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Repeated dose toxicity: inhalation

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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other: Study conducted in compliance with GLP and OECD guideline, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
GLP compliance:
yes (incl. QA statement)
TNO Triskelion, Utrechtseweg 48, P.O. Box 360, 3700 AJ Zeist, The Netherlands
Limit test:

Test material

Constituent 1
Test material form:
gas under pressure: liquefied gas

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River, Deutschland, Sulzfeld, Germany
- Age at study initiation: 7 weeks
- Mean weight at study initiation: 295 g for males and 199 g for females
- Fasting period before study: No
- Housing: Macrolon cages with a bedding of wood shavings (Lignocel, Type ¾) and strips of paper (Enviro-dri) as environmental enrichment. Initially, all animals were housed five per cage, separated by sex. However, from 2 July 2010 onwards, because of their increased weight, male animals were housed 2 or 3 animals per cage.
- Diet: Rat and Mouse No. 3 Breeding Diet RM3 (SDS Special Diets Services, Witham, England) was available ad libitum, except during periods of exposure to test substance
- Water: Tap water was available ad libitum via polypropylene bottles, except during periods of exposure to test substance.
- Acclimation period: 8 Days

- Temperature (°C): 22-24
- Humidity (%): 45 - 65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: gas
Type of inhalation exposure:
nose only
Details on inhalation exposure:
- Exposure equipment: Animals were exposed to the test atmosphere in nose-only exposure units. Each unit consisted of a cylindrical PVC column with a volume of ca. 70 litres, surrounded by a transparent hood. The test atmosphere was introduced at the bottom of the central column, and was exhausted at the top. Each column included three rodent tube sections and each rodent tube section accommodated 20 ports for animal exposure. Additional ports were used for test atmosphere sampling, measurement of oxygen concentration, temperature and relative humidity. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column (males and females alternated). The remaining ports were closed. Only the nose of the rats protruded into the interior of the column. The units were illuminated externally by normal laboratory TL-lighting.

- The inhalation equipment was designed to expose the rats to a continuous supply of fresh test atmosphere. To generate the test atmosphere, a flow of cooled liquid test material controlled by a peristaltic pump was allowed to evaporate in a flow of humidified air (mass flow controlled). The air was supplemented with mass flow controlled oxygen for the mid and high concentrations to ensure a sufficiently high and equal oxygen concentration. Total test atmosphere flow was between 27 and 28 L/min for all groups except during the period from 7 June to 1 July 2010 (the period when also animals of a prenatal developmental inhalation toxicity study were exposed) when it was between 54 and 55 L/min and on 28 June 2010 where it was between 29 and 30 L/min. At all times the flow was higher than 1 L/min/animal with regard to the amount of animals present. The exposure unit for control animals was supplied with a mass flow controlled stream of humidified compressed air only.
- The measured concentrations were used in a PI feedback system to control the peristaltic pumps. The feed back system took into account the proportional (P) and the integrated deviations (I) of the concentrations from the set point.
- The settings of the mass flow controllers were checked each morning at the start of the generation and subsequently at regular intervals during exposure (approximately bi-hourly, i.e. three times a day).

- The temperature and the relative humidity of the test atmospheres were measured continuously and recorded every minute during exposure using a CAN transmitter with temperature and relative humidity probes.
- Mean temperature (± standard deviation) during exposure was 23.8 (± 0.4), 23.5 (± 0.5), 23.4 (± 0.5) and 23.6 (± 0.5)°C for the control, low, mid and high concentration exposure conditions, respectively. Measured minimum and maximum temperatures were 20.2 and 25.1°C, respectively. The temperature ranged between 25.0 and 25.1°C during 70 minutes on 11 June for the control group, during 23 minutes on 9 July for the low concentration group and during 10 minutes on 9 July for the high concentration group.
- Mean relative humidity (± standard deviation) during exposure was 40% (± 3), 40% (± 3), 38% (± 2) and 37% (± 3), respectively. Measured minimum and maximum relative humidity was 28 and 74%, respectively. Relative humidity was less than 30% (minimum 28%) during periods ranging from 1 to 9 minutes on 27 May, 1, 15, 20 and 21 July and 19 and 26 August for the high concentration group and on 1 July for the control group. Relative humidity was higher than 70% for group 1 on 1 June (duration 5 min, maximum 74%), for group 3 on 6 July (duration 3 min, maximum 71.5%) and for group 4 on 23 July (duration 1 min, maximum 71%) and on 5 August (duration 5 min, maximum 72.5%.

Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The actual concentration of the test substance in the atmospheres was measured by total carbon analysis. The test atmospheres were sampled from the exposure units at the animals’ breathing zone and were passed to total carbon analyzers. The response of the analyzers was recorded on a pc every minute using a CAN transmitter. The daily mean response was calculated by averaging values read every minute.
The total carbon analyzers were calibrated in the period 18-21 May 2010. Calibrations were done by sampling from 3 concentrations (in duplicate) in a range including the target concentration. The concentrations of the test material used to calibrate the total carbon analyzer were prepared in a sample bag by injecting known amounts (by mass) of the (cooled) test material. The calibrations were checked weekly by measuring the concentration from a freshly prepared sample bag with a concentration around the target concentration. The measured concentrations from the sample bags did not deviate more than 5% from the calculated concentration in the sample bags during the study (maximum difference 2.1%, 4.4% and -2.5% for the low, mid and high concentration, respectively). At the end of the study calibration was also checked with a sample bag with a concentration around the target concentration. Results were 0.5%, 5.7% and -0.7% deviations for the low, mid and high concentration, respectively. Because the deviation of the mid concentration was above 5%, the measurement was repeated with a newly prepared sample bag. The result was a deviation of the calculated concentration of 3.7%.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week (i.e., 65 exposure days over a 91-day study period)
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 4000, 10000 or 15000 ppm
other: target concentrations
Doses / Concentrations:
0, 3987, 9974 or 14903 ppm
analytical conc.
No. of animals per sex per dose:
Control animals:
Details on study design:
- Dose selection rationale: Doses were selected based on results of a sub-acute (28-day) inhalation toxcity study with this test substance in rats.
- Group 1: 0 ppm, Group 2: 4000 ppm, Group 3: 10000 ppm, Group 4: 15000 ppm.


Observations and examinations performed and frequency:
Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A group-wise observation was made halfway during each exposure day. On working days, all cages were checked again in the afternoon. At weekend days and public holidays only one check per day was carried out.

The body weight of each animal was recorded once during the acclimatization period (one or two days before the start of the study; nominal day -1/-2 for males and females respectively), at initiation of treatment prior to the first exposure (Nominal day 0), and weekly thereafter. In addition, animals were weighed before sacrifice for calculation of relative organ weights..

Food consumption of the main groups was measured per cage, over successive weekly periods (starting on Nominal day 0). The results were expressed in g per animal per day.



Ophthalmoscopic examinations were made prior to the first exposure in all animals and during the last week of exposure in animals of the control and high concentration group (groups 1 and 4). Because no treatment-related changes were observed in animals of the high concentration group, the eye examinations were not extended to the animals of the intermediate concentration groups (groups 2 and 3). Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.

At scheduled necropsy (Nominal day 91) blood samples of all surviving animals were taken from the abdominal aorta of (overnight) fasted rats whilst under pentobarbital anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count. The following parameters were calculated: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC).

Clinical chemistry determinations were conducted on blood plasma of all surviving animals. Blood samples were collected in heparinized plastic tubes at the same time blood samples for haematology were collected. Plasma was prepared by centrifugation. The following measurements were made: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, fasting glucose, bilirubin total, cholesterol, triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl), inorganic phosphate

On the day before necropsy, animals were transferred to metabolism cages (one animal per cage) for overnight urine collection. During urine collection the animals were deprived of food (approximately 16 hours) but not of water. The following determinations were carried out in individual samples: volume, density, appearance, dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen), microscopic examination of the sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, worm eggs).

