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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1976
Report Date:
1976

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The study was conducted before the time of adoption of OECD Test Guidelines on carcinogenicity and combined chronic toxicity/carcinogenicity.
6-month exposure period of 120 rats per dose level, with consecutive observation of most of the animals for their whole lifespan (maximum 30 months), and pathological as well as histopathological examination of organs after necropsy
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
The test sample of vinylbenzyl chloride was 98.49% pure with 0.14% of vinyl toluene, 0.20% of alpha chlorinated vinyl toluene, 0.47% of beta chlorinated vinyl toluene, 0.23% of dichloroethyl toluene and 0.24% of unknown a impurities.
The sample contained 67.91% meta- and 30.58% para-isomers of vinylbenzyl chloride.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rats were bred by Spartan Research Animals, Haslett, Michigan, U.S.A.
Sex:
male

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Details on inhalation exposure:
Exposures were conducted in 3.7 m3 stainless steel chambers under dynamic airflow conditions. The exposure atmosphere in each chamber was generated by metering liquid vinylbenzyl chloride into a heated glass vaporizer at a calculated rate. The vapors emerging from the vaporizer were diluted with room air at a rate to provide the desired test material concentration. The nominal concentration in the chamber atmosphere was the ratio of the rate of vinylbenzyl chloride delivery to the rate of total chamber airflow (the volume of air ejected from the vaporizer plus the volume of make-up air).
Analytical verification of doses or concentrations:
yes
Remarks:
by gas chromatography, two times on 124 or 125 days
Details on analytical verification of doses or concentrations:
20 L of air from the exposure chamber were drawn at the rate of 1 L/min through 25 mL of cold methanol in a fritted bottom bubbler. The vinylbenzyl chloride (VBC) dissolved in methanol was extracted with 10 mL of carbon disulfide. The quantity of VBC in a 2 µL sample of VBC-CS2 solution was analyzed by means of a gas chromatographic technique. The concentration of VBC in each sample was derived by interpolation from a curve prepared with standard VBC solutions in CS2.

There were some day-to-day variations in chamber VBC concentration. However, 80-90% of the total exposure days were within +50% of the designated levels. There was no overlapping of concentration between the high and low range throughout the exposure period.
Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
5 days per week (months 1-6 of study)
Doses / concentrationsopen allclose all
Dose / conc.:
0.1 ppm (nominal)
Dose / conc.:
1 ppm (nominal)
No. of animals per sex per dose:
120
Control animals:
yes, concurrent vehicle
Details on study design:
In total, animals were exposed on 131 days in 6 months. Food and water were removed from all animals during the exposure period. Even though controls were not placed in chambers, food and water were withdrawn during the exposure period. After the 6 month exposure period, the rats were kept under ambient conditions and observed for the duration of their lifespan (maximum of 30 months).

Examinations

Observations and examinations performed and frequency:
Animals were observed throughout the study for changes and demeanor. Body weights were recorded once a week for 3 months of the study and monthly thereafter.
Sacrifice and pathology:
Pathological examination of rats after the exposure period of six months
Rats killed for pulmonary exfoliative cytologic evaluation and for cytogenetic evaluation of bone marrow cells (see below) were also used for pathological evaluation as described in this section.

Each rat was subjected to a complete gross examination. The heart, brain, liver, kidneys, and testes were removed from each rat and weighed. Organ to body weight ratios were calculated. The trachea and lungs were removed as a unit and fixed by intratracheal infusion of 10% formalin. Fixation with formalin occurred after washing of the tracheobronchial tree with saline in animals used for pulmonary exfoliative cytologic examination. Representative portions of thoracic lymph nodes, thymus, trachea, thyroid, parathyroid, heart, liver, kidney, testes, adrenal, accessory sex glands, spleen, pancreas, spinal cord, brain, eye, stomach, small intestine, large intestine, olfactory epithelium,
urinary bladder, accessory sex glands, external nasal mucosa, pituitary gland and all gross nodules were routinely preserved in 10% formalin. Following fixation, standard histologic procedures or decalcification and tissue processing were used to prepare hematoxylin and eosin stained sections for histologic examination.

Hematology from sacrificed rats
From the rats killed on day 1 and day 5 of the post-exposure period (week 27 of the study), blood samples were collected from the cervical blood vessels at time of decapitation into heparinized test tubes to conduct hematologic determinations on these blood samples.

