Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 January 1979 to 27 February 1980
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
The study predates the appropriate OECD test guideline and GLP, but was conducted in compliance with standards used in this laboratory at that time. Nevertheless, the study was conducted similar to the OECD TG 413 with acceptable restrictions. The restrictions were that the use of a whole-body inhalation system was not justified in the study report, no detailed clinical and ophthalmoscopic observations were made, no food and water consumptions were determined, and neuronal behaviour was not checked. However, extended satelite groups were maintained for up to 90 days post-exposure to ensure the detection of latent effects and to investigate the reversibility of treatment-related effects.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Version / remarks:
(2009)
Deviations:
yes
Remarks:
(the use of a whole-body inhalation system was unjustified in the study report, no detailed clinical and ophthalmoscopic observations were made, no food and water consumption were determined, and neuronal behaviour was not checked)
GLP compliance:
no
Remarks:
The study predates GLP, but was in compliance with internal laboratory standards of that time.

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Strain: CDF (Fischer 344-derived)- Source: Charles River Breeding Laboratory, Portage, Michigan- Age at study initiation: 8 weeks- Housing: in standard rat cages, 2 per cage- Diet: Purina Rat Chow, ad libitum (except during exposure and overnight prior to necropsy)- Water: tap water, ad libitum (except during exposure and overnight prior to necropsy)- Acclimation period: 2 weeksENVIRONMENTAL CONDITIONSNo details are given in the study report.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION- Exposure apparatus: three 4.3 m³ Rochester-type chambers- Method of holding animals in test chamber: 10 rats per sex were placed in 15''x16''x12'' inhalation cages and 4 cages each (2 cages with males and 2 cages with females) were closely grouped in the center of the exposure chambers on the top shelf. Control rats were similarily transferred into inhalation cages, but were not placed in an inhalation chamber. - Source and rate of air: The main air supply was directly from the building air conditioning ducts. - Method of conditioning air: see above- System of generating vapour: Vapour was generated by counter-current exchange between air and liquid test item in a 15-plate perforated plate distillation column, which was water jacketed and thermostated to a few degrees below ambient room temperature in order to provide freedom from fluctuations caused by variability of room temperature, and to ensure that vapour being transferred stayed below saturation. Because the target concentrations required such small amounts of test material vapour, the airflow through the column that would have resulted from only the demand to supply the chambers would have been too low for steady operation of the system. Therefore, the generating system was so arranged that air was continuously pumped through a closed loop containing the distillation column and a large (5 L) plenum flask. The apparatus was designed to hold constant at a level of 100 ppm the concentration of the test item in the plenum flask; this was found to be achieved when the water jacket temperature was 14.6°C. (Therefore, the vapour pressure of the test material at 14.6°C was 100 x 10E-6 x 760 = 0.76 mmHg.) Stability of the system was ensured by monitoring the concentration of the plenum flask continously (i.e. every 4 min) by a semi-automated gas chromatographic method. Variation of the test item concentration in the plenum flask throughout the entire study, except for an occasional malfunction, was smaller than could be detected analytically. To reach target the concentrations of the test item in the inhalation chamber air was pumped from the plenum flask at an appropriate rate into a "Y"-joint in the main room air input duct of each chamber, using FMI fluid metering pumps. Minor adjustments in pumping rate were made from time to time, as indicated by analyses of chamber air, to hold the concentration in each chamber as close to the target value as possible. - Temperature, humidity, pressure in air chamber: 72±2°F (equivalent to 22.2-23.3°C), 50±10% relative humidity, slight negative pressure of 0.05'' H2O- Air flow rate: dynamic airflow, 700 L/min- Air change rate: approximately 10/hTEST ATMOSPHERE- Brief description of analytical method used: An on-line analytical method was employed by connecting an MDA paper tape detector, specific for isocyanates, to each chamber, providing continuous record of the test item concentration. Due to rapid and continuous measurement but insufficient accuracy, this method was only determined for adjustment of the chamber concentration during animal exposure. - Samples taken from breathing zone: no (The lines, which sampled chamber air for analysis (2/chamber) were placed with their opening just above the center of the group of 4 cages; presumably what is measured, then, is the concentration of the test item in air just before it reached the rats, since the airflow in the chamber was from top to bottom.)