Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Qualifier:
according to
Guideline:
EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)
Qualifier:
according to
Guideline:
EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE experimental toxicology and ecology
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 1,2,3,4-Tetrahydroisoquinoline
- Purity: 93.2 area-% (analytical report 08L00186)
- Test substance number: 05/0797-2
- Batch number: 0640710217
- Homogeneity: given
- Storage stability: expiry date: 18 June 2010
- Date of production: Feb 2006
- Physical state / appearance: Liquid / yellowish, clear
- Storage conditions: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Age at arrival: about 7 weeks
- Weight at initiation of treatment: males 251-254 g, females 177-179 g (group mean weight ranges)
- Housing: The rats were housed together (5 animals per cage) in H-Temp polysulfonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2); dust-free wooden bedding ; enrichment: wooden gnawing blocks (Typ NGM E-022), supplied by Abedd® Lab. and Vet. Service GmbH, Vienna, Austria.
- Diet: mouse/rat maintenance diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Switzerland) ad libitum (no access to diet during exposure)
- Water: tap water ad libitum (no access to water during exposure)
- Acclimation period: the animals arrived about 2 weeks before initiation of treatment
- Adaptation to the exposure conditions: 2 days (on study days -2 and -1)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24;
- Humidity (%): 30-70;
- Air changes (per hr): fully air-conditioned;
- Photoperiod (hrs dark / hrs light): 12/12 (6.00 am - 6.00 pm/ 6.00 pm - 6.00 am);

Administration / exposure

Route of administration:
other: vapour for test groups 1 and 2, mixture of vapour and liquid aerosol for test group 3
Type of inhalation exposure:
nose/head only
Vehicle:
other: Air and ethanol. The mean measured concentrations of ethanol in the test atmosphere were 339.1, 299.2, 325.6 and 248.3 ppm for the control, low-, mid- and high-concentration, respectively.
Remarks on MMAD:
MMAD / GSD: The sample of test group 3 showed most (86.9 %) of the substance in impactor on the back up filter. As the EACD (effective aerodynamic cutoff diameter 50%) of the last impactor stage is 1.2 μm, MMAD was below 1.2 μm, indicating that the aerosol was highly respirable.
Details on inhalation exposure:
GENERATION OF THE INHALATION ATMOSPHERES
- Generator systems:
• Piston metering pumps (Sarstedt DESAGA)
• Two-component atomizers (stainless steel, Schlick mod. 970)
- Generation procedure:
The test substance was diluted as followed
• Group 1: 1 part test substance + 99 parts ethanol absolute (w/w)
• Group 2: 1 part test substance + 24 parts ethanol absolute
• Group 3: 1 part test substance + 7 parts ethanol absolute
For each concentration the test substance was supplied to a two-component atomizer at a constant rate by means of a metering pump. The aerosol was generated with compressed air mixed with conditioned dilution air and passed into the inhalation system. The solvent ethanol evaporated completely. Below the saturation concentration of the test substance, the aerosol droplets evaporated very fast so that the animals were exposed to vapour only. At high concentration above the saturation concentration, the aerosol droplets evaporated partly so that the animals were exposed to a mixture of vapour and liquid aerosol.
In the control group, solvent was sprayed at the pump rate used for the high concentration.
The following test pump rates and air flows were scheduled: pump rate (mL/h) ethanol feed 5-15 (control group) ; pump rate (mL/h) substance feed 5-15 (test groups 1-3); supply conditioned air (m3/h) 4.2-4.8 (all groups); supply compressed air (m3/h) 1.2-1.8 (all groups); exhaust air (m3/h) 5.1-5.7 (all groups). Conditioned supply air is activated charcoal filtered air conditioned to about 50% ± 20% relative humidity and 22°C ± 2°C. Compressed air is filtered air pressurized to about 6 bar.

HEAD-NOSE EXPOSURE SYSTEM
The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 60, BASF SE) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
NOMINAL CONCENTRATION: not determined.

ANALYTICAL DETERMINATION OF CONCENTRATIONS
Ethanol:
At the tested concentration, ethanol is expected to evaporate completely. Thus, the solvent ethanol was determined by infrared spectroscopy in all groups. The infrared spectroscopy was carried out as a separate study at the Competence Center Analytics Department of BASF SE, Ludwigshafen, Germany under the responsibility of the study director of this facility. The study was carried out in compliance with the principles of Good Laboratory Practice.

