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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study design comparable to OECD Guideline 413
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
not applicable
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
Lot/batch No.: BNW 13871-4
Purity: 99.5 %

Test animals

Fischer 344
Details on test animals or test system and environmental conditions:
Male and female F344/N rats were obtained from Taconic Laboratory Animals and Services (Germantown, NY). On receipt, the rats were approximately 4 weeks old. Animals were quarantined for 11 - 12 days and were approximately 6 weeks old on the first day of the studies. Before the studies began, five male and five female rats were randomly selected for parasite evaluation and gross observation for evidence of disease.

NTP-2000 irradiated pelleted diet, available ad libitum (except during animal exposure and urine collection periods); changed weekly

Tap water via automatic watering system, available ad libitum

Stainless steel, wire bottom, changed weekly

Untreated paper cage pan liner, changed daily

Stainless steel, excreta pan at each of six levels, chambers changed weekly, excreta pans changed daily

Chamber Air Supply Filters
Single HEPA changed annually, charcoal new at study start, Purafil new at study start

Chamber Environment
Temperature: 75 +/- 3 degrees F
Relative humidity: 55 +/- 15 %
Room fluorescent light: 12 hours/day
Air changes: 15 +/- 2/hour

Administration / exposure

Route of administration:
Type of inhalation exposure:
whole body
other: clean air
Details on inhalation exposure:
Alpha-methylstyrene was held in an 8-gallon stainless steel chemical reservoir. Alpha-methylstyrene was pumped through a pre-heater and into the top of a heated glass column filled with glass beads to increase the surface area for evaporation. Heated nitrogen entering the column from below vaporized the chemical as it conveyed it out of the generator. The vapour was transported to the exposure room at an elevated temperature to prevent condensation. In the distribution manifold cabinet, the vapour was mixed with additional heated air before it entered a short vapour distribution manifold. The pressure in the distribution manifold was fixed to ensure constant flow through the manifold and into the chambers as the flow of vapour to each chamber was adjusted. Electronically actuated metering valves controlled the flow to each chamber. In addition, an exposure-shutoff valve controlled vapour delivery to each chamber. Vapour was diverted to the exposure chamber exhaust until the generation system was stable and exposures were ready to proceed. To start the exposure, the valves were opened to allow the flow of vapour to reach the chamber-metering valves and move into individual temperature-controlled delivery lines to each chamber. The vapour was then injected into the chamber inlet duct where it was diluted with conditioned chamber air to achieve the desired exposure concentration. The study laboratory designed the inhalation exposure chamber so that uniform vapour concentrations could be maintained throughout the chamber with the catch pans in place. The total active mixing volume of each chamber was 1.7 m3. A condensation particle counter was used to count the particles in all chambers before and during generation. No particle counts greater than 200 particles/cm3 were detected.

The alpha-methylstyrene concentrations in the exposure chambers were monitored by an online GC. Samples were drawn from each exposure chamber approximately every 20 minutes during each 6-hour exposure period using a 16-port stream select valve. The online GC was checked throughout the day for instrument drift against an online standard of alpha-methylstyrene in nitrogen supplied by a diffusion standard generator. The online GC was calibrated monthly by a comparison of chamber concentration data to data from grab samples, which were collected with graphitized carbon black sampling tubes, extracted with toluene containing butylbenzene as an internal standard, and analyzed by an offline GC. The volumes of gas were sampled from each chamber at a constant flow rate ensured by a calibrated critical orifice. The offline GC was calibrated with gravimetrically prepared standard solutions of alpha-methylstyrene containing butylbenzene as an internal standard in toluene.

Build-up and decay rates for chamber vapour concentrations were determined with and without 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 vapour generation (T90) and the time for the chamber concentration to decay to 10 % of the target concentration after vapour generation was terminated (T10) was approximately 12.5 minutes. Based on experimental data, a T90 value of 12 minutes was selected for the studies. The uniformity of alpha-methylstyrene vapour concentration in the inhalation exposure chambers without animals was evaluated before each of the studies began; concentration uniformity with animals present in the chambers was also measured once during the 3-month studies. The vapour concentration was measured using an online GC. Chamber concentration uniformity was maintained throughout the studies. The persistence of alpha-methylstyrene in the chamber after vapour delivery ended was determined by monitoring the concentration with animals present in the 1000 ppm chambers in the 3-month studies. In the 3-month studies, the concentration decreased to 1 % of the target concentration within approximately 46 minutes in both 1000 ppm chambers.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Samples of alpha-methylstyrene were collected, with and without animals present, from the distribution line, 75 and 1000 ppm exposure chambers, generator reservoir, and vapour trap and analyzed by GC. No evidence of degradation was detected, and no impurities were detected that were not present in the bulk material. HPLC was used to determine the concentration of 4-tert-butyl catechol in exposure chambers; none was detected. Polymer concentration was determined using UV/Vis; the concentration was less than 10 ppm. These results indicated that alpha-methylstyrene was stable for up to 7 weeks in the generator reservoir.
Duration of treatment / exposure:
14 weeks
Frequency of treatment:
6 hours plus T90 (12 minutes) per day on 5 days/week
Doses / concentrations
Doses / Concentrations:
nominal: 0, 75, 150, 300, 600 or 1000 ppm; analytical: 0, 75 +/-2. 150 +/- 3, 300 +/- 7, 603 +/- 11 or 1002 +/- 30 ppm

No. of animals per sex per dose:
10 m / 10 f

Additional clinical pathology groups of 10 male and 10 female rats were exposed to the same concentrations for 23 days. One additional exposure day was scheduled during the last exposure week to give male rats at least two consecutive days of exposure before terminal sacrifice.
Control animals:
yes, concurrent vehicle


Observations and examinations performed and frequency:
All animals were observed twice daily. Clinical findings were recorded weekly. Animals were weighed initially, weekly, and at the end of the studies.