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Primary analysis of chamber air was made with a liquid chromatographic procedure. In this method, an aliquot of the chamber air (approximately 20 L) was bubbled in through an impinger into a solution containing p-nitrobenzyl propyl amine. The test item reacts quantitatively with this material to form the urea derivate of the isocyanato group. An aliquot of the solution containing this urea derivate, together with unreacted p-nitrobenzyl propyl amine, was analysed by HPLC withn UV detector. The method was reported to be sensitive to 2 ppb test item in the air. In order to ensure a recovery as complete as possible of the test item in an aliquot chamber air sample, several modifications of gas sampling procedures and standardisation procedures had to be made, for very dilute solutions of the test item in solvents or air are not analytically stable for more than approximately 1 h. The maximum recovery that could be achieved was just over 90%, and it could not be determined whether this was inherent in the analytical system or in the difficulty of preparing known concentrations of the test item at levels of 250 ppb and below. Finally, it was accepted that the lack of quantitative recovery results from the inability to prepare gas standards accurately, and the analytical results should be taken at face value. If this assumption was wrong, the end result would be that the actual chamber concentration would be 10% above the reported value, and thus, if anything, actual chamber concentrations were understated. The concentrations of the test item in the 3 exposure chambers could only be determined at most 3 times each per exposure day by this precise HPLC method. As the study proceeded and experience gained in the behaviour and control of the apparatus, each chamber was analysed twice daily by this method. For each day the arithmetic means of the analytical results of each chamber was calculated; these daily mean values were averaged, and the standard deviation was calculated.
Duration of treatment / exposure:
6 h plus 0.5 h time period for chamber ventilation at the conclusion of the exposure
Frequency of treatment:
5 days/week, 13 consecutive weeks
Doses / concentrationsopen allclose all
Dose / conc.:
25 other: ppb (nominal)
Remarks:
24.2±2.6 ppb (analytical)
Dose / conc.:
80 other: ppb (nominal)
Remarks:
79.2±3.8 ppb (analytical)
Dose / conc.:
250 other: ppb (nominal)
Remarks:
250±15 ppm (analytical)
Dose / conc.:
1.583 mg/m³ air
Remarks:
nominal conc., calculated on the basis of c(mg/m³) = (((155.1513 g/mol / 24.5 L) x concentration in ppm) / 1000)
Dose / conc.:
0.507 mg/m³ air
Remarks:
nominal conc., calculated on the basis of c(mg/m³) = (((155.1513 g/mol / 24.5 L) x concentration in ppm) / 1000)
Dose / conc.:
0.158 mg/m³ air
Remarks:
nominal conc., calculated on the basis of c(mg/m³) = (((155.1513 g/mol / 24.5 L) x concentration in ppm) / 1000)
No. of animals per sex per dose:
20
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: previous toxicity studies with the test item- Rationale for animal assignment: random- Rationale for selecting satellite groups: In order to assess the reversibility of treatment-related effects and to identify potential latent effects of exposure, 10 animals per sex per dose (half group size) were maintained for further observation. - Post-exposure recovery period in satellite groups: 30 and 92 days following the last exposure

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: at the end of each day's exposure DETAILED CLINICAL OBSERVATIONS: NoBODY WEIGHT: Yes- Time schedule for examinations: on days 1, 2, 4, 12, 19, 26, 33, 40, 47, 54, 62, 75, 82, and 89. In addition, terminal fasted body weights were determined. FOOD CONSUMPTION:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: NoFOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: NoWATER CONSUMPTION: NoOPHTHALMOSCOPIC EXAMINATION: NoHAEMATOLOGY: Yes- Time schedule for collection of blood: on day 87, just prior to exposure- Animals fasted: No- How many animals: all animals which were scheduled for necropsy after the last exposure (10 rats/sex/dose)- Parameters checked: erythrocyte count (RBC), haemoglobin (Hb), packed cell volume (PCV), white blood cell count (total and differential)CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: on day 92 at necropsy- Animals fasted: Yes- How many animals: all animals which were scheduled for necropsy after the last exposure (10 rats/sex/dose)- Parameters checked: blood urea nitrogen (BUN), glucose, serum glutamic pyruvic transaminase (SGPT), serum alkaline phosphatase (AP)URINALYSIS: Yes- Time schedule for collection of urine: on day 87- Animals fasted: No- Parameters checked: specific gravity, bilirubin, glucose, ketones, occult blood, pH, protein, urobilirubinNEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes- Organs checked: accessory sex glands, adrenal glands, adipose tissue, aorta, bile duct, brain, epididymides, eyes, heart, kidneys, large intestine, larynx, liver, lung, lymph nodes (thoracic, mesenteric), mammary tissue, muscle (skeletal), nasal turbinates, peripheral nerve (sciatic n.), oesophagus, ovary, oviduct, pancreas, parathyroides, pituitary gland, salivary glands (submandibular), skin, small intestine, spinal cord (thoracic,vertebral column), spleen, sternum, stomach, testes, thymus, thyroid, tongue, trachea, urinary bladder, uterusHISTOPATHOLOGY: Yes- Organs checked (all animals): gross lesions, larynx, lung, nasal turbinates, trachea- Additional organs checked (control and high dose animals as well as all low and mid dose animals with grossly described lesions): adrenal glands, adipose tissue, aorta, brain, epididymides, eyes, heart, kidneys, large intestine, liver, lymph nodes (thoracic, mesenteric), mammary tissue, muscle (skeletal), peripheral nerve (sciatic n.), ovary, oviduct, pancreas, parathyroides, pituitary gland, salivary glands (submandibular), seminal vesicles, skin, small intestine, spleen, stomach, thymus, thyroid, tongue, urinary bladderORGEN WEIGHTS: Yes- Organs checked: brain, heart, kidneys, liver, testes, thymus
Statistics:
Body weights, clinical chemistry, haematology, and urinalysis parameters were evaluated for differences between control and treatment group means using an analysis of variance and Dunnett's t-test. The level of significance in all cases was chosen as p≤0.05. Terminal fasted body weights, organ weights, and organ-to-body weight ratios were analysed by Barlett's t-test for homogeneity of variances (p<0.01). A parametric analysis of variance (ANOVA) was subsequently performed when Bartlett's t-test was not significant (variances were homogenous); otherwise a non-parametric ANOVA was performed. Dunnett's test or Wilcoxon'n signed-rank test (p<0.05) were conducted in order to delinate group differences when the F-ratio for the parametric or non-parametric ANOVA, respectively, was significant at p<0.1.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
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:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hyperplasia of the respiratory epithelium lining the nasal turbinates in 4/10 males and 5/10 females of the high dose group and 3/10 males of the mid dose group. The effect was reversible, since it was not observed in any of the recovery groups.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITYNo signs of any effects resulting from the test material exposure were seen in any of the animals throughout the study. On day 2 of exposure one male was found dead. Necropsy revealed internal physical damage due to a handling accident. On day 16, some rats in all groups showed signs of incipient salivary gland infections. Rats of a concomitant study showed the same signs earlier, indicating that this disease spread from them to the rats in this study. By day 20 the signs had begun to resolve and by day 23 the signs disappeared. All groups, including controls, appeared to be equally affected. BODY WEIGHT AND WEIGHT GAINNo treatment-related effects on body weights were observed throughout the study.On day 19 the mean body weight of the females exposed to 250 ppb was statistically significant greater that the mean body weight of the controls. At later weighings, this effect was not observed. On day 158, the mean body weight the mean body weight of the females exposed to 80 ppb was statistically significant less than the control mean. No such effect was observed in the high dose group. At the subsequent terminal weighing this effect was no longer observed. HAEMATOLOGY, CLINICAL CHEMISTRY, AND URINALYSISOnly very few statistically significant differences between mean values of treated groups and and controls were observed, and none of these showed a dose-dependency, indicating that the observations were not treatment-related.Haematology data showed a statistically significant increase n the total mean white blood cell count for all females as compared to the controls. However, mean white blood cell counts values obtained at this sampling interval for the control group was substantially lower than the historical control values of rats of the same strain, age, and sex. Furthermore, the values of the exposed animals were within the normal range of historical controls, the absence of a dose-dependency, and the absence of effects on white blood cell counts in previous studies with the test item make the significance of the effect