Document Rj9XZb8kkjxq9zLm1ox4Rey0n

f EPL EXPERIMENTAL PATHOLOGY LABORATORIES, INC. HU Cfi) ROUGH DRAFT PATHOLOGY REPORT ON THE BRAINS FROM MICE, HAMSTERS, AND RATS EXPOSED TO VINYL CHLORIDE IN IBT STUDY 663-03222 i Submitted to: Chemical Manufacturers Association Washington, D. C. 20009 May 14, 1980 VC4452 EPL EXPERIMENTAL PATHOLOGY LABORATORIES, INC. PATHOLOGY REPORT ON THE BRAINS FROM MICE, HAMSTERS, AND RATS EXPOSED TO VINYL CHLORIDE IN IBT STUDY 663-03222 I. INTRODUCTION At -the request of the Chemical Manufacturers Association, Experimental Pathology Laboratories, Inc. conducted a pathology evaluation of the available brains from mice, hamsters, and rats either exposed to vinyl chloride or used as controls in Industrial Bio-Test (IBT) Study 663-03222. This inhalation toxicology study was conducted in the IBT facilities at Decatur, Illinois, and was initiated on September 10, 1973. The experimental design for this study is as follows: Group Control Tl* T2* T3* T4** Number of Animals MiceRatsHamsters MF M" F MF 100 100 100 100 100 100 100 100 _- 100 100 100 100 100 100 100 100 100# 100 100 100 100 100 100 100 100 -- Exposure Levels _i 50 ppm Vinyl Chloride 200 ppm Vinyl Chloride 2500 ppm Vinyl Chloride 2500 ppm Vinyl Chloride *No food, water, or bedding in cages during exposure **Food, water, and bedding to remain in cages during exposure #A11 rats of same sex, either sex permitted The protocol as amended called for the inhalation exposure of the above groups six hours per day, five days per week. Mice were to be exposed for nine months; rats and hamsters for twelve months. These groups were originally scheduled to be held for equal additional periods of time (nine or twelve months); but, subsequently it was agreed that VC4453 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. they would be kept for their lifetime or sacrificed if moribund. All animals were to be necropsied either at the time of death or sacrifice, and a relatively complete set of tissues was to be preserved in 10% neutral buffered formalin. These tissues were to be examined from all animals in the control, T2, and T3 groups. If exposure related lesions were detected in the organs of the T2 or T3 groups, these same organs from the T1 group were to be examined. An EPL pathologist (Dr. William Busey) and four histology/ pathology technicians traveled to IBT at Northbrook, Illinois, where the material from the study is archived. At IBT, facilities were provided for examination of the residual wet tissue, paraffin blocks, anjd microscope slides. In addition, facilities were provided for the histologic processing of brains recovered from the residual wet tissue. H. MATERIALS AND METHODS A. Residual Wet Tissue All of the bags of residual fixed wet tissue from the mice, hamsters, and rats were examined for brain material. If brain material was discovered in the bag, it was recovered, examined grossly for any alterations, and trimmed into three to five sections for histologic processing. B. Paraffin Blocks All of the paraffin blocks from the mice, hamsters, and rats were examined for the presence of embedded brain material. If brains were discovered, the blocks were set aside for microtoming and staining. -2- VC4454 EXPERIMENTAL PATHOLOGY LABORATORIES, INC C. Microscope Slides (I8T Prepared) All of the microscope slides from the mice, hamsters, and rats were examined for brains. All brain slides were examined microscopical.ly at IBT by the EPL pathologist. D. Pathology Sheets The original pathology sheets from the mice, hamsters, and rats were examined for gross descriptions of brain abnormalities. The descriptions of any brain abnormalities were recorded along with the date of death of any animal with a recorded gross or microscopic brain lesion. In addition to the gross description of any brain ab normality, the microscopic findings of the IBT pathologist were also recorded. In the animals for which brain material was recovered from either the residual wet tissue, paraffin blocks, or microscope slides, an attempt was made to verify the animal identification. This was done by comparing the animal number and date of death recorded on the pathology sheet with the animal numbers and date of death recorded on the bags of residual wet tissue and with the animal numbers embedded in the paraffin blocks or recorded on the