Sacrifice and pathology:
At the end of the exposure period, all surviving animals were killed in such a sequence that the average time of killing was approximately the same for each group. Animals were killed on Nominal day 91. The animals were killed by exsanguination from the abdominal aorta under pentobarbital anaesthesia and then examined grossly for pathological changes. The following organs were weighed (paired organs together) as soon as possible after dissection (to avoid drying): adrenals, brain, heart, kidneys, liver, spleen, testes, thymus, thyroids (with parathyroids), lungs with trachea and larynx, ovaries, uterus, epididymides

Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde (10% solution of formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to insure fixation. adrenals, aorta, axillary lymph nodes, brain (brain stem, cerebrum and cerebellum), caecum, colon, epididymides, eyes (with optic nerve), exorbital lachrymal glands, femur with joint, heart, kidneys, liver, lungs/trachea/larynx, lungs/trachea/larynx, mammary glands (females), cervical lymph nodes, nasopharyngeal tissue (with nasal associated lymphoid tissue and teeth), nerve-peripheral (sciatic nerve), oesophagus, olfactory bulb, ovaries, pancreas, parathyroids, pharynx, parotid salivary glands, pituitary, prostate, rectum, seminal vesicles with coagulating glands, skeletal muscle (thigh), skin (flank), small intestines (duodenum, ileum, jejunum), spinal cord (three levels), spleen, sternum with bone marrow, stomach (glandular, non-glandular), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, tongue, tracheobronchial (mediastinal) lymph nodes, ureter, urethra, urinary bladder, uterus (with cervix), all relevant gross lesions.

- Preparation of slides: All tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin.
- Histopathological examination: The organs of the list above were examined in animals of the high concentration group (group 4) and control group (group 1). The nasal tissues were examined at 6 levels, the larynx at 3 levels, the trachea at 3 levels (including the bifurcation), and each lung lobe at 1 level. A few gross lesions and the hearts of the animals of the low and mid concentration groups were also examined microscopically.
- Body weight data, clinical pathology data measured on continuous or semi-continuous scales, and organ weights: data were analysed using one-way analysis of variance (Anova), after checking for homogeneity of variance (Bartlett test) and normality of data distribution (Shapiro-Wilks test). If variances were not homogeneous or data not normally distributed, the data were stepwise log or rank transformed prior to the Anova. If the Anova yields a significant effect (p<0.05), intergroup comparisons with the control group were made by Dunnett’s multiple comparison test.
- Food consumption: no statistics were applied on food intake (only two cages per sex).
- Incidences of histopathological changes: Fisher’s exact probability test.
Tests generally performed as two-sided tests with results taken as significant where the probability of the results is p<0.05 or p<0.01. Because numerous variables were subjected to statistical analysis, the overall false positive rate (Type I errors) is greater than suggested by a probability level of 0.05. Therefore, the final interpretation of results was based not only on statistical analysis but also on other considerations such as dose-response relationships and whether the results are significant in the light of other biological and pathological findings.

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:
no effects observed
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:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
- The individual observations in the mornings, before the start of each day’s exposure did not indicate exposure-related clinical abnormalities. The group-wise observations during each day’s exposure did not reveal abnormalities.

- No significant differences in body weights between the groups were seen.

- Food consumption was similar among the groups throughout the study period.

- Near the end of the exposure period no abnormalities were seen in groups 1 and 4, therefore the animals of groups 2 and 3 were not investigated.

- The concentration of thrombocytes was statistically significantly decreased in the female animals of the low and mid concentration group. Since a concentration-response relation was not seen, these small differences were not considered related to the treatment. Other parameters associated with the red blood cells were not different among the groups. The relative concentration of the lymphocytes was decreased in male animals of the high concentration group. The decrease in the relative concentration of the lymphocytes and the increase of the relative concentration of the neutrophils in female animals of the low concentration group was considered not related to the treatment because of the absence of a relation with the concentration.

- The concentration of ASAT and ALAT was significantly increased in plasma of the male animals of the high concentration group. The isolated small significantly decreased concentration of ASAT in male animals of the low concentration group was not considered to be treatment-related. In female animals of the mid and high concentration groups, glucose and urea were significantly increased. Potassium was significantly increased and triglyceride was slightly, but significantly decreased in female animals of the high concentration group.

