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Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-study equivalent to OECD guideline 413.
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
Reference Type:
study report
Title:
Unnamed
Year:
2006

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Principles of method if other than guideline:
See below
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of divinylbenzene and ethylstyrene
EC Number:
910-757-7
Cas Number:
N/A
Molecular formula:
Divinylbenzene: C10H10 Ethylstyrene: C10H12
IUPAC Name:
Reaction mass of divinylbenzene and ethylstyrene
Details on test material:
Divinylbenzene-HP (80% divinylbenzene with 20% ethylvinylbenzene) was obtained from Dow Chemical Company (Midland, MI) in two lots (LJ31012V18 and ND13012V23). Lot LJ31012V18 was used in the 2-week and 3-month studies, and lot ND13012V23 was used during the 2-year studies. Identity and purity analyses were conducted by the analytical chemistry laboratory, Research Triangle Institute (Research Triangle Park, NC); Chemir/Polytech Laboratories, Inc. (Maryland Heights, MO); and the study laboratory, Battelle Northwest Operations (Richland, WA).

Lots LJ31012V18 and ND13012V23, pale, strawcolored liquids with a hydrocarbon odor, were identified as divinylbenzene-HP using infrared and proton nuclear magnetic resonance (NMR) spectroscopy and gas chromatography/mass spectrometry (GC/MS). The infrared, proton NMR, and GC/MS spectra were consistent with reference and literature spectra of divinylbenzene-HP.

The purity of both lots was determined using GC with flame ionization detection (FID). For both lots, elemental analyses and moisture analyses by Karl Fischer titration were performed, and concentrations of 4-tert-butylcatechol added as a polymerization inhibitor were measured using GC, high-performance liquid chromatography (HPLC), or ultraviolet/visible (UV/Vis) spectroscopy. Polymer concentrations were measured in both lots using a UV/Vis turbidity assay.

For lot LJ31012V18, elemental analyses for carbon and hydrogen were in agreement with the theoretical values for divinylbenzene-HP. Karl Fischer titration indicated a moisture content of 87 ± 5 ppm. Polymer content and 4-tert-butylcatechol concentration were well within the specifications of <20 ppm and >600 ppm, respectively. GC/FID and GC/MS detected four major peaks that were identified as the meta- and para-isomers of divinylbenzene and ethylvinylbenzene; the percent total area of the divinylbenzene isomers was 79.3%. GC/FID and GC/MS, using different systems, detected four major peaks and two minor impurity peaks; the minor peaks had areas of approximately 0.1% of the total peak area. The percent total area of the divinylbenzene isomers was 80.2%. Measured as the sum of the meta- and para-isomers of divinylbenzene, the overall purity of lot LJ31012V18 was determined to be approximately 80%.

For lot ND13012V23, elemental analyses for carbon, hydrogen, nitrogen, and sulfur were in agreement with the theoretical values for divinylbenzene-HP. Karl Fischer titration indicated a moisture content of approximately 200 ppm. Polymer content and 4-tert-butylcatechol concentration were well within the specifications. GC/FID and GC/MS, using different systems, detected four major peaks that were identified as the meta- and para-isomers of divinylbenzene and ethylvinylbenzene; the percent total area of the divinylbenzene isomers was 81.2%. GC/FID indicated a purity exceeding 99.9% relative to a reference standard. GC/FID and GC/MS, using different systems, detected four major peaks and one minor impurity peak having an area percent of 0.13%; the retention time of this minor peak matched that of naphthalene. The percent total area of the divinylbenzene isomers was 81%. Measured as the sum of the meta- and para-isomers of divinylbenzene, the overall purity of lot ND13012V23 was determined to be approximately 81%.

The bulk chemical was stored in its original shipping containers, 5-gallon metal pails, at approximately –20° C. Periodic reanalyses of area percent purity and purity relative to a reference standard stored at –70° C were conducted during the 3-month and 2-year studies with GC/FID. Periodic reanalyses of polymer content and 4-tert-butylcatechol concentration were conducted using GC/FID and HPLC during the 3-month and 2-year studies, respectively. No degradation of the bulk chemical was detected, and polymer content and 4-tert-butylcatechol concentration remained within the specifications.

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female F344/N rats were obtained from Taconic (Germantown, NY). On receipt, the rats were 4 weeks old. Animals were quarantined for 13 or 14 days and were 6 weeks old on the first day of the studies. Feed was available ad libitum except during exposure periods; water
was available ad libitum. All animals were housed individually.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
other: unchanged (no vehicle)
Remarks on MMAD:
MMAD / GSD: None
Details on inhalation exposure:
Concentrations of 0, 25, 50, 100, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks

Preheated divinylbenzene-HP was pumped onto glass beads in a heated glass column where it was vaporized. Heated air flowed through the column and carried the vapor out of the generator. Generator output was controlled by the delivery rate of the chemical metering pump.

Buildup and decay rates for chamber vapor concentrations were determined with animals present in the chambers. At a chamber airflow rate of 15 air changes per hour, the theoretical value for the time to achieve 90% of the target concentration after the beginning of vapor generation (T90) and the time for the chamber concentration to decay to 10% of the target concentration after vapor generation was terminated (T10) was approximately 12.5 minutes. Based on experimental data, a T90 value of 12 minutes was selected for all studies.