microscope slides. EPL EXPERIMENTAL PATHOLOGY LABORATORIES, INC. The brains recovered from the residual wet tissue were processed, embedded, microtomed, and mounted on microscope slides at IBT. The paraffin blocks containing brain material, previously embedded by IBT, were microtomed and mounted on microscope slides. Duplicate slides were prepared from each paraffin block containing brain. One set of slides was left at IBT and the other set was returned to EPL, stained with hematoxylin and eosin, and evaluated by an EPL pathologist. IH. RESULTS A. Number of Recovered Brains The following table details the number of brains recovered from either the residual wet tissue or paraffin blocks previously prepared by IBT: Number of Brains Recovered from Residual Wet Tissue or IBT Paraffin Embedded Blocks Group Untreated Control T1 TZ T3' T4 Mi ce MF 54 13 8 37 12 14 _ Hamsters MF 43 84 14 10 13 6 __ Rats MF 18 20 15 13 23 11 23 17 _ 12 -4- VC4456 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. B. Pathology 1. Mice and Hamsters With the exception of focal encephalitis in Group T3 male hamster-No. 2208, no gross or microscopic alterations were seen in any of the brains examined from the mice and hamsters. 2. Rats Grossly detected changes in the brain were described at the time of necropsy by IBT in four male rats from the T3 group. These gross observations are as follows: Animal No. / Gross Observation 641 1 cm. mass located in front of brain in skull leaving a compression in brain. 664 A gelatinous-!ike focus beginning on the anterior frontal half of the brain beneath the meninges compressing the adjacent brain tissue. Mid sagittal section of the brain reveals an 8 mm white firm focus involving olfactory region and compression of adjacent cerebrum. 667 A soft tan mass was found in the right frontal region in the skull cap compressing the front quarter of the brain. Mass located between the meninges and the skull cap 1 cm. in diameter. 672 Front portion of brain was gelatinous and very soft. In addition to the above four gross brain abnormalities, the following brain with a gross abnormality was detected by EPL personnel: -5- VC4457 EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Animal No. __________ Gross Observation 649 0.2 x 0.5 tan mass attached to the left olfactory bulb. The detailed histologic findings for the microscopic evaluation of the brains are presented in Table II. A summary of the findings is presented in Table I. Microscopic evaluation of the brains from the control and vinyl chloride exposed male and female rats revealed olfactory neuro blastomas (esthesioneuroblastoma, esthesioneuroepithelioma, medulloepithelioma) in five T3 males (641, 649, 664, 667, and 672), one T3 female rat (761), and one T4 female rat (2413). A single glioblastoma multiforme was detected in the brain from T2 male rat No. 412, and a single astrocytoma was detected in the brain of T2 female rat No. 566. The histological features of the seven olfactory neuroblastomas were remarkably similar. They were characterized by a proliferation of deeply basophilic cylindrical cells with abundant cytoplasm supported by small blood vessels. Characteristic pseudo-rosettes were present along with numerous mitotic figures. In most instances these neoplasms compressed the anterior portion of the cerebrum; and, in some cases, infiltration under the meninges was evident. The glioblastoma multiforme was characterized by a proliferation of a mixture of highly pleomorphic cells containing large nuclei. Numerous giant cells and mitotic figures were also evident. The astrocytoma was characterized by a proliferation of well differentiated astrocytes infiltrating the neuropile, forming no particular structural pattern. A few mitotic figures were eivdent. -6- VC4458 EPL EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Scattered instances of meningoencephalitis, suppurative meningitis, encephalomalacia, and focal encephalitis were detected in a few rats from both the control and vinyl chloride exposed groups. The incidence of these inflammatory lesions was low and not related to any treatment group. IV. DISCUSSION AND CONCLUSION The presence of seven olfactory neuroblastomas in the brains from vinyl chloride exposed rats indicates a causal relationship between the exposure and the development of these neoplasms. Olfactory