Test material:
Atmospheric concentration of 1,2,3,4-Tetrahydroisoquinoline was determined by gas chromatography analyses of absorption samples. Gas chromatographic analyses were carried out at the Analytical Chemistry Laboratory of the Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. Daily means were calculated based on 2 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived. At 75 mg/m3 a mixture of liquid aerosol and vapour are present. Aerosol and vapour fraction was determined separately at 75 mg/m3. At 5 and 15 mg/m3 majority of the test substance is present as vapour, thus no differentiation was made for these two lower concentrations.
Collection of samples for gas chromatography:
Equipment: Air sampler GS 312 (DESAGA); Absorption solvent: ethanol per analysis; Glass sampling probe (internal diameter: 7 mm) with quartz wool plug, 2 glass absorption vessels for the first sampling and 1 glass absorption vessel for further sampling.
Sampling frequency: as a rule, 2 samples per concentration and exposure
Sampling site: immediately adjacent to the animals' noses
Sampling flow rate: 3 L/min
Sampling velocity: 1.25 m/s
Sampling volumes: 90 L for test group 1, 18 L for test group 2 and 6 L for test group 3 (volumes were adjusted to achieve suitable amounts of the test substance in the samples in reference to the calibration of the analytical method).
The samples were drawn through the absorption vessels connected in series, each of which was filled with ethanol per analysis as absorption solvent. For test groups 1 and 2, the content of the probe with quartz wool plug and all absorption vessels except the last one in series was eluted and pooled into a 50 mL graduated flask for individual analysis. The content of the last absorption vessel was transferred separately to a 50 mL graduated flask and analyzed to check for the absorbing efficiency of sampling. For test group 3, the content of the probe and quartz wool plug was analyzed to determine the aerosol concentration, and the absorption vessels were analyzed to determine the vapor fraction.

Real time monitoring of constancy of concentrations:
In test group 1-3 the vapor fraction was continuously analyzed by on-line infrared spectroscopy.
In test group 3 the constancy of concentration was measured by scattered light photometers RAM1 (Mie, USA). The measurements were recorded using line recorders and transferred to the automated measuring system.

Particle size analysis (test group 3 only):
The particle size analysis was carried out with a cascade impactor. However, as the test material evaporated from the impactor during sampling, the measured aerodynamic diameter did not reflect the particle size in the test atmosphere. For this reason, only one sample was taken.
Equipment: Stack sampler Mark III (Andersen) without preimpactor; Vacuum compressed air pump (Millipore); Limiting orifice 3 L/min (Millipore); Sampling probe internal diameter 6.9 mm; Absorption solvent: ethanol per analysis.
Sampling: A backup particle filter (glass fiber) was placed into the cascade impactor and a sample of 540 L was taken at a sampling velocity of 1.25 m/sec from the breathing zone of the animals.
The deposits in the probe and the wall losses in the impactor were also determined by eluting the respective parts of the impactor and analysis by gas chromatography (same method as used for concentration analysis).

Duration of treatment / exposure:
6 hours per day
Frequency of treatment:
each workday, total of 65 exposures
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
test group 1 (target 5 mg/m3): 5.2±0.9 mg/m3
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
test group 2 (target 25 mg/m3): 24.7±2.5 mg/m3
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
test group 3 (target 75 mg/m3): 75.1±9.2 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
10
Control animals:
other: yes, exposed to ethanol as solvent control
Details on study design:
The selection of the concentration is based on the results of a 14-day range finding study in which atmospheric concentrations of 5.4 ± 1.8, 31 ± 8 and 150 ± 25 mg/m3 were tested. At 150 mg/m3, decreased body weight gain was found in male animals on study day 7 and 13 and in females on study day 7. The decreased body weight gain in male rats was accompanied by decreased food consumption: -48 % on study day 2 and -25 % on study day 9. Decreased food consumption was also found in female animals, though in a less severe grade (-30 % on study day 2 and -19 % on study day 9). The findings were concentration-related and indicated a general distress induced by inhalation exposure to the test article. Moreover, histological examination of the respiratory tract revealed strong irritation of the upper respiratory tract. Degeneration (some animals with ulceration) of the squamous epithelium was observed in level I of nasal cavity. In level I of the larynx epithelial alteration, mucous cell metaplasia and lymphoid cell infiltration was noted. Moreover, mild mucous cell hyperplasia in the large bronchi was present in most of the exposed animals. At 31 mg/m3, mucous cell hyperplasia was still observed in one female animal. Based on the histological findings, the No Observed Adverse Effect Concentration (NOAEC) was 5 mg/m3 in the range finding study.
Considering the considerably decreased body weight gain and the long exposure period of 90 days, the following concentrations were selected for the 90-day study: 5 mg/m³ as low concentration and expected NOAEC, 25 mg/m³ as mid concentration, 75 mg/m³ as high concentration causing toxic effects.

