
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The unpublished review article is a draft version, but reliable in that it is derived from a reliable study series.

Data source

Reference
Reference Type:
secondary source
Title:
Unnamed

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: EPA 1998 and OECD 1983 and 2001
GLP compliance:
yes
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
The test substance, DCBD (CAS Registry No. 1653-19-6) was a clear to straw colour liquid. Because of the duration of the reproductive and developmental toxicity studies and the reactive nature of the test substance, three separate production lots of DCBD (98.2-98.6% pure) were used to minimise degradation of the sample due to spontaneous polymerization or dimerization. DCBD containing polymerization inhibitors was shipped on ice and stored under nitrogen at -20°C.

Analysis of purity, stability and DCBD dimer content was performed on all lots of DCBD prior to and following the last inhalation exposure. Mass spectral identification of impurities was performed on a separate lot of DCBD that was used for the dose range-finding studies; results of this analysis were compared to chromatograms of the three lots used on this study. Inhibitor concentrations in the DCBD as shipped were below 0.1% when initially analyzed and are not reported. For perspective, based on pilot studies, the measured airborne inhibitor concentration was 1.2 ppb at a DCBD air concentration of 150 ppm.

Test animals

Species:
rat
Strain:
other: Crl:CD®(SD)IGS BR Sprague-dawley rats
Sex:
male/female
Details on test animals and environmental conditions:
Rats of the Crl:CD®(SD)IGS BR strain were obtained from Charles River Breeding Laboratories, Inc. (Raleigh, North Carolina)

For the developmental toxicity study, nulliparous, time-mated females were received from Charles River Breeding Laboratories, Inc. at either GD 1, 2, or 3, were approximately 63 days of age, and were acclimated for at least 3 days. The rats used in the reproductive toxicity study were approximately 43 days old, and were acclimated for at least 6 days; males and females were non-siblings.
All rats were housed individually except during breeding, in stainless steel, wire-mesh cages suspended above cage boards. Females in the reproductive toxicity study that mated were housed in polycarbonate pans with bedding from GD 20 until sacrifice; females that did not mate were also housed in this manner during the post-cohabitation phase until sacrifice.
Animal rooms were illuminated artificially from 6:00 AM to 6:00 PM daily; with controlled temperature (18º-26ºC) and humidity (30%-70%) levels. All rats were given PMI® Nutrition International, Certified Rodent LabDiet® 5002 pellets and tap water from United Water Delaware ad libitum.

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: conditioned room air
Details on exposure:
In the developmental toxicity study, four groups of 22 time-mated females each were exposed to DCBD at atmospheric concentrations of 0, 1, 10, or 50 ppm (whole body exposure) for 6 hours per day on GD 6-20 and sacrificed on GD 21.
In the reproductive toxicity study, four groups of rats (24/sex/concentration) were exposed to DCBD at atmospheric concentrations of 0, 1, 5, or 50 ppm (whole body exposure). Rats were exposed (6 hours/day) during premating (8 weeks; 5 days/week), cohabitation of mating pairs (up to 2 weeks, 7 days/week), postcohabitation for males and nonpregnant females (approximately 7 days, 7 days/week) and conception to implantation (GD 0-7) for a total of 10-11 weeks. Males were sacrificed at the end of the exposure period (test days 71-73). The gestation exposure period was followed by a recovery period (GD 8-21) for presumed pregnant females, which were sacrificed on GD 21.
In both studies, the control groups were exposed to conditioned air alone according to the same exposure regimen as the other groups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the purity and stability analysis, approximately 30 to 50 mg of undiluted DCBD was transferred into a 1.5-mL glass vial and mixed with methanol (1.0 – 1.5 mL). Triplicate analyses were performed by capillary gas chromatography (GC) using a HP 6890 Plus GC with flame ionization detection (FID). Samples were chromatographed using a ramped temperature and pressure program and equipped on a DB-5 column (30 m X 0.53 mm X 1.50 ¿m; J & W Scientific). Normalized area percent purity values for DCBD and the major impurities (toluene, DIB-1, and DIB-2) were based on response factors supplied by DuPont Performance Elastomers LLC. Normalized percent values were determined by applying individual response factors to each of the GC peak areas (except the solvent peak) to compensate for changes that occur in detector sensitivity for different sample components.
For dimer quantification by GC, approximately 1.0 mL of undiluted DCBD was transferred into a 20-mL glass scintillation vial to which 5.0 mL of internal standard (hexadecane; 0.02% vol.:vol. in acetone) was added. DuPont Performance Elastomers LLC supplied the individual response factors that were used for quantification of the three major dimers (Stewart, 1971). Duplicate analyses were performed by GC with FID.
Mass spectral identification was performed for all peaks > 0.1 normalized area percent. The sample of DCBD was prepared in the same manner as the sample purity analysis. A single analysis was performed by gas chromatography/mass spectroscopy (GC/MS) using a HP 6890 Plus GC operating with a ramped temperature program and equipped with a DB-5MS column (30m X 0.25mm X 1.00¿m; J & W Scientific).
Component identification GC/MS was performed using the HP Enviroquant Chemstation (version B.01.00), which searches the Wiley Library for best spectral fit. A spectral fit of 85% or better was considered to be a probable match for component identification.
Duration of treatment / exposure:
Test atmospheres were generated by low temperature vaporization. Nitrogen was swept over liquid DCBD contained in a glass flask (one per chamber) that uses partially immersed in a constant temperature, cold-water bath (approximately 16ºC and 7ºC for the 1 ppm and 5 50 ppm chambers, respectively). Vaporization at this low temperature helped to control the DCBD evaporation rate and reduce dimerization and polymerization. Chamber concentrations of DCBD were controlled by varying the nitrogen flow over the test substance. Concentrated DCBD vapour was diluted with conditioned, filtered air as it entered the exposure chamber. Nitrogen flow was metered using a mass flow controller. Test atmospheres were generated dynamically with a total airflow sufficient to achieve approximately 10 air changes per hour in the exposure chamber. Exposure chambers were operated at a slight negative pressure and test atmospheres were discharged directly into an exhaust stack.
After every 5 exposures, any DCBD remaining in the generation flask was discarded and replaced with fresh DCBD. The amount of DCBD added to the generation flasks was sufficient to generate test atmospheres for the greatest number of days while ensuring that the amount of spontaneously formed DCBD-dimers remaining in the flask was within acceptable levels (< 1000 ppm).
The 1.4 m³ cubical NYU-style exposure chambers were constructed of stainless steel and glass with a tangential feed at the chamber inlet to promote vapour mixing and uniform distribution of the test atmosphere. Homogeneous distribution of DCBD vapour for this type of exposure chamber was verified during prestudy method development.
Animals were placed in stainless steel wire mesh modules inside the exposure chamber. Animals were housed individually except during the cohabitation period, when animals were housed as mating pairs in the modules. The total body volume did not exceed 5% of the chamber volume.
Control animals:
yes, sham-exposed
Details on study design:
Developmental toxicity study:
On GD 6, females selected for the study were ranked by their GD 0 body weights and assigned to control or experimental groups (22/group) by random sampling from strata established within ranked lists. The randomization resulted in mean body weights on GD 0 that were statistically similar between groups. To the extent possible, each exposure group contained approximately the same number of rats from each breeding lot. Body weights were recorded daily. Food consumption was measured on all even-numbered gestation days, and on GD 21. Individual clinical observations were recorded once each day before the onset of the exposure period and at study termination on GD 21. During the exposure period (GD 6-20), clinical observations were recorded once in the morning before exposure and again in the afternoon. Cage-side examinations to detect moribund or dead rats and abnormal behaviour and appearance among rats were conducted at the time of loading the animals into the chamber on exposure days, immediately following each exposure, and at least once daily on non-exposure days. Corpora lutea were counted and the number was recorded for each ovary. Live fetuses were weighed, sexed, and examined for external alterations. The first live fetus and thereafter, every other fetus in each litter were decapitated and examined by dissection for visceral alterations. All externally malformed fetuses were also examined for visceral alterations.