highly questionable and was concluded to be not treatment-related.ORGAN WEIGHTSSeveral statistically significant differences between mean values of treated and control animals for organ weights and organ/body weight ratios were evident. However, in all cases these differences were considered sporadic in nature due to the absence of consistent effects with respect to specific organ or time periods, the absence of any apparent dose-response relationship, and the absence of other evidence (particularly histological alterations) suggestive of a treatment-related effect of exposure to the teat material of any organ or tissue other than the nasal turbinates. Thus, the toxicological significance of the changes is questionable and therefore they were not considered attributable to the test material exposure. GROSS PATHOLOGYThere were no recognisable changes in any of the sacrifice intervals (following 91 days of exposure, 30 days post-exposure, or 92 days post-exposure), which were interpreted as being related to exposure to the test item. HISTOPATHOLOGY non-neoplasticThe only change identified histologically as being directly exposure-related was slight to very slight, multifocal hyperplasia of the respiratory epithelium lining the nasal turbinates. This change was noted in 4/10 males and 5/10 females at the 250 ppb level and in 3/10 males at the 80 ppb level and was generally more evident at the higher level. No treatment-related effects were seen in the females at the 80 ppb level or in either sex at the 25 ppb level. In rats of the recovery groups, sacrificed on days 30 and 92 post-exposure, there were no hyperplastic lesions in the nasal turbinates or any residual effects of them, and no evidence of latent treatment-related lesions were observed. In affected animals the respiratory epithelium was focally stratified (3-5 cells thick), and there was some change in nuclear polarity as the cells became polygonal, however, the squamous differentiation was lacking in the superficial cell layers. In some animals rounded cells appeared to exfoliate from the surface. The location of these lesions appeared to be quite specific occuring only in the most rostral of the four sections and only on the tips and lateral aspects of the nasoturbinates and maxilloturbinates and the lateral aspect of the nasal cavity at the level of the middle meatus. These areas are presumably adjacent to the mainstream airflow through the nasal cavity. Simple columnar hyperplasia was not identified due to the normal accentuation of the pseudo-stratified columnar pattern in these areas. A variety of other lesions occurred in the turbinate mucosa, including subepithelial accumulations of lymphoid cells and focal increases in goblet cells, however, none of these changes occurred in a manner which suggests a dose-response relationship or in a frequency significantly different from the control group. Some degree of multifocal, chronic, interstitial inflammation of the lungs, probably of viral origin, was present in all animals at the 91-day sacrifice and most of the post-exposure animals to a lesser extent. Since these foci were generally small, discrete, subpleural, and perivascular rather than peribronchiolar in location, the interpretability of potential treatment-related effects in the lungs was not compromised. Nor was there exacerbation of the inflammatory process as a result of exposure to the test item. Residual effects of sialodacryoadentitis viral infection were noted in the lacrimal glands of many of the sacrificed rats after 91 days of exposure. A result of a change in histologists, lacrimal glands were generally not present in the most caudal plane of section of the nasal cavity in slides prepared from the two post-exposure groups and, consequently, were not examined. HISTOPATHOLOGY neoplasticThree benign neoplastic lesions occurred during the course of the study: an endometrial stromal polyp (25 ppb, female, 92-day post-exposure), a pituitary adenoma (80 ppb, female, 92-day post-exposure), and an adrenal ganglioneuroma (25 ppb, male, 30-day post exposure). The two former tumours are relatively frequent in the strain of rat used. The ganglioneuroma, in contrast, is a relatively infrequent lesion and was diagnosed in a fairly young animal (approximately 230 days). However, this tumour has beeen diagnosed sporadically in this laboratory since the introduction of the Fischer 344 strain, and the occurrence of a solitary tumour at 30 days post-exposure does not warrant an interpretation of a treatment-related association.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Effect level:
250 other: ppb (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: reversible hyperplasia of the respiratory epithelium lining the nasal turbinates in the 250 ppm (high dose) group
Dose descriptor:
NOEC
Effect level:
80 other: ppb (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion