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Repeated dose toxicity: inhalation

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short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1979-06-11 - 1979-07-12
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
It was not compliant with GLP. Due to an accident, the mid dose group had to be terminated and the respective exposure was repeated in a following experiment.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
equivalent or similar to
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
(longer exposure duration (6.5 h) and no detailed information on test material purity)
GLP compliance:
Limit test:

Test material


Test animals

Details on test animals and environmental conditions:
- Source: Bio-Breeding Laboratories Ltd., Ottawa, Ontario, Canada (received on 1979-05-24)
- Age at study initiation: 10-14 weeks
- Weight at study initiation: 200-250 g
- Housing: individually in in wire mesh-bottomed metal cages
- Diet: standard commercial laboratory diet (Purina) ad libitum (no food was available for the animals during exposure)
- Water: tab water available through a water sipper system ad libitum (no water was available for the animals during exposure)
- Acclimation period: 2 weeks
- Subsequent to arrival the animals received a qulified vetinary aid to ensure "normal" health status.

- Temperature (°C): animal room: 20.0-23.9, inhalation room: 21.4-23.6

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
other: clean, dry air
Remarks on MMAD:
MMAD / GSD: not applicable
Details on inhalation exposure:
- Exposure apparatus: stainless steel whole body exposure chambers (400 l volume)
- Method of holding animals in test chamber: in a stainless steel wire mesh compartmentalised cage (each compartment measuring 7 x 4 x 3 inches)
- System of generating aerosols: Delivery of the test article into the chambers was accomplished by forcing air through gas generators fittet with sintered glass frits and containing the test item, and metering vapours into the chamber.
- Temperature, humidity, pressure in air chamber: Airflow through each chamber was measured in the exhaust line of the chamber by reading the pressure drop across an orifice plate on a magnehelic gauge. These gauges were calibrated against conventional ball-type flow meters. The magnitude of negative pressure within each chamber with respect to the outside atmosphere was always less than 0.5 inches of water. Temperature and relative humidity within both the inhalation chamber and the inhalation room were measured at hourly intervals during each exposure period. Chamber temperature and relative humidity were measured by means of remote sensors placed within each inhalation chamber. A mean daily chamber temperature of 22-26 °C was maintained throughout the study with the exception of study days 0.1 and 2.1 when higher temperatures were recorded in Chambers 1 to 2 and 3, respectively. Inhalation chamber relative humidity during exposure was generally 35-65% A higher relative humidity was observed in Chamber 1 (air controls) compared with the inhalation room, and a decrease in chamber relative humidity tended to occur as chamber test substance concentration increased.
- Air flow rate: One gas generator was used for Chamber 2 with an airflow of 6 l/min and two generators in parallel with an airflow of 30 l/min for Chamber 3. In the case of Chamber 4 a total airflow of 40 l/min was passed through two parallel generators partially immersed in a water bath at approximately 65 °C. The two generators were each serially connected to a pair of generators maintained at room temperature. The outputs from the vapour generation systems were each diluted with room air to a total chamber airflow of 45 l/min.

- Brief description of analytical method used: Nominal chamber concentrations were calculated from the total airflow and the weight loss from the generator(s) during the exposure. Actual chamber test material concentrations were measured hourly using a MIRAN 1A Infrared gas analyser at an analytical wave length of 9.2 µm and a path length of 0.75 m. The Infrared gas analyser had been previously calibrated by injection of known microliter quantities of the test article into a closed loop system and allowing the liquid to vaporise.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Target chamber concentrations for Groups 2 and 3 were generally achieved by the end of the 30 min equilibration time. In case of Group 4 approximately two hours of equilibration were required before the chamber concentration reached the targeted value. This was concluded by the study authors to be possibly related to the low volatility of the test material.
Mean daily actual chamber concentrations were close to the target concentrations on all exposure occasions except for Chamber 3 on study day 1.4 when the mean test article concentration reached a recorded maximum of 134 ppm. This overexposure was found to result from a technical error. The measured concentration in Chamber 1 (control chamber) was approximately 2 ppm throughout the study although no test article was introduced into this chamber. The possible reason for this spurious value was concluded to be caused by infrared absorbance at the analytical wavelength by vapours emanating from rat faeces and urine in the chambers. Consequently, the chamber test article concentrations should be considered as 2 ppm higher than the true concentrations existing in the chambers.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6.5 h/day, 5 days/week
Each daily exposure period was 6.5 hours in duration, starting after the 30 min equilibration time when 95% of the target concentration was established within the chamber. After 6.5 hours of exposure at the desired concentration, delivery of the test article into the chamber was stopped and the chamber was allowed to equilibrate with room air for 30 min prior to the removal of the animals. Thus, the animals remained in the chamber for 7.5 hours/day.
Doses / concentrationsopen allclose all
Dose / conc.:
10 ppm (nominal)
Dose / conc.:
50 ppm (nominal)
Dose / conc.:
80 ppm (nominal)
Dose / conc.:
11.5 ppm (analytical)
mean analytical conc. over the whole study period
Dose / conc.:
54.4 ppm (analytical)
mean analytical conc. over the whole study period
Dose / conc.:
78.5 ppm (analytical)
mean analytical conc. over the whole study period
Dose / conc.:
9.5 ppm (analytical)
corrected mean analytical cnc. over the whole study period due to additional absorbance of vapours emanating from rat faeces and urine in the chambers
Dose / conc.:
52.4 ppm (analytical)
corrected mean analytical cnc. over the whole study period due to additional absorbance of vapours emanating from rat faeces and urine in the chambers
Dose / conc.:
76.5 ppm (analytical)
corrected mean analytical cnc. over the whole study period due to additional absorbance of vapours emanating from rat faeces and urine in the chambers
Dose / conc.:
77 mg/m³ air (analytical)
corrected mean analytical conc. over the whole study period due to additional absorbance of vapours emanating from rat faeces and urine in the chambers; values calculated with (molecular weight [g/mol] x mean corrected exposure conc. [ppm])/24.45
Dose / conc.:
425 mg/m³ air (analytical)
corrected mean analytical conc. over the whole study period due to additional absorbance of vapours emanating from rat faeces and urine in the chambers; values calculated with (molecular weight [g/mol] x mean corrected exposure conc. [ppm])/24.45
Dose / conc.:
620 mg/m³ air (analytical)
corrected mean analytical conc. over the whole study period due to additional absorbance of vapours emanating from rat faeces and urine in the chambers; values calculated with (molecular weight [g/mol] x mean corrected exposure conc. [ppm])/24.45
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
Group 1: vehicle control, Group 2: 10 ppm exposure, Group 3: 50 ppm exposure, Group 4: 80 ppm exposure

During exposure an incident occured in Group 3. The line supplying suppressed air to the Chamber 3 gas generators was improperly connected resulting in test article being sprayed over ten animals in the chamber. Four rats were thoroughly soaked with test article, and the chamber concentration of the test substance increased to 134 ppm. Two rats were semi-conscious and ataxic within 45 min, whereas one female rat died after approximately 8 hours. Ten rats were observed to have wet fur on their ventral surface post-treatment and two of them were semi-conscious and lethargic. Three rats were found dead the next morning, and the deaths, as well as the lethargy and the ataxia, were concluded by the study authors to be attributed to an acute toxic effect of the dermal absorption of the test article.
As a consequence of this accident a second group of twenty rats were treated with the test article at 50 ppm and the data generated was presented as an addendum to this report (Bio-Research Laboratories No. 1981-I-0065-1573-6 (Addendum), 1981).


Observations and examinations performed and frequency:
- Time schedule: daily; druing the treatment period after each exosure, and additionally during exposure for signs of overt toxicity


- Time schedule for examinations: Individaul body weights were measured twice weekly during both acclimatisation and treatment periods.

- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

- 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: No



- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: red blood cell count, haemoglobin concentration, packed cell volume, and total and differential leucocyte count

- Time schedule for collection of blood: at the termination of the treatment period
- Animals fasted: Yes
- How many animals: all
- Parameters checked: fasting serum glucose, blood urea nitrogen (BUN), serum alkaline phospahtase (SAP), serum glutamic oxalacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT)

- Time schedule for collection of urine: during the night prior to terminal necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters checked: volume, colour, odour, transparency, specific gravity, occult blood, ketones, glucose, protein, pH, bilirubin, and microscopic examination of urinary sediment
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (complete gross pathology of all animals)
HISTOPATHOLOGY: Yes (brain (cerebrum, cerebellum, medulla, and pons), spinal cord, olfactory nerve and/or olfactory bulb, both eyes and optic nerves, pituitary gland, thyroide, thymus, adrenal glands, heart, spleen, thoracic aorta, oesophagus, submandibular salivary gland, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, liver, pancreas, gonads, lymph nodes (mesenteric and mediastinal), uterus, prostate, both kidneys, urinary bladder, sciatic nerve, skeletal muscle, bone marrow section (sternum), skin, tongue and buccal mucosa (bilateral), nasal and paranasal sinuses with vestibulum and adjacent bone, nasal septum, larynx, trachea, and major bronchi)
ORGAN WEIGHTS: Yes (left and right adrenal glands, brain, left and right gonads, heart, left and right kidneys, liver, pituitary gland, spleen, lungs and thyroid)
Other examinations:
MYELOGRAMS: Femoral bone marrow smears and sections from all control and high dose animals were examined by a qualified haematologist.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
No abnormal clinical observations were noted for the animals of Groups 1-4 during exposure except for the animals of Group 3 as a consequence of the accident as described above. The majority of abnormal clinical observations recorded during the daily physical examinations consisted of signs of pulmonary congestion. Both treated and control groups exhibited a low incidence of congestion throughout the study, suggesting that this finding was unrelated to the treatment with the test item. Incidental observations included reddish brown fur discolouration of the perinasal and periorbital regions. Additionally, Groups 2 and 3 showed wet fur. The low incidence of these observations coupled with the absence of a wet fur in Group 4 animals post treatment and the presence of reddish brown fur discolouration perinasally and periorbitally in three control rats pretreatment suggests that exposure to the test item at concentrations up to 80 ppm had no adverse effect.

Statistical analysis (Dunnett’s t test), comparing the mean body weights of treated and control animals, did not reveal any significant differences between groups which could be attributed to treatment with the test article. Body weight of treated and control rats increased in a similar fashion throughout the study with the exception of several animals, which lost weight for a short period of time: one control male, one female of the 10 ppm dose group, and one male and female each of the high dose group, whereas the two latter animals showed congestion during these periods and the effect might be related to the observed weight loss. Due to a lack of time- and dose-dependence of the observed weight loss and the additional occurrence in the control group, it is suggested that the effects are not treatment related.

No significant differences were observed between the absolute food consumption of treated and control groups. With respect to relative food consumption, significantly greater food intake was observed in Group 4 females during week 3 compared with the control group. A review of the individual data reveals that this is mainly contributed to one female animal of Group 4. This period of high food intake followed several weeks of reduced food consumption during which the body weight had decreased.
Infrequent periods of reduced food intake were noted in several rats of both the control and the high dose group and occurred at the same time as the body weight loss previously mentioned. The authors conclude that the effects seen are not related to the test material exposure.

No significant differences in blood parameters existed between treated and control groups. Lymphocyte count was slightly elevated in some Group 4 animals, while segmented neutrophil count was slightly lower in Group 4 males and females as compared to controls. These small differences were not considered to be of any biological importance and the test material was concluded not to cause any adverse haematological effect in rats.