Reproductive toxicity study:
Rats of each sex were ranked by their most recently recorded body weight and assigned to control and experimental groups (24/sex/group) by random sampling from strata established within the ranked lists. The randomization resulted mean body weights that were statistically similar between groups within a sex. Individual clinical observations, body weights and food consumption were recorded weekly, except during cohabitation (males and females) and post-cohabitation (males), when food consumption was not recorded. Cage-side examinations were performed as described for the developmental toxicity study. After approximately 8 weeks (40 exposures), on test day 56, each female was continually housed on a 1:1 basis with a randomly selected male of the same dose level in the male's cage. On the day copulation was confirmed, the female was transferred back to individual cage housing. Mating pairs were co-housed until evidence of copulation was observed (designated as GD 0), or until 2 weeks elapsed. The presence of an intravaginal or extruded copulation plug was considered evidence of copulation.
Vaginal smears were collected from all female rats in order to evaluate the estrous cycle (% days in estrus, diestrus, proestrus; cycle length). Vaginal smears were collected daily beginning 3 weeks prior to cohabitation with males, and continuing until copulation was confirmed, or the cohabitation period ended.
All parental rats were given a gross pathological examination. Reproductive organs and other selected organs, including potential target organs, were weighed and collected for all rats. The right cauda epididymis was weighed and sperm was collected for evaluation of motility and morphology.
Statistics:
The litter (i.e., the proportion of affected fetuses per litter or the litter mean) was considered the experimental unit for statistical evaluation (Haseman and Hogan, 1975). All tests were applied at the 0.05 significance level. Endpoints measured on a continuous scale (e.g. body weight) were assessed for normality (Shapiro and Wilk, 1965) and variance homogeneity (Levene, 1960). Pairwise comparison procedures including Dunnett’s test (Dunnett, 1964;1980; Tamhane, 1979) and Dunn’s test (Dunn, 1964) were used. Incidence data (pregnancy, clinical observations) were analyzed using the Cochran-Armitage test (Snedecor and Cochran, 1967). Then mean percent affected fetuses per litter was calculated for each alteration and was analyzed using the Exact Mann-Whitney test with a Bonferroni-Holm adjustment (Agresti, 1992; Mehta et al., 1984). An analysis of covariance was applied (Hsu, 1992; Milliken and Johnson, 1984) to test for the effect of exposure concentration on fetal weight while quantitatively accounting for the variance contribution of overall litter size and sex ratio. Fetal sex ratio was similarly analyzed; the potential impact of litter size on this variable was accounted for, while testing for any effect of dose.

Results and discussion

Any other information on results incl. tables

The test substance was considered stable over the duration of the studies and of acceptable purity (98.1-98.6%). The total DCBD dimer concentration in the generation flask ranged from 413-586 ppm. The mass spectral analysis identified three other components besides DCBD in the samples: 2,4,4-trimethyl-1-pentene (DIB-1), 2,4,4-trimethyl-2-pentene (DIB-2) and toluene. Quantification of these three components indicated normalized percent purity values ranging from 0.1% to 1.0%, which was considered acceptable for this study. Chamber concentrations were at the targeted levels and were consistent throughout both studies, with minimal day-to-day variability.

Development toxicity

No test substance-related maternal mortality occurred at any exposure concentration; all animals on study survived to the scheduled sacrifice. At 50 ppm, gasping and laboured breathing were observed in some rats, but these signs were transient occurring only during the first and third days of exposure, respectively. These signs were not observed when the animals were returned to the home cage following exposure, and they were not observed during subsequent exposure periods. In addition, there were no test substance-related clinical signs of toxicity observed during the non-exposure period for any exposure concentration.Maternal body weight was significantly decreased throughout gestation at 50 ppm and was primarily due to weight loss during the first few days of exposure (mean weight loss of 16.8 grams at 50 ppm compared to a mean weight gain of 10.1 grams at 0 ppm on GD 6-8) and the result of a slower rate of weight gain during the remainder of the gestation period. Net maternal weight gain (weight gain minus the weight of the uterine contents) for the entire exposure period (GD 6-21) at 50 ppm was 72% lower than the control value. Maternal weight gain was significantly lower than control on GD 6-8 at 10 ppm (48% lower than control; data not shown) but was not considered adverse because the reduction was transient, had no impact on overall weight gain and no other corroborative evidence of maternal toxicity was observed at this concentration. Daily maternal food consumption was decreased throughout gestation resulting in a significant decrease in food consumption for the entire exposure period (GD 6-21) at 50 ppm. Maternal gross post-mortem findings were limited to one dam in the control group that had bilateral, distended, renal pelvis. A statistically significant decrease in mean fetal body weight occurred at 50 ppm (6.2% lower than control mean). The number of resorptions and live fetuses, and the fetal sex ratio were comparable across groups. No test substance-related fetal alterations (malformations or variations) were observed. Any observed malformations were either not dose-related, occurred in the control group or occurred at single incidences. The number and type of fetal variations was similar for all groups, and the observed variations were either common to this strain and developmental age, present at single incidences and/or not dose-related.

Reproductive Toxicity

No test substance-related mortality occurred at any exposure concentration; all animals on study survived to the scheduled sacrifice. As noted in the developmental study, gasping and labored breathing were observed transiently at 50 ppm, but salivationand closed eyeswere also observed in some rats during the first exposure only;no other test substance-related clinical observations were observed in males or females at any concentration for the remainder of the study.

In males, overall weight gain for the exposure period (Days 1-71) at 50 ppm was 17% lower than the control group (19% lower for the premating period [days 1-50]), mainly as a result of lower weight gain during the first 2 weeks of exposure (43% and 24% lower mean weight gain than controls on days 1-8 and 8-15) and later during exposure (21% and 59% lower than controls on days 29-36 and 50-57, respectively). Weekly mean body weights at 50 ppm were lower than controls throughout the exposure period in accordance with the lower weekly body weight gains at this concentration. At 5 ppm, lower body weight gain was limited to the second week of exposure (18% lower than controls for days 8-15) and weight gain was generally comparable to controls for the rest of the exposure period. In addition, there were several instances where weekly body weights at 5 ppm were slightly but significantly lower than controls (approximately 5% lower) after the second week of exposure, which is consistent with the lower body weight gain during the second week at this concentration. The lower weekly body weights and weight gain at 5 ppm were not considered adverse since these changes were small, transient, and did not have a significant impact on final body weight or overall weight gain.

In females, overall weight gain for the premating period (days 1-50) at 50 ppm was 22% lower than the control group, mainly as a result of lower weight gain during the first 2 weeks of exposure (33% and 23% lower mean weight gain than controls on days 1-8 and 8-15, respectively), as well as intermittently later during exposure (43% lower than controls on days 22-29). Weekly mean body weights at 50 ppm were lower than controls throughout the premating period in accordance with the lower weekly body weight gains during premating at this concentration (Fig. 4). Mean body weights at 50 ppm were also lower than controls on GD 0 and 7 (Fig. 5), however, weight gain for this period (GD 0-7) was slightly higher than controls (data not shown). During the post-exposure period (GD 7-21), weekly mean body weight gains at 50 ppm were slightly higher than controls, which resulted in overall maternal weight gain and net maternal weight gain (weight gain minus the weight of the uterine contents) during gestation (GD 0-21) that was comparable to controls at this concentration (data not shown).