Throughout the studies, concentration uniformity, persistence and stability of the chemical, and degradation impurities were monitored in the chambers. Chamber concentration uniformity was maintained; no degradation was observed, and no impurities other than those in the bulk chemical were observed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations of divinylbenzene-HP in the exposure chambers were monitored by an on-line gas chromatograph equipped with FID. Samples were drawn from each exposure chamber approximately every 36 minutes.
Duration of treatment / exposure:
14 weeks
Additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 23 days
Frequency of treatment:
6 hours plus T90 (12 minutes)per day, 5 days per week for 14 weeks
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 50, 100, 200, or 400 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
10 male and 10 female rats
Additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 23 days.
Control animals:
yes
Details on study design:
Groups of 10 male and 10 female rats were exposed to divinylbenzene-HP at concentrations of 0, 25, 50, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks; additional groups of 10 male and 10 female clinical pathology study rats were exposed to the same concentrations for 23 days.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
Clinical findings were recorded twice daily. Core study animals were weighed initially, on day 10 or 11, weekly thereafter, and at the
end of the studies.
Sacrifice and pathology:
Animals were anesthetized with carbon dioxide, and blood was collected from the retroorbital sinus of clinical pathology rats on days 3 and 23 and from core study rats at study termination for hematology and clinical chemistry analyses. Packed cell volume; hemoglobin concentration;
erythrocyte, platelet, and leukocyte counts; mean cell volume; mean cell hemoglobin; and mean cell hemoglobin concentration were determined. Manual hematocrit values were determined. A Miller disc was used to determine reticulocyte counts from blood smears. Hematology: automated and manual hematocrit; hemoglobin concentration; erythrocyte, reticulocyte, and platelet counts; erythrocyte and platelet morphology; mean cell volume; mean cell hemoglobin; mean cell hemoglobin concentration; and leukocyte count and differentials. Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, globulin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and total bile acids.

Necropsies were performed on all core study animals. Organs weighed were heart, right kidney, liver, lung, right testis, and thymus.

Complete histopathology was performed on 0 and 400 ppm rats. In addition to gross lesions and tissue masses, the following tissues were examined: adrenal gland, bone, brain, clitoral gland, esophagus, eye, gallbladder (mice only), heart and aorta, large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung and mainstem bronchi, lymph nodes (bronchial, mandibular, mediastinal,
mesenteric), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, seminal vesicle, skin, spleen, stomach (forestomach and glandular), testis with epididymis and seminal vesicles, thymus, thyroid gland, trachea, urinary bladder, and uterus. The lung and nose were examined in all remaining groups of rats, and other tissues in rats were examined to a no-effect level.
Other examinations:
At the end of the studies, sperm samples were collected from male rats in the 0, 100, 200, and 400 ppm groups for sperm motility evaluations. The following parameters were evaluated: spermatid heads per testis and per gram testis, spermatid counts, and epididymal spermatozoal motility and concentration. The left cauda, left epididymis, and left testis were weighed. Vaginal samples were collected for up to 12 days during the last 2 weeks of the study from female rats in the 0, 100, 200, and 400 ppm groups for vaginal cytology evaluations. The percentage of time spent in the various estrous cycle stages and estrous cycle length were evaluated.
Statistics:
None

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
All rats survived to the end of the study. There were no biologically significant changes in body weight in either sex. Nasal/eye discharge was noted in 400 ppm males and 100 ppm females. Kidney and liver weights of exposed groups of males and of 400 ppm females were generally greater than those of the chamber controls. In addition, the relative weights of the heart and testis were significantly increased in 200 and 400 ppm males. Incidences of degeneration of the olfactory epithelium in 200 and 400 ppm rats and basal cell hyperplasia of the olfactory epithelium in rats exposed to 100 ppm or greater were significantly increased.

Effect levels

open allclose all
Dose descriptor:
NOEC
Effect level:
50 ppm
Sex:
male/female
Basis for effect level:
other: Overall effects
Dose descriptor:
NOAEC
Effect level:
100 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
All rats survived to the end of the study. There were no biologically significant changes in body weight in either sex. Nasal/eye discharge was noted in 400 ppm males and 100 ppm females. Kidney and liver weights of exposed groups of males and of 400 ppm females were generally greater than those of the chamber controls. In addition, the relative weights of the heart and testis were significantly increased in 200 and 400 ppm males. Incidences of degeneration of the olfactory epithelium in 200 and 400 ppm rats and basal cell hyperplasia of the olfactory epithelium in rats exposed to 100 ppm or greater were significantly increased. However, the nasal effects observed in rats after inhalation exposure to DVB-HP are not considered relevant for human risk assessment. Therefore, a NOEAC of 100 ppm will be taken forward for the risk characterization.
Executive summary:

Groups of 10 male and 10 female rats were exposed to divinylbenzene-HP at concentrations of 0, 25, 50, 100, 200, or 400 ppm for 6 hours plus T90 (12 minutes) per day, 5 days per week for 14 weeks. All rats survived to the end of the study. There were no biologically significant changes in body weight in either sex. Nasal/eye discharge was noted in 400 ppm males and 100 ppm females. Kidney and liver weights of exposed groups of males and of 400 ppm females were generally greater than those of the chamber controls. In addition, the relative weights of the heart and testis were significantly increased in 200 and 400 ppm males. Incidences of degeneration of the olfactory epithelium in 200 and 400 ppm rats and basal cell hyperplasia of the olfactory epithelium in rats exposed to 100 ppm or greater were significantly increased.