- Significant differences in urinary volume and density were not seen. Measurements indicated that the amount of occult blood was significantly increased in male animals of the high concentration group. Other parameters or microscopic observations of urine did not show significant changes.

- The relative and absolute weight of the heart was significantly decreased in male animals of the high concentration group. In female animals of that group relative weight was also significantly increased. In male animals of the high concentration group, relative liver weight was slightly, but significantly increased. The significant increase in relative kidney weight of the male animals of the low concentration group was considered an isolated incidental finding, not related to the treatment. Other significant differences in organ weight were not found.

- Macroscopic examination at necropsy did not reveal treatment-related gross changes.

- Microscopic examination revealed treatment related histopathological changes in the hearts of the treated females and males. The changes comprised very slight to moderate multifocal (diffuse in one high-dose male) mononuclear cell infiltrates in the ventricular part of the heart muscle. Several animals showed very slight to slight focal mononuclear cell infiltrates in the heart, but this was not considered treatment related because this finding is a common background change and occurred also in some control animals. The multifocal inflammatory infiltrations were observed in 9/10 high-dose males, in 5/10 high-dose females, in 7/10 mid-dose males and in 1/10 low-dose males. In several cases the inflammatory changes were accompanied by vacuolisation of cardiac muscle cells. The males were clearly more affected than the females. Because of the presence of moderate multifocal mononuclear cell infiltrates with concomitant muscle cell vacuolation in the heart of one male animal of the low-concentration group, a No Adverse Effect Level could not be established. The other histopathological changes observed are common findings in rats of this age and strain. Furthermore, they were about equally distributed amongst the groups or they occurred in only one or a few animals.

Effect levels

Dose descriptor:
Effect level:
4 000 ppm
Based on:
test mat.
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Under the conditions of the test, a NOAEL in rats exposed to the test substance for 6 hours/day, 5 days/week for a period of 90 days could not be derived. Therefore the lowest concentration was considered to be the LOAEC: 4000 ppm (21240 mg/m3).
Executive summary:

In a 90-day repeated dose toxicity study, performed according to OECD Guideline 413 and under GLP, the test substance was administered by inhalation 6 hours/day on 5 days/week at concentrations of 4000, 10000, and 15000 ppm to groups of 10 Sprague-Dawley rats per sex each. The actual concentrations, as measured by total carbon analysis, were close to the target concentrations. Daily observation of the animals did not reveal treatment-related clinical abnormalities. Treatment-related differences in body weight gain or food consumption were not seen. Ophthalmoscopic examination near the end of the exposure period did not reveal any abnormalities. Treatment-related effects in red blood cell parameters were not seen. In male animals of the high concentration group the relative concentration of lymphocytes was decreased. No other treatment-related effects were seen in any of the other haematology parameters. In the high concentration group, the concentration of ASAT and ALAT in blood plasma was significantly increased in male animals. In female animals of the high and mid concentration groups glucose and urea were increased. Female animals of the high concentration group also showed increased potassium and decreased triglyceride levels. Other treatment-related changes in clinical chemistry parameters were not seen. Occult blood in urine was significantly increased in male animals of the high concentration group. The relative and absolute weight of the heart was significantly decreased and the relative weight of the liver was significantly increased in male animals of the high concentration group. Relative weight of the heart was also significantly decreased in female animals of the high concentration group. No other treatment-related changes in absolute or relative organ weights were detected. Macroscopic examination at necropsy did not show exposure-related gross changes. Microscopic examination revealed that exposure to the test substance induced multifocal mononuclear cell infiltrates in the ventricular part of the heart muscle. It was seen in males and females of the high-concentration group and with a lower incidence in male animals of the mid and low concentration groups. Because the histopathological changes in the heart of one male animal of the low concentration group was considered to be related to the exposure, a NOAEL can not be given and the lowest concentration tested was considered to be the LOAEC: 4000 ppm (21240 mg/m3).