In males, overall food consumption for the premating period (days 1-50) at 50 ppm was 7% lower than the control group (Fig. 6), as a result of lower weekly food consumption during most of the premating period, particularly during the first week of exposure (13% lower mean food consumption than controls for days 1-8). Overall food efficiency for the premating period (days 1-50) at 50 ppm was 12% lower than controls, as a result of lower weekly food efficiency during the first 2 weeks of exposure (34% and 16% lower mean food efficiency than controls for days 1-8 and 8-15, respectively), which is consistent with the lower body weight gain for these intervals. At 5 ppm, food consumption was significantly decreased on the fifth week of exposure (7% lower than controls for days 29-36) and was slightly lower than controls (not statistically significant) for the rest of the premating period. Food efficiency was lower than controls during the second week of exposure (15% lower than controls on days 8-15), which is consistent with the lower body weight gain for this interval. The lower weekly food consumption and food efficiency at 5 ppm were not considered adverse since these changes were small, transient, and did not have a significant impact on overall food consumption or food efficiency.

In females, overall food consumption for the premating period (days 1-50) at 50 ppm was 7% lower than the control group (Fig. 6), as a result of lower weekly food consumption during most of the premating period, particularly during the second and third week of exposure (11% and 15% lower mean food consumption than controls for days 8-15 and 15-22, respectively). Overall food efficiency for the premating period (days 1-50) was 18% lower than controls at 50 ppm, as a result of lower weekly food efficiency during the first 2 weeks of exposure (33% and 14% lower mean food efficiency for days 1-8 and 8-15, respectively), which is consistent with the lower body weight gain for these intervals. There were no test substance-related effects on food consumption or food efficiency during gestation at any concentration.

The reproductive outcome was comparable across all groups and there were no external alterations in fetuses at any concentration. Estrous cycle and sperm parameters were comparable across groups.

There were no test substance-related gross observations in males or females. Mean absolute, mean relative (organ wt./body wt.), and mean relative (organ wt./brain wt.) liver and kidney weights were increased in males at 50 ppm. The 16% increase in the mean kidney wt./body wt. ratio was largely due to the 9% decrease in final body weights. Although the increase in kidney weight at 50 ppm was slight, it was considered biologically significant. Organ weight parameters in females were comparable across groups.

Minimal to mild degeneration and regeneration of the olfactory nasal epithelium and associated minimal to moderate atrophy of Bowman’s glands was observed at 50 ppm in males and females. The olfactory nasal lesion was characterized by multifocal degeneration, necrosis, and depletion of the sensory cells, enlargement of basal cells, with occasional areas of olfactory mucosal hypercellularity and degeneration of sustentacular cells. The nasal lesions, which were observed in nasal turbinate levels II, III, and IV, were morphologically similar between males and females; however, the olfactory degeneration in females was generally less severe than in males. In males, olfactory degeneration/regeneration was generally graded as mild (grade 2 of 4) and Bowman’s gland atrophy was usually graded as moderate (grade 3 of 4). The typical affected olfactory mucosa consisted of a normal sustentacular cell layer, a depleted sensory cell layer, enlargement of basal cells within the single-cell basal layer, atrophy of Bowman’s glands, and normal nerve cell bundles. Sensory cell layers that would normally be 6 to 8 nuclei thick were 3 to 4 nuclei thick. The presence of degenerate and necrotic sensory cells within the depleted sensory cell layer and enlarged basal cells suggests that olfactory cell degeneration/regeneration was ongoing and may have reached an equilibrium state. In females, olfactory degeneration/regeneration was generally graded as minimal (grade 1 of 4) and Bowman’s gland atrophy was usually graded as mild (grade 2 of 4). Although minimal sensory cell necrosis and degeneration was present, depletion of the sensory cell layer was uncommon. Sustentacular cells were normal except in scattered areas of hypercellular regeneration where they were degenerate. In some areas, olfactory epithelial cysts were formed due to both dilatation of the ducts of Bowman’s glands and involution of sustentacular cells as a result of underlying sensory cell loss. These cysts were much more common in the females than in the males. As in males, the single-cell basal layer was prominent, Bowman’s glands were atrophied, and nerve cell bundles were normal. There were no gross or microscopic findings in any other organ examined, including reproductive organs of males and females.