Clinical Pathology
Blood was collected from the retroorbital sinus of mice at the end of the study for haematology.
Haematology: haematocrit; packed red cell volume; haemoglobin; erythrocyte, reticulocyte, and platelet counts; Howell-Jolly bodies; mean cell volume; mean cell haemoglobin; mean cell haemoglobin concentration; and leukocyte counts and differentials.
Clinical chemistry: urea nitrogen, creatinine, total protein, albumin, globulin, alanine aminotransferase, alkaline phosphatase, creatine kinase, sorbitol dehydrogenase, and total bile acids
Urinalysis: creatinine, glucose, protein, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase, gamma-glutamyltransferase, N-acetyl-beta-D-glucosaminidase, volume, and specific gravity

Blood was collected from the retroorbital sinus of clinical pathology rats on days 3 and 23 and from core study rats at the end of the study for haematology and clinical chemistry analyses.
After three (males) or four (females) consecutive exposure days during week 12, core study rats were placed in metabolism cages, and urine was collected over ice for 16 hours. During collection, the animals had access to water but not to food. After collection, the appearance, volume, and specific gravity of the samples were determined and recorded. A microscopic analysis of the urinary sediment was then performed.
Sacrifice and pathology:
Necropsies were performed on all core study animals. Organs weighed were heart, right kidney, liver, lungs, right testis, and thymus.

Complete histopathology was performed on all core study chamber control and 1000 ppm mice. In addition to gross lesions and tissue masses, the following tissues were examined to the no-effect level: adrenal gland, bone with marrow, brain, clitoral gland, oesophagus, eyes, gallbladder, harderian gland, heart and aorta, large intestine (caecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidney, larynx, liver, lung (with mainstem bronchus), lymph nodes (mandibular, mesenteric, bronchial, mediastinal), mammary gland, nose, ovary, pancreas, parathyroid gland, pituitary gland, preputial gland, prostate gland, salivary gland, skin, spleen, stomach (forestomach and glandular), testis (with epididymis and seminal vesicle), thymus, thyroid gland, trachea, urinary bladder, and uterus.

The left kidney was removed from all male and female core study rats at terminal necropsy, sectioned in half longitudinally, placed in a cassette, and fixed with 10 % neutral buffered formalin for approximately 24 hours. After fixation, one half of the left kidney was processed and embedded in paraffin. Analysis of alpha2u-globulin in supernatants prepared from kidney homogenates was conducted using a competitive indirect enzyme-linked immunosorbent assay (ELISA). The amount of alpha2u-globulin was measured by comparing the relative fluorescent signal intensity in the study samples to that observed with known amounts of alpha2u-globulin present in calibration standards. Calibration standards and ELISA control standards (negative and positive) were plated in predetermined wells on 96-well microtiter plates. Calibration standards and study samples were assayed in triplicate.
Other examinations:
Sperm Motility and Vaginal Cytology
Epididymal sperm concentration and motility; spermatid heads/testis; and left cauda, epididymis, and testis weights were evaluated in males from the 0, 300, 600, and 1000 ppm exposure groups at terminal sacrifice. For 12 consecutive days prior to scheduled terminal sacrifice, vaginal cytology slides were prepared for all surviving females in the 0, 300, 600, and 1,000 ppm exposure groups. Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain oestrous cycle stage (i.e., dioestrus, prooestrus, oestrus, and metoestrus).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed

Effect levels

Dose descriptor:
Effect level:
300 ppm
Basis for effect level:
other: Kidney & liver

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

From this study a NOAEL of 300 ppm for rats was derived.
Executive summary:

In this study (design comparable to OECD Guideline 413), groups of 10 male and 10 female rats were exposed by whole-body inhalation to alpha-methylstyrene at concentrations of 0, 75, 150, 300, 600 or 1000 ppm for 6 hrs per day and 5 days per week for 14 weeks. Additional clinical pathology groups of 10 male and 10 female rats were exposed to the same concentrations for 23 days. All rats survived to the end of the study, and mean body weights of all exposed groups were similar to those of the chamber controls. Kidney weights were significantly increased in 1000 ppm males and at >= 600 ppm females. Statistically significant increases in liver weights occurred at >= 150 ppm males and at >= 600 ppm females. The incidences of renal hyaline droplet accumulation were similar between exposed groups and chamber control groups, but the severity of hyaline droplet accumulation in >= 600 ppm males was greater than in chamber controls. Consistent with the hyaline droplet accumulation, an exposure related increase in alpha2u-globulin was detected in the kidneys of males exposed to alpha-methylstyrene. Morphologic changes were not detected in the liver.

The effects observed in male rats at concentrations of 300 and below are not considered to be relevant for human health and a the relevant NOAEL of 300 ppm has been derived.