Pulmonary Exfoliative Cytology
Four rats from each group were subjected to pulmonary exfoliative cytologic examination on day 1 of the post-exposure period. Following deprivation of food overnight, each of these rats was anesthetized with methoxyflurane; the trachea was exposed prior to clamping with a hemostat; and the trachea and lungs were removed as a unit following decapitation. A saline wash was used to collect cells from the tracheobronchial system. Cells in the saline washings of the tracheobronchial system were sedimented by centrifugation and then resuspended in 0.5 mL of the supernatant. Two drops of the cell suspension were spread on glass slides and fixed by application of an aerosol hair spray. After drying, a fixed smear from each rat was stained with Giemsa's stain and another was stained by the Papanicolaou method. The prepared slides were examined microscopically using the oil immersion objective. The cell population on slides from each rat was evaluated for atypical or neoplastic cells and for altered cellular distribution resulting from inflammatory or other disease processes. In addition, a representative number (100) of cells on the Papanicolaou stained smear was classified according to morphological type.

Cytogenetic evaluation of bone marrow cells (for details, see genetic toxicity endpoint)
On day 5 of the post-exposure period 4 rats from each group were submitted for cytogenetic evaluation of bone marrow cells.

Examination of rats held for life-time observation
Remaining rats were held for the duration of their natural lifespan subsequent to the completion of the six month period of exposure. All rats dying spontaneously, as well as all rats killed in a moribund condition were subjected to necropsy examinations, with tissues preserved in 10% buffered formalin fixative. The lungs were routinely distended with formalin fixative. Unless contraindicated by the presence of advanced autolysis, sections of the following tissues were routinely preserved in the fixative:
multiple lobes of lung, thoracic lymph nodes, thymus, trachea, thyroid, parathyroid, heart, liver, kidney, testes, accessory sex glands, urinary bladder, brain, stomach, small intestine, large intestine, mesenteric lymph nodes, pituitary gland, pancreas, adrenals, salivary gland, spleen, eyes and any abnormal tissue masses or other lesions.
The maxilla and adjacent tissues were routinely stripped of soft tissue and split down the mid-line to allow inspection of nasal turbinates (and ear canals) prior to preservation in formalin fixative. Following fixation, standard histologic procedures (including decalcification of nasal turbinates) were used to prepare hematoxylin and eosin stained sections from all rats for histologic examination.
Other examinations:
Hematology
During week 12 of the exposure period non-heparinized blood samples were obtained from the tail veins of 10 rats from each of the control and the 1.0 ppm exposure groups. These blood samples were used in determination of the packed cell volume (PCV), total red blood cell count (RBC), hemoglobin (Hb), total and differential white blood cell count (WBC). During weeks 52 and 104 of the study non-heparinized blood samples were obtained from five rats/exposure level for evaluation of the same hematologic parameters.

Statistics:
Statistical analysis of hematological parameters, body weights and organ weights were carried out using an analysis of variance and Dunnett's Test. The mortality data and tumor incidence were analyzed with Fisher's Exact Probability Test. The level of significance in all statistical analysis was p < 0.05.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
During the exposures, the rats exposed to 1 ppm vinylbenzyl chloride exhibited slight transient eye and nasal irritation.. The signs of irritation disappeared soon after termination of exposure to vinylbenzyl chloride each day.

Mortality:
mortality observed, non-treatment-related
Description (incidence):
All groups of rats survived the 6 month exposure period with very few deaths. The incidence of mortality as a function of time was not significantly different in rats exposed to vinylbenzyl chloride vapors compared with control rats.

Details on the mortality in the post-exposure observation period are given in the section 'histopathological findings, non-neoplastic'.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The monthly mean body weights of the survivors or the treated animals were not significantly different from those of the control.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean hematologic values of 10 male rats during week 12 of exposure to 1.0 ppm vinylbenzyl chloride (VBC) were comparable to controls. Hematologic values on 4 rats/group on day 1 post-exposure and day 5 post-exposure revealed no alterations considered related to VBC exposure. In determinations done on post-exposure day 1, the total RBC counts of rats in both treatment groups were significantly higher than the control values but were well within the expected range for rats of this age, sex and strain. The total WBC counts of all rats appeared to be lower on both post-exposure day 1 and day 5 due to the collection of the blood sample into a test tube containing excess heparin at time of decapitation. The reversed neutrophilic-lymphocytic ratio noted in the WBC differential on post-exposure day 5 was due to the administration of colchicine which caused mitotic arrestment of the lymphocytic elements of the body. Hematologic examination of 5 rats/group at 52 and 104 weeks of the study revealed no differences from the controls.
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no alteration in the weights of the total body, liver, kidneys, brain, heart or testes of the treated rats.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Week 1 post-exposure:
The rats, which were examined on day 1 or day 5 post-exposure or later, revealed no gross pathological lesions considered related to vinylbenzyl chloride exposure.