Applicant's summary and conclusion

Conclusions:
The NOEL for reproductive toxicity was 50 ppm. The NOEL for systemic toxicity in the reproduction study was 5 ppm based on adverse effects on body weight and food consumption parameters and nasal olfactory epithelial toxicity at 50 ppm in parental rats.

The NOEL for maternal and developmental toxicity was 10 ppm based on reduced maternal weight gain and food consumption and reduced foetal weight at 50 ppm in the developmental toxicity study.

The alleged reproductive effects (reduced sperm motility, testicular atrophy, pre- and post-implantation embryomortality) reported in two earlier inhalation exposure studies (Khechumov et al., 1972; Vardanyan et al., 1976) were not reproduced in the current comprehensive evaluation of the developmental and reproductive toxicity of DCBD. Unlike the current studies, the experimental methodology, including chamber atmosphere generation and characterization, test substance purity, stability and storage conditions, and quantification of DCBD dimer and impurities were not well described in those studies. This is especially important for reactive substances like DCBD, which can degrade if improperly stored. In the current studies, considerable effort was spent to eliminate contamination of the test atmospheres with DCBD decomposition or reaction by-products.

In the current developmental toxicity study, maternal toxicity evident as clinical signs of toxicity, body weight loss, and decreased weight gain and food consumption was observed at 50 ppm, the highest concentration tested. Developmental toxicity occurred at 50 ppm and was limited to a decrease in fetal body weight. Therefore, the no-observed-adverse-effect level (NOEL) for both maternal and developmental toxicity was 10 ppm.
In the current reproductive toxicity study, the NOEL for reproductive toxicity was 50 ppm, the highest concentration tested. The NOEL for systemic toxicity in parental rats was 5 ppm based on adverse effects on body weight and food consumption parameters, and nasal olfactory epithelial toxicity at 50 ppm.
Executive summary:

Inhalation developmental and reproductive toxicity studies were conducted with 2,3-dichloro-1,3-butadiene (DCBD), a monomer used in the production of synthetic rubber.

In the reproductive toxicity study, Crl:CD®(SD)IGS BR rats (24/sex/group) were exposed whole body by inhalation to 0, 1, 5, or 50 ppm DCBD (6 hr/day) for approximately 10-11 weeks total, through premating (8 wks; 5 days/wk), cohabitation of mating pairs (up to 2 wks, 7 days/wk), post-cohabitation for males (~7 days) and from conception to implantation (gestation days 0-7 [GD 0-7]), followed by a recovery period (GD 8-21) for presumed pregnant females. Oestrous cyclicity was evaluated during premating (last 3 wks) and cohabitation. Reproductive organs and potential target organs, sperm parameters, and GD 21 foetuses (viability, weight, external alterations) were evaluated.

In the developmental study, pregnant Crl:CD®(SD)IGS BR rats (22/group) were exposed whole body by inhalation to 0, 1, 10, or 50 ppm DCBD (6 hr/day) on GD 6-20; dams were necropsied on GD 21 (gross post-mortem only) and foetuses were evaluated (viability, weight, and external, visceral and skeletal exams).

During the in-life portion of both studies, body weight, food consumption, and clinical observation data were collected.

At 50 ppm, gasping and laboured breathing occurred in both studies during the first few exposures; body weight and food consumption parameters were affected in parental animals from both studies, but were more severely affected in the developmental study. Foetal weight was decreased in the developmental study at 50 ppm. Degeneration of the nasal olfactory epithelium was observed in the reproduction study at 50 ppm. There were no effects on reproductive function, embryo-foetal viability or foetal weight, and no increases in foetal structural alterations in either study.

The NOEL for reproductive toxicity was 50 ppm. The NOEL for systemic toxicity in the reproduction study was 5 ppm based on adverse effects on body weight and food consumption parameters and nasal olfactory epithelial toxicity at 50 ppm in parental rats.

The NOEL for maternal and developmental toxicity was 10 ppm based on reduced maternal weight gain and food consumption and reduced foetal weight at 50 ppm in the developmental toxicity study.