neuro blastomas rarely, if ever, occur spontaneously in the rat. Three of these neoplasms were initially diagnosed by IBT as neuroepitheliomas. Subsequent review of the slides indicates the classification of olfactory neuroblastoma to be appropriate for these neoplasms. The glioblastoma multi forme detected in T2 male rat No. 412 was also initially diagnosed as a neuroepithelioma by IBT. Subsequent evaluation of this neoplasm indicates that this lesion is more properly classified as a mixed glioma or glioblastoma multiforme. The olfactory neuroblastomas detected in these vinyl chloride exposed rats are apparently identical to the neuroblastomas reported by Maltoni in 1975, 1 and again in 1977. 2 In 1975, Maltoni reported at the New York Academy of Sciences Symposium on the toxicity of vinyl chloride- ^Carcinogenicity Bioassays of Vinyl Chloride: Current Results, Maltoni, C. and Lefemine, G., Annual New York Academy of Science, Vol. 246 (1975), 195-218. 2 Vinyl Chloride Carcinogenicity: An Experimental Model for Carcinogenesis Studies, Maltoni, C., Origins of Human Cancer, Cold Spring Harbor Conferences on Cell Proliferation, Vol 4, 1977. -7- VC445<9 EPL EXPERIMENTAL PATHOLOGY LABORATORIES, INC. poly vinyl chloride. The results of his BT1 experiment revealed seven neuroblastomas in rats exposed to 10,000 ppm of vinyl chloride, three neuroblastomas in rats exposed to 6,000 ppm, and five neuroblastomas in rats exposed to 2,500 ppm of vinyl chloride. In 1977, at the Cold Spring Harbor Conference on Cell Proliferation, he again reported the results of his BT1 experiment in addition to the results of his BT3 and BT7 experiments where neuroblastomas were induced in rats exposed to vinyl chloride at 10,000, 6,000, and 2,500 ppm. The findings of olfactory neuroblastomas in the T3 male and female rats and a T4 female rat confirms the findings of Maltoni of an oncogenic effect on the rat brain from vinyl chloride exposure. / The shortcomings of this study have been previously documented. Regardless of these, the rarity of this neurological neoplasm coupled with the fact that it has only been reported in rats exposed to known carcinogens indicates a relationship between their presence in this study and exposure to vinyl chloride. As mentioned previously, the finding of these neoplasms confirms previously reported results from vinyl chloride exposures by Maltoni. Since the exposure conditions in this study have not been confirmed, the levels of vinyl chloride inducing these neoplasms cannot be stated at this time. Similarly, because of the large number of missing brains from the control and exposed rats, the true incidence of these neoplasms in IBT Study 663-03222, resulting from vinyl chloride exposure, cannot be determined. Pathologist WMB/sfh -8- VC4460 EPL EXPERIMENTAL PATHOLOGY LABORATORIES, INC. Cti IBT STUDY 663-03222 TABLE I SUMMARY INCIDENCE TABLES VC4461 IBT Study 663-03222 Vinyl Chloride Male Mice BRAINS (NO. EXAMINED) SUMMARY INCIDENCE TABLE Untreated Control (5) T1 (13) T2 T3 (3) L (12) ' 1 .... | ( Npi'rimt'Ptrtl P.iihfilo^iv I iilioMtnncv Int 1 i | VC446? IBT Study 663-03222 Vinyl Chloride Female Mice BRAINS (NO. EXAMINED) SUMMARY INCIDENCE TABLE Untreated Control . TI (4) (8) T2 T3 (7) (14) ------------------- 1 / EPl t xpprimentiil Pathology Liborntoripv ln< -10- IBT Study 663-03222 Vinyl Chloride Male Hamsters BRAINS (NO. EXAMINED) Focal Encephalitis SUMMARY INCIDENCE TABLE Untreated Control (4) T1 (8) T2 (14) T3 (13) 1 / / t EPL f xpi'rimental Pathnlonv Laboratories, Im -11- IBT Study 663-03222 Vinyl Chloride Female Hamsters BRAINS (NO. EXAMINED) SUMMARY INCIDENCE TABLE Untreated Control (3) T1 (4) T2 (10) T3 (6) - -- EPL experimental Pathology Laboratories Im VC446>3 IBT Study 663-03222 Vinyl Chloride Male Rats SUMMARY INCIDENCE TABLE BRAINS (NO. EXAMINED) Meningoencephalitis Suppurative Meningitis Encephalomalacia Focal Encephalitis Olfactory Neuroblastoma Glioblastoma Multiforme Untreated Control (18) 1 1 Tl (15) 1 T2 (23) 1 1 T3 (23) 1 5 / EPL Experimental P.itholonv I ibor.itonf"; Iru VC4464 IBT Study 663-03222 Vinyl Chloride Female Rats SUMMARY INCIDENCE TABLE BRAINS (NO. EXAMINED) Meningoencephalitis Astrocytoma Olfactory Neuroblastoma Pituitary Carcinoma - Untreated Control Tl (30) (13) 1 T2 (11) 1 1 T3 (17) 1 T4 (12) 1 / < ipi Fxperimental Pathology laboratories. Inc VC4465