Examinations

Observations and examinations performed and frequency:
MORTALITY:
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) on working days and once a day (in the morning) on Saturdays, Sundays and public holidays.

CAGE SIDE OBSERVATIONS: Yes
The clinical condition of the test animals was recorded once during the preflow period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible.

DETAILED CLINICAL OBSERVATIONS: Yes
Detailed clinical observations were performed in all animals prior to the exposure period and thereafter in weekly intervals. The findings were ranked according to the degree of severity, if applicable. The animals were transferred to a standard arena (50 x 37.5 cm with sides of 25 cm high). The following parameters were examined: abnormal behavior during handling; fur; skin; posture; salivation; respiration; activity/arousal level; tremors; convulsions; abnormal movements; impairment of gait; lacrimation; palpebral closure; exophthalmus; feces (appearance/consistency); urine; pupil size.

BODY WEIGHT: Yes
The body weight of the animals was determined at the start of the preflow, at the start of the exposure period and then once a week as well as prior to gross necropsy. As a rule, the animals were weighed at the same time of the day. Body weight change was calculated.

FOOD CONSUMPTION: Yes
Food consumption was determined weekly per cage and calculated as mean food consumption in grams per animal per day.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
Before the start of the exposure period (day -2 males; -3 females) the eyes of all animals, and on study day 75 the eyes of the animals of the control group and the high concentration group were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, FRG) after administration of a mydriatic (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

HAEMATOLOGY: Yes
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: all
- Parameters examined: haemoglobin, red blood cells, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, reticulocytes, total white blood cells, differential white blood cells (absolute and relative), thrombocytes, prothrombin time

CLINICAL CHEMISTRY: Yes
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: all
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamuyl transferase, total bilirubin, total protein, albumin, globulins (calculated), glucose, total cholesterol, triglycerides, creatinine, urea, electrolytes (calcium, chloride, potassium, sodium, inorganic phosphate, magnesium)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time of examination: study day 75 (the animals were not exposed on this day)
- Dose groups that were examined: all groups, 5 males and 5 females per group
- Battery of functions tested (FOB and motor activity): home cage and open field observations, sensorimotor tests, reflexes, motor activity
Sacrifice and pathology:
SACRIFICE
The animals were killed under Narcoren anesthesia by exsanguination from the abdominal aorta and vena cava.

GROSS PATHOLOGY
All animals, including the animal (no. 21 exposed to 25 mg/m3) that died intercurrently, were necropsied and assessed by gross pathology.

ORGAN WEIGHTS AND TERMINAL BODY WEIGHT
At scheduled sacrifice, the anesthetized animals and the following organs were weighed: adrenal glands, brain, epididymides, heart, kidneys, liver, lung, ovaries, spleen, testes, thymus, thyroid glands, uterus.

TISSUE PRESERVATION
The following organs/tissues of all animals were preserved in neutral buffered 4% formaldehyde solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Colon, Duodenum, Epididymides, Esophagus, Extraorbital lacrimal glands, Eyes with optic nerve, Femur with knee joint, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lungs, Lymph nodes (mediastinal, mesenteric and axillary lymph nodes), Mammary gland (female), Nose (nasal cavity), Ovaries, Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Seminal vesicle with coagulation glands, Sciatic nerve, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cords), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid glands, Trachea,Urinary bladder, Uterus, Vagina.