The mean blood serum glucose level of Group 2 (10 ppm) females was significantly higher than that of the control group. Since no such effects were observed at higher doses, this effect was considered to be not treatment related. Although some individual values fell outside the range of normal values (e. g. increased BUN in one male of the control group, increased SAP in one male of the low dose group, increased SGOT in one female of the control group and one male of the high dose group (the latter in combination with an increased SGPT level)) none of these findings was considered to be exposure related.

Large intersubject variations existed in each group with respect to urine volume and specific gravity. However, the range for these parameters was similar in both treated and control groups. The high urine volume and associated low specific gravity in a proportion of the animal was considered possibly to be due to leakage of water from the water sipper in each cage. Hence, the test item was concluded not to cause any effect on urinary parameters measured in this study.

The left adrenal gland (but not the right) of Group 4 (80 ppm) males and the spleens of Group 2 (10 ppm) males were significant lighter than those of control rats. There were no differences compared with the controls for the spleens of the high dose males. Hence, the slight effects observed were considered not to be treatment related. Individual data revealed that one male rat of the high dose, which had lost weight in the last week of the study, had high relative lung and kidney weights. One male each of the control group ant the low dose group had small left thyroide lobes and on male rat of the low dose group had small testes in terms of both absolute and relative weight. One female of the high dose group had small ovaries, one female of the low dose group had a small spleen, and two males of the low dose group had large lungs in terms of relative weight. Since these observations were isolated, they were not considered treatment related.

The most commonly observed gross pathological findings were pulmonary congestion, enlargement and/or congestion of the lymph nodes and enlargement and/or fluid in the uterus and surrounding the ovaries of females. Each of these findings appeared as frequently in the treated groups as in the control group with the exception of fluid surrounding the ovary. The frequency of the latter finding was not dose related, and hence, gross pathological findings were concluded not to be treatment related. Further incidental findings not associated with the treatment were small testes or spleen, focal to general discolouration of the lung, liver, and kidney, and congestion of the thymus and salivary glands.

Pulmonary lesions were observed in both treated and control animals and consisted of lymphatic infiltration about air passages and blood vessels, some bronchiectasis, interstitial fibrosis, neutrophilic exudation, and alveolar macrophages. Similar lymphatic inflammatory was observed in the trachea, the larynx, and air passages. In most cases, lymphocytic aggregates were focal or multifocal in distribution and substantial amount of pulmonary parenchyma was intact and uninvolved. These changes were possibly the result of a mild to moderate murine respiratory mycoplasmosis. In some animals of both control and treated groups there was evidence of pulmonary oedema with accumulation of an eosinophilic proteinaceous material in alveolar spaces. This change was considered to be of agonal and post mortem occurrence. Hence, the test material was concluded not to produce any pathological changes.

MYELOGRAMS: There were no differences in cell/fat and myeloid/erythroid ratios between control and treated animals. Megakaryocytes were plentiful in all rats and there was no evidence of tumour or granuloma infiltration. Bony spicules were normal in treated and untreated animals alike. The eosinophilia observed in one high dose female could be associated with extramedullary haematopoiesis in the liver of this animal, however, the eosinophilia, neutrophilia, and lymphocytosis observed in other control or treated animals cannot be correlated with any histological changes. Hence, the test material was concluded not to cause effects on bone marrow haematopoiesis.

Effect levels

open allclose all
Dose descriptor:
Effect level:
>= 80 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: highest dose tested
Dose descriptor:
Effect level:
>= 620 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: highest dose tested

Target system / organ toxicity

Critical effects observed:

Applicant's summary and conclusion

The test item was tested for repeated inhalation toxicity similar or equivalent to the OECD TG 412, but not in compliance with GLP. The test article did not exhibit any systemic toxicity after repeated exposure to concentrations up to 620 mg/m³. Hence, the NOAEC was set at >= 620 mg/m³, which was the highest dose tested.