Duration of study (the incidence of tumors is disscussed separately for the carcinogenicity endpoint):
Respiratory System:
During the latter stages of the 6 month period of exposure, pathologic examination of animals found dead or moribund revealed lesions indicative of a respiratory infection in those groups of rats placed in the chambers for exposure to 1.0 or 0.1 ppm of vinylbenzyl chloride. This was grossly evident as variable degrees of pulmonary congestion and consolidation, and sometimes pleural adhesions, hydrothorax and nasal exudate.
Neuropathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Various lesions usually inflammatory, degenerative or tumorous in nature occurred in a few isolated rats, with no evidence to indicate a treatment-related effect.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Respiratory system:
Microscopic examination of hematoxylin and eosin stained paraffin sections disclosed increased numbers of inflammatory cells in the mucosa and submucosa of the nasal turbinates from 2 of 4 rats exposed to 1.0 ppm vinylbenzyl chloride (VBC) and killed on post-exposure day 1. On this basis, these inflammatory changes appeared to be related to treatment. However, as similar lesions were not observed in comparable rats killed on post-exposure day 5, these inflammatory changes should be considered as reversible in nature.

Animals found dead or moribund during exposure showed characteristic findings of a bronchopneumonia, typically caseopurulent to necrotic in type, sometimes accompanied by fibronecrotic pleuritis, inflammation of the thoracic lymph nodes or purulent pericarditis/myocarditis. The bacterium Corynebacterium Kutscheri was repeatedly cultured from these cases of pneumonia. During the initial 6-month period, this infection was noted in 2 rats from each of the groups being exposed to 1.0 of 0.1 ppm VBC. The control group of rats, which at the time were being held in an adjacent holding room rather than being placed in comparable exposure chambers, did not have any deaths due to this bacterial respiratory infection during this initial 6-month time period.

During months 6-12 of the study (initial 6 months of post-exposure observation period) there was only one additional death in a rat of the 1.0 ppm group, due to this bacterial pneumonia. During months 13-18 (second 6-month post-observation period), the bacterial pneumonia was noted in 6 rats of the 1.0 ppm group, 7 rats of the 0.1 ppm group and 3 rats of the control group. At this time period, there were a few cases in which the bacterial infection had disseminated to other organs, such as liver and kidney where similar inflammatory reactions were noted adjacent to the bacterial organisms. During months 19-24 (third 6-month time period of post-exposure observation) 22, 10 and 7 rats died with lesions of a bacterial pneumonia in the groups of rats exposed to 1.0, 0.1 and 0 ppm of VBC, respectively.

From month 25 to the end of the observation period, there were 2, 1, and 0 cases of bacterial pneumonia in rats exposed to 1.0, 0.1 or 0 ppm of VBC, respectively. It is assumed that the higher incidence of bacterial pneumonia in the groups exposed to VBC vapors was more likely associated with the physical stress of the exposure regimen than with the effect of VBC upon pulmonary tissues.

In addition to the respiratory lesions associated with the bacterial pneumonia, a wide range of inflammatory and/or degenerative changes of the respiratory system occurred in a scattered pattern among the control and exposed rats; none of these changes appeared to be related to exposure to VBC. In this group, the most frequent gross observations were circumscribed dark-red or pale foci which histologically were associated with small inflammatory reactions and alveolar histiocytosis, respectively. Another common observation noted upon histologic examination of lungs of both control and exposed rats was the presence of focal alveolar fibrosis and increased exfoliation of alveolar macrophages; this was typically seen in terminal cases of spontaneous geriatric cardiovascular/renal insufficiency.

Cardiovascular system:
All groups, treated and control, had rats with age related focal myocardial degenerative changes, aortic and thoracic vessel mineralization, myocardial vascular changes and periarteritis of principally mesenteric vessels. Thrombosis of the left atrial ventricle was the cause of death of some rats.

Liver:
All groups, treated and control, had rats with variable degrees of focal inflammation, fatty metamorphosis, degeneration, necrosis, bile duct hyperplasia, focal pericholangiolar or periportal inflammation or hyperplastic nodule formation. Additional lesions occurred in a scattered distribution with no evidence of alterations related to exposure to VBC.

Spleen:
An increased level of hematopoietic activity and an increased amount of hematogenous pigment was noted in rats of all groups. Reticuloendothelial hyperplasia was also noted in a few rats of all groups.

Urinary system:
An age-related chronic progressive nephrosis was commonly noted in a majority of all rats of all groups. This was frequently accompanied by secondary parathyroid hyperplasia and mineralization of various tissues, such as the gastric mucosa. A few rats had a chronic inflammatory process of the urinary bladder which, in some cases, included a hyperplasia of the mucosal lining. It appeared as if some of these inflammatory processes originated as a descending purulent inflammation from the kidney.

Male reproductive system:
An age-related decrease in testicular spermatogenesis was noted in rats of both control and exposed rats. Also, the accessory sex glands were sometimes found to have a decreased content of secretory material. Focal inflammation of the testes, prostate or other accessory sex glands was noted in a few cases.

Thyroid and parathyroid glands:
Thyroid hyperplasia was noted in some of the rats from both control and exposed groups.
The most frequent alteration in the parathyroid glands was hyperplasia occurring secondary to the age-related chronic nephrosis.

Adrenal glands:
Hyperplastic changes were observed in the adrenal cortex and medulla of rats from both control and exposed groups of rats. Hematocyst formation and vacuolization of cortical cells were noted in some rats from all groups.

Lymphoreticular system:
Thoracic lymph nodes of some of the rats with the bacterial pneumonia had a purulent inflammatory process. Some of the rats dying as a result of age-related heart failure had edema of the thoracic lymph nodes. A few rats had lymph nodes with inflammatory or hyperplastic changes. A few lymph nodes had increased amounts of hematogenous pigment.

Gastrointestinal tract:
The most common observation was mineralization of the gastric mucosa or muscularis, secondary to age-related chronic nephrosis. Various other inflammatory or degenerative lesions were noted in some cases, with no indication of being related to the exposure to VBC.

Pancreas:
Focal atrophy of some of the pancreatic acinar structures was the most common observation. This was sometimes accompanied by focal fibrosis of the atrophic area. Hyperplastic lesions involving the pancreatic acini or islets were noted in rats of all groups.

Central nervous system:
Various lesions usually inflammatory and degenerative lesions occurred in a few isolated rats, with no evidence to indicate a treatment-related effect.

Pituitary gland:
Common lesions were hyperplastic proliferation of chromophobe cells of the pituitary gland some of these cases were associated with cyst formation or accumulation of hematogenous pigment.

Musculoskeletal system:
A few rate had degenerative or inflammatory changes of the skeletal musculature; one rat had a demineralization of the skull.

Miscellaneous:
A few rats had variable degrees of focal or diffuse keratitis; additional lesions were found to be scattered among rats from all groups.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid and parathyroid glands:
Thyroid tumor formation (see carcinogenicity endpoint) was noted in some of the rats from both control and exposed groups.

Adrenal glands:
Tumorous changes were observed in the adrenal cortex and medulla of rats from both control and exposed groups of rats.

Lymphoreticular system:
Some rats showed lymph nodes with tumour changes.

Gastrointestinal tract
Tumorous lesions were noted in some animals.

Pancreas:
Tumorous lesions involving the pancreatic acini or islets were noted in rats of all groups.

Central nervous system:
Tumorous lesions were observed in a few animals.

Pituitary gland:
Tumorous proliferation of chromophobe cells of the pituitary gland was found in some animals.

Subcutaneous tissues and integument
The most frequently observed lesions were tumorous in nature (see carcinogenicity endpoint); the specific type of tumor was varied. All groups were affected, and there was no indication of a relationship to the exposure to VBC.

Muscuskeletal system:
A single rat showed a tumorous proliferation involving a thoracic rib.


Other effects:
no effects observed
Description (incidence and severity):
Pulmonary exfoliative cytology:
Examination of the prepared smears revealed no abnormal appearing cells and no alterations in the distribution of morphological cell-types. The cellular populations consisted primarily of normal-appearing macrophages and respiratory epithelial cells.

Effect levels

Key result
Dose descriptor:
LOAEC
Effect level:
ca. 1 ppm (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 ppm (nominal)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

Applicant's summary and conclusion

Conclusions:
The results of this study are interpreted to indicate that lifetime observation of rats inhaling 0.1 or 1.0 ppm vinylbenzyl chloride for 6 hours/day, 5 days per week for 6 months had no discernible ill effects, including carcinogenesis, except for reversible eye and nasal irritation noted in animals exposed to the 1.0 ppm level of VBC.
Executive summary:

Groups of 120 male rats were exposed to atmospheres containing 0, 0.1 and 1.0 ppm of vinylbenzyl chloride (VBC) 6 hours/day, 5 days/week for 6 months and subsequently observed for their natural lifespan. There was evidence of slight reversible eye and nasal irritation during the exposure to 1 ppm VBC. Pathologic examination at the end of the 6-month exposure period revealed slight inflammation of the nasal passageways of the rats exposed to 1.0 but not 0.1 ppm VBC. There were no other alterations in the rats with respect to body weight gain, mortality, hematology, pulmonary exfoliative cytology or cytogenetic examination of bone marrow cells. Gross and histopathologic examination of tissues of all rats maintained for the duration of their natural lifespan revealed no

lesions, including tumor formation, considered related to exposure to 1.0 or 0.1 ppm VBC. The results of this study are interpreted to indicate that lifetime observation of rats inhaling 0.1 or 1.0 ppm VBC for 6 hours/day, 5 days per week for 6 months had no discernible ill effects, including carcinogenesis, except for reversible eye and nasal irritation noted in animals exposed to the 1.0 ppm level of VBC.