HISTOPATHOLOGY
The hematoxylin-eosin stained slides were assessed by light microscopy. Histopathology was conducted on the preserved tissues (except for the cervix, axillary lymph nodes and coagulation glands) of all animals of the control group and the high-concentration group. In addition, gross lesions were examined in the affected animals of the intermediate concentration groups and histopathology of the larynx (affected level I only) was extended to the low- and mid-concentration groups.
Statistics:
- Body weight / body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means.
- Fecal pellets, rearing, grip strength forelimbs, grip strength hindlimbs, footsplay test, motor activity: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
- Clinical pathology parameters: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians.
- Narcotized animal body weight and organ weights: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each test group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS (INCLUDING DETAILED CLINICAL OBSERVATIONS) AND MORTALITY
One male rat exposed to 25 mg/m³ died on study day 7 before exposure. Because of the absence of clinical signs in this animal and no mortality occurred in test group 3, the dead of the animal was regarded as incidental.
During the exposure period, the animals of the control group and low concentration (5 mg/m³) showed no clinical signs and findings different from normal except 1 female of the low dose group showing salivation beginning on study day 49.
During the exposure period, the animals of the mid and high concentration (25 and 75 mg/m³) showed following abnormal clinical signs: salivation (8/10 male and female animals in test group 2 beginning day 42; all animals of test group 3 beginning study day 33) and reduced fur care (9/10 male and all female animals in test group 2 beginning on study day 42; all animals in test group 3 beginning on study day 29). Additional all male and female animals of test group 3 attempted to escape beginning on study day 5. One animal of test group 3 showed local irritation (swelling) of the right ear beginning on study day 69.

BODY WEIGHT AND WEIGHT GAIN
The body weight of the animals of the low concentration (5 mg/m³) was not statistically significantly different from the control group.
The body weight of the male animals of the mid (25 mg/m³) and high concentration (75 mg/m³) was statistically significantly decreased beginning on study day 28 and 21 for test group 2 and 3, respectively. In average the body weight was only slightly lower than the concurrent control group (between -5.6 and -7.7% for mid concentration animals and between -5.7 and -8.4% for high concentration animals).
The body weight of the female animals of the mid and high concentration (25 and 75 mg/m³) was statistically significantly decreased about 5.1 to 6.5 % on days 35-49 in test group 2 and day 7 and 42 in test group 3.
In accordance to the reduced body weight, the body weight change of male animals of the mid (25 mg/m³) and high (75 mg/m³) concentration group was significantly decreased throughout the exposure period. The body weight change of female animals was significantly decreased at the mid concentration (25 mg/m³) on study day 7 through to study day 63, and at the high concentration (75 mg/m³) on the study days 7 and 42.

FOOD CONSUMPTION
In male animals food consumption was slightly lowered compared to controls at the mid- and high concentration throughout the study. In female animals no changes in food consumption was observed.

OPHTHALMOSCOPIC EXAMINATION
Spontaneous findings such as remainders of the pupillary membrane, corneal stippling or striation of lens were observed in several animals of all test groups and the control group without any concentration-response relationship.

HAEMATOLOGY
No treatment-related changes were measured among hematological parameters.
In males of test group 2 and 3 (25 mg/m3 and 75 mg/m3) platelet counts were statistically significantly lower compared to controls. This decrease was not dose-dependent and was not measured in dosed female rats. Therefore, this alteration was regarded as incidental rather than treatment-related.
Relative counts of large unstained cells (LUC) were higher in males of test group 2 (25 mg/m3) compared to controls. This change was not dose dependent, no other alteration in the differential blood cell count was measured, and the median of the relative LUC counts in the mentioned dose group was within the historical control range (0.2 – 0.6 %). Therefore, this change was regarded as incidental and not treatment-related.
In females of test group 3 (75 mg/m3) the prothrombin time (HQT) was prolonged compared to controls, but the median prothrombin time was within the historical control range (26.3 – 37.9 seconds). Therefore, this alteration was regarded as incidental and not treatmen-trelated.

CLINICAL CHEMISTRY
- In male and female rats of test groups 2 and 3 (25 and 75 mg/m3) total serum protein levels were decreased correlating with the decrease of globulin concentrations in these animals. Additionally, in males of test group 1 (5 mg/m3) the globulin values were statistically significantly lower than the controls. These lower globulin levels were not accompanied by a decrease of the total protein levels or any other relevant clinical pathology parameter. Therefore, lower globulin levels in males of test group 1 may be treatment-related, but they were not regarded as an adverse effect.
- Glucose concentrations in rats of both sexes of test group 3 (75 mg/m3) were decreased compared to controls.
- Inorganic phosphate concentrations were higher in rats of both sexes of test group 3 (75 mg/m3) and additionally in males of test groups 1 and 2 (5 and 25 mg/m3). At least in the latter dose groups phosphate levels were not dose-dependently increased and the medians were within the historical control range (1.51 – 1.96 mmol/L). Therefore, higher phosphate levels in males of test group 1 and 2 were regarded as not treatment-related.
- Chloride concentrations in males of test group 3 (75 mg/m3) were statistically significantly lower compared to controls. This was the only altered electrolyte level in these animals and the median was within the historical control range (100.2 – 107.4 mmol/L). Therefore, this change was regarded as incidental and not treatment-related.

NEUROBEHAVIOUR
Due to lethality of one male animal in test group 2 (25 mg/m³), FOB and MA were performed only in 4 male animals of this group. This does not impair the significance of this study.
FOB: Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental.
Overall motor activity (summation of all 12 intervals): There were no statistically significant deviations from the control group.
Single motor activity intervals (5 min/interval): No abnormalities were detected.

ORGAN WEIGHTS AND TERMINAL BODY WEIGHT
- Absolute weights:
Compared to control group (= 100%), the following mean absolute weights were statistically significantly increased or decreased: terminal body weight male animals group 2 (93%**) and 3 (92%**); thymus weight male animals group 1 (119%*) and 3 (82%*) and thymus weight female animals group 3 (84%*). All other mean absolute weight parameters did not show significant differences when compared to the control group.
- Relative weights:
Compared to control group 0 (= 100%), the following mean relative weights were statistically significantly increased or decreased: adrenal glands male animals group 2 (112%*), brain male animals group 2 (107%**) and group 3 (110%**), heart male animals group 1 (114%*) and group 3 (114%*), kidney male animals group 2 (108%*) and group 3 (116%**), testes male animals group 3 (118%**) and thymus male animals group 1 (122%**). All other mean relative weight parameters did not show significant differences when compared to the control group.
Only the statistically significant decrease of terminal body weight in treated males of test group 2 (25 mg/m³) and 3 (75 mg/m³) is regarded a test-substance related effect.
All other deviations in absolute and/or relative organ weights of treated male and female animals are regarded incidental and not related to treatment, either due to a missing dose-response relationship (thymus, adrenal glands, heart) and/or due to the decreased terminal body weight (thymus, brain, heart, kidneys, testes) and of no further biological relevance.

GROSS PATHOLOGY
All gross lesions observed in test animals occurred singularly. They are considered to be spontaneous lesions in origin and are not related to treatment.

HISTOPATHOLOGY
The level I of larynx (level: base of the epiglottis) revealed focally at the base of epiglottis a minimal to slight epithelial alteration, a minimal squamous metaplasia and a lymphohistiocytic inflammation in a concentration-dependent manner.
Regarding the epithelial alteration (characterized by a focal flattening of epithelial cells) the number of affected males was comparable between control animals (4 animals) and animals of test group 2 (5 animals), additionally no animal of test group 1 showed this finding. The number of affected males in test group 3 (7 animals) was slightly increased. The number of affected females was increased in test group 2 (8 animals) and 3 (9 animals) accompanied by an increased severity compared to control females (3 animals) and test group 1 females (1 animal). A minimal focal squamous metaplasia at the base of epiglottis was seen in 3 males of test group 3. Additionally, 2 males and 9 females of test group 3 showed a minimal to moderate submucosal lymphohistiocytic inflammation, which was found also in 1 control female animal.
Additionally, one male animal of test group 3 revealed a minimal focal epithelial alteration in level II of larynx.
Taken together, the histopathological findings in the larynx (mainly level I) indicate test substance-/concentration-related findings in the male animals of test group 3 (75 mg/m³) and in females of test group 2 (25 mg/m³) and 3 (75 mg/m³).
The epithelial alteration in males of control group and test group 2 and in females of control group and test group 1 are considered spontaneous lesions in origin and not related to treatment.
All other findings noted were single observations either, or were similarly in distribution pattern and severity in control rats compared to test groups. All of them are considered to be incidental and/or spontaneous in origin and without any relation to test substance administration.

Effect levels

Dose descriptor:
NOAEC
Effect level:
5 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on clinical signs indicative of irritation, slightly decreased growth rate and food consumption, some biochemical alterations and histopathological changes at the base of the epiglottis at higher concentrations.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion