Registration Dossier

Administrative data

Description of key information

In a subchronic oral toxicity study in Wistar rats with administration of the test substance in drinking water for 3 months, the NOAEL was determined to be 64 mg/kg/day based on reduced water consumption at this dose level but without any corroborating changes in-life or pathologically (BASF SE, 2002).
In a subacute inhalation study in Wistar rats with administration of the test substance as liquid aerosol for 28 days a NOAEC could not be established for local irritation effects based on histopathology findings in larynx, trachea and lung (LOAEC = 3 mg/m³). For systemic effects the NOAEC was determined to be 30 mg/m³ (Triazine Task Force, 2011).
In a subchronic dermal toxicity study in rats there were no indications of systemic toxicity in any test group, whereas dermal reactions were observed at 50 and 250 mg/kg bw/day. Skin lesions included hair loss and scab formation and microscopically appeared as incidences of epidermal hyperplasia, scab formation, and ulceration of application sites (cited in EPA, 2008).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-09-11 to 2002-02-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 1157
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Wistar rats CrIGIxBrIHan
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 d
- Weight at study initiation: males 146.1 - 165.4 g(group mean: 154.9 g); females 116.5 - 131.8 g(group mean: 124.7 g.)
- Housing: single
- Cage type: DK III stainless steel wire mesh cages (Becker & Co., Castrop-Rauxel, Germany.)
- Diet (ad libitum): ground Kliba maintenance diet (Provimi Kliba SA, Kaiseraugst, Switzerland.)
- Water (ad libitum): demineralized drinking water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: drinking water
Vehicle:
water
Details on oral exposure:
DOSING SOLUTIONS AND EXPERIMENTAL PROCEDURE:
For each concentration, the test substance was weighed out in a beaker, transferred to a 10-litre can, and demineralized water was added to the desired volume. Subsequently, the preparations were mixed with a magnetic stirrer for about 5 minutes. The mixtures were prepared twice a week.
The test substance was administered daily in demineralized water for about 3 months. Control animals received demineralized water only . At the end of the 3-month administration period all animals were sacrificed (withdrawal of food for about 16 - 20 hours).

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analyses of the test substance preparations were carried out as a separate study. The stability of the test substance in water over a period of up to 96 days at room temperature was tested prior to the start of the study with a comparable batch. Concentration control analyses of the test substance preparations were performed with samples of all concentrations at the start and at the end of the administration period. Homogeneity analyses of the test substance preparations were not necessary as the preparations were aqueous solutions.
Duration of treatment / exposure:
3 months
Frequency of treatment:
continuously
Dose / conc.:
200 ppm
Remarks:
14 mg/kg bw/day in males; 21 mg/kg bw/day in females
Dose / conc.:
1 000 ppm
Remarks:
64 mg/kg bw/day in males; 91 mg/kg bw/day in females
Dose / conc.:
5 000 ppm
Remarks:
285 mg/kg bw/day in males; 339 mg/kg bw/day in females
No. of animals per sex per dose:
10
Control animals:
yes
Positive control:
None
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS:
The animals were examined for evident signs of toxicity or mortality twice a day (in the morning and in the late afternoon) from Mondays to Fridays and once a day (in the morning) on Saturdays, Sundays and public holidays. Additionally, further clinical examinations were carried out daily.

BODY WEIGHT:
Individual body weights were recorded weekly.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined weekly and calculated as mean food consumption in grams per animal and day. The mean daily intake of test substance (group means) was calculated based upon individual values for body weight and water consumption.

OPHTHALMOSCOPIC EXAMINATION:
One day prior to the start of the administration period the eyes of the animals of the all dose groups were examined for any changes using an ophthalmoscope (HEINE OPTOTECHNIK, Herrsching, Germany) after administration of a mydriatic (Pharma Stulln GmbH, Stulln, Germany). On day 91, only animals of the high dose group and controls were examined.

HAEMATOLOGY, CLINICAL CHEMISTRY:
Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anesthesia for examination of haematological and clinical chemical parameters. Following parameters were considered.

- Haematological parameters:
Leukocytes, erythrocytes, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelets and differential blood count.

- Clinical chemical parameters:
Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, serum-γ-glutamyltransferase, sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol and magnesium.

URINALYSIS:
The individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. The urine samples were evaluated in a randomized sequence.
The following examinations were carried out:
Volume, color, turbidity, pH, protein, glucose, ketones, urobilinogen, bilirubin, blood, specific gravity and sediment.

SPERM PARAMETERS:
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals. The following parameters were determined:
Sperm motility, sperm morphology, sperm head count (cauda epididymis) and sperm head count (testis).

NEUROBEHAVIOURAL EXAMINATION:
A functional observational battery (FOB) was performed in all animals at the end of the administration period. The FOB started with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotortests as well as reflex tests. Motor activity was measured on the same day as FOB was performed. The measurement was performed in the dark using the Multi-Varimex-System. During the measurement the animals were kept in Polycarbonate cages with absorbent material and received no food and no water.
Sacrifice and pathology:
At study termination, the animals were sacrificed by decapitation under CO2 anesthesia. The exsanguinated animals were necropsied, assessed by gross pathology, and organs were weighed. Tissues were collected and fixed for further histopathological examinations.

ORGAN WEIGHTS:
Liver, kidneys, adrenal glands, testes, epididymides, ovaries, uterus, spleen, brain, heart and thymus.

HISTOPATHOLOGY:
The following organs were fixed in 4 % formaldehyde solution:
All gross lesions, salivary glands (Glandula mandibularis and Glandula sublingualis), esophagus, stomach (forestomach and glandular stomach), duodenum/jejunum/ileum, cecum/colon/rectum, liver, pancreas, brain, pituitary gland, sciatic nerve, spinal cord (cervical, thoracic and lumbar cord), eyes, adrenal glands, thyroid glands, parathyroid glands, trachea, lungs, pharynx, larynx, nose (nasal cavities), aorta, heart, bone marrow (femur ), lymph nodes (mandibular and mesenteric), spleen, thymus, kidneys, urinary bladder, oviducts/uterus/vagina, prostate gland, seminal vesicles, female mammary gland, skin, skeletal muscle, sternum with marrow, femur with knee joint and extraorbital lacrimal glands. The left testis and the left epididymis as well as both ovaries were fixed in Bouin's solution and embedded in paraplast, thereafter.
Statistics:
Means and standard deviations of each test group were calculated for the variables of terminal body weight and of absolute and relative organ weights (related to terminal body weight) of the animals in each test group. Statistical analyses were performed using the Kruskal-Wallis test. The Wilcoxon test was used additionally for the hypothesis of equal medians.
Details on results:
CLINICAL SIGNS AND MORTALITY:
Neither mortalities nor clinical symptoms of toxicity were observed. Appearance and behavior of the animals showed no treatment-related changes.

BODY WEIGHT AND WEIGHT GAIN:
Body weight was significantly reduced in high dose males. Body weight change values of this group were significantly reduced during the entire treatment period. This was assessed as being treatment-related. No significant effects were seen in females.

FOOD CONSUMPTION:
Food consumption was significantly reduced in high dose males as well as in high dose females during the entire treatment period. The values were up to 13.3 % (males) and 18.9 % (females) below controls. This was assessed as being treatment-related.

FOOD EFFICIENCY:
Food efficiency was significantly reduced in high dose males and in mid dose males. Due to the isolated occurrence and the lack of a clear dose-response relationship, this was assessed as being incidental.

WATER CONSUMPTION:
Water consumption was significantly reduced in high dose males and females during the entire treatment period . The values were up to 35.7 % (males)and 50.4 % (females) below controls. Also in mid dose males and females, water consumption was reduced with statistical significance several days in males or most of the days in females, the values being up 23.7 % (males) and 27.1 % (females) below controls. This was assessed as being treatment-related .

OPHTHALMOSCOPIC EXAMINATION:
No substance-related effects were obtained. All findings were spontaneous in nature and equally distributed between treated animals and controls.

HAEMATOLOGY:
There are no treatment-related changes in the hematological parameters measured.

CLINICAL CHEMISTRY:
Compound-related differences in serum enzyme activities were not evident at any dose level in either males or females. Blood chemistry examinations revealed significantly increased urea concentrations in the serum of the high dose animals of either sex. No treatment-related changes were found in the other blood chemistry parameters examined.

URINALYSIS:
At the end of the study high dose males and females produced decreased amounts of urine with increased specific gravity. In the other urine parameters no treatment-related findings were observed.

ESTROUS CYCLE DETERMINATION:
In the estrus cycle determinations conducted from day 63 to day 91, no substance-related effects were obtained.

SPERM ANALYSIS:
There are no treatment-related changes in the sperm parameters measured.

NEUROBEHAVIOUR:
No substance-related effects were observed.

ORGAN WEIGHTS:
In female rats, the mean weight of the kidneys was significantly increased in the high dose group. The mean weight of the heart was significantly decreased in female rats of the mid dose group. In the high dose group, the mean heart weight of female rats was also significantly decreased, however, only slightly and without a dose-response relationship. In males of the high dose group, the mean weights of brain and adrenal glands were significantly increased in males of the high dose group. The other mean absolute weight parameters did not show significant differences when compared to the control group.

GROSS PATHOLOGY:
Only a few gross lesions were noted in the glandular stomach (erosion/ulcer), epididymides (abscess), ovaries (cyst) and skin (sparse hair). With one exception (sparse hair in two control females) these gross lesions occurred only once per group, with no indication of a relationship to treatment. They were hence all interpreted to have developed spontaneously and unrelated to treatment.

HISTOPATHOLOGY:
With the exception of the areas of sparse hair in the skin of two control female rats and of a low dose male rat, all gross lesions could be correlated with a meaningful histopathologic correlate. However, regardless of whether or not they had a microscopic correlate, all the gross lesions were considered to have developed spontaneously and to be unrelated to treatment. Histopathology failed to correlate the significantly increased mean absolute (high dose group) and relative kidney weights (mid and high dose groups) of female rats with meaningful histologic finding. A treatment-related effect could, however, not be excluded, especially for the high dose group. Further, no histologic correlate was obtained for the significantly increased mean relative weights of brain and adrenal glands in males of the high dose group. These mean weight increases were regarded to be incidental in nature and were most likely related to the slight although not significant decrease of the mean terminal body weight. Finally, also no morphologic correlate was obtained for the significantly decreased absolute heart weight noted in females of the high dose group. This was also most likely related to the slightly although not significantly decreased mean terminal body weight. This assumption was supported by the value of the mean relative heart weight that was almost identical in the high dose group as compared to the control group. The same mechanism was assumed for the significantly decreased mean absolute heart weight noticed in females of the mid dose group, as also the mean terminal body weight was slightly although not significantly decreased. Moreover, there was no indication of a dose-response relationship for the recorded mean absolute and relative weight deviations. All microscopic findings recorded were either single observations, or they were recorded at a low incidence, or they occurred in control animals only, or at comparable incidence and graded severity in control and high dose males and/or females.
Dose descriptor:
NOEL
Effect level:
ca. 14.1 - <= 21.1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 64.1 - <= 91 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Dose descriptor:
LOAEL
Effect level:
ca. 285.2 - <= 338.6 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
Key result
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
64 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 001175MCAO

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature; avoid temperature >40°C
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
- Age at study initiation: about 8 weeks
- Weight at study initiation: mean ± SD males: 263 ± 8; females: 179 ± 7
- Housing: 5 animals per cage in Polysulfon cages (H-Temp [PSU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm²)
- Diet: mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: All measurements of particle size resulted in MMADs between 2.1 and 2.7 μm with GSDs around 1.9. The calculated mass fractions of particles below 3 μm aerodynamic size ranged between 56.6 and 70.7 %. Thus the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs. The remaining fractions may have reached the upper respiratory tract and been deposited there.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 60 volume V ~ 90 L, BASF SE) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The exposure systems were located in exhaust hoods in an air conditioned room.
- Method of holding animals in test chamber: The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol.
- Method of conditioning air: activated charcoal filtered air conditioned to about 50 ± 20 % relative humidity and 22 ± 2 °C
- System of generating particulates/aerosols: The test substance was diluted 1 part test substance + 9 parts highly deionized water (w/w = 10 % solution). 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 and mixed with conditioned dilution air and passed into the inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres in test groups 1 - 4 were analyzed by gravimetry. Daily means were calculated based on 3 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. In these groups, the constancy of concentrations in the inhalation system in each chamber was continuously monitored using scattered light photometers. Additionally, Monoethanol amine (MEA) concentrations were determined 2 times during exposure period by gas chromatography. Formaldehyde (FA) in the test atmosphere was monitored two times during the exposure period in each test group using a Formaldehydemeter HAL-HFX105 (HAL Technology, LLC, USA)
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling for gravimetric analyses: Sampling velocity: 1.25 m/s; Flow rate of sampling: 3 L/min; Sample volumes: Test group 1: 270L; Test group 2: 90 L; Test group 3: 30 L; Test group 4: 12 / 18 L; Sampling site: immediately adjacent to the animals' noses at a separate spare port; Sampling frequency: as a rule, three samples per exposure and concentration group.
To confirm the identity of the test item during the exposure one separate filter sample per concentration group was drawn from the inhalation atmosphere and examined by IR spectrometry in addition. These samples were drawn against the end of the exposure.
Gravimetric measurement: A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the dust/aerosol was drawn through the filter. The aerosol concentration in mg/m³ was calculated from the difference between the weight of the preweighed filter and the weight of the filter after sampling with reference to the sample volume of the inhalation atmosphere.

Real time monitoring of constancy of concentrations: Scattered light photometers (VisGuard (Sigrist) in test groups 1-4 were used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. The measurements were recorded using line recorders.

Sampling for gas chromatography of Monoethanol amine: To trap the test substance aerosol, a filter was switched between the inhalation chamber and the sampling probe. Sampling velocity: 1.25 m/s; Flow rate of sampling: 3 L/min; Sample volumes: 1080 L; The sample volumes were adjusted to achieve amounts of test substance in the samples within the calibration range of the analytical method; Sampling site: immediately adjacent to the animals' noses at a separate spare port; Sampling frequency: as a rule, 2 samples per concentration.
The samples were drawn through the absorption vessels connected in series, each of which was filled with Acetonitrile as absorption solvent. After the sampling, the content of the absorption vessels was eluted into a 500 mL graduated flask for individual analysis.

Determination of formaldehyde in test atmospheres: Equipment: FA concentrations in the test atmosphere was determined using a Formaldehyde-meter HALHFX105 (HAL Technology, LLC, USA); Sampling site: immediately adjacent to the animals' noses at a separate spare port; Sampling frequency: measured and recorded three times per exposure, two times during exposure period.

Duration of treatment / exposure:
6 hours per day, on 5 consecutive days per week for 4 weeks (20 exposures)
Remarks:
3, 10, 30 and 100/50 mg/m³ air. Due to severe clinical findings and premature death in the high dose group test concentration was reduced to 50 mg/m³ after two (males) or one (female) exposures and was stopped after five (females) or six (males) exposures.
Remarks:
3.0 ± 0.3, 10.2 ± 0.4, 30.2 ± 1.3, 104.8/49.2 ± 2.2 mg/m³ air
(analytical conc.)
Remarks:
6.1, 17.4, 50.6, 172.4/86.2 mg/m³ air (nominal conc.)
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
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.

CLINICAL OBSERVATIONS
The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days.

BODY WEIGHT DATA
The body weight of the animals was determined at the start of the pre-exposure, at the start of the exposure period and then, as a rule, twice a week as well as prior to gross necropsy. Body weight change was calculated as the difference between body weight on the respective exposure day and body weight on the day of the first exposure. Group means were derived from the individual differences.

FOOD CONSUMPTION
Food consumption was determined at the start of exposure and weekly thereafter and calculated as mean food consumption in grams per animal and day. The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only two cages per sex, no statistical evaluation of food consumption is possible.

OPHTHALMOLOGY
Before the start of the exposure period (day -2) the eyes of all main group animals, and at the end of the study (day 26) the eyes of the animals of test group 0 (control group) and test group 3 (high intermediate concentration) were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, Germany) after administration of a mydriatic (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

CLINICAL PATHOLOGY
In the morning blood was taken from the retroorbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The following examinations were carried out in 10 animals per test group and sex:
- Hematology: The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
- Clinical chemistry: An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the following clinicochemical parameters: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG).
Sacrifice and pathology:
- Necropsy: The animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and assessed by gross pathology. Due to the severe clinical findings and premature death of high concentration group animals, test group 4 (100/50 mg/m³) was terminated early and the histological examination of the full panel of organs and tissues was performed in animals of test group 3 (30 mg/m³).
- Organ weights: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Spleen, Testes, Thymus, Thyroid glands, Ovaries, Uterus.
- Organ/tissue fixation: The following organs or tissues were fixed in 4% buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain with olfactory bulb, Cecum, Colon, Duodenum, Epididymides, Esophagus, Extraorbital lacrimal gland, Eyes with optic nerve and eyelid (modified Davidson’s), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lung, Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes), Mammary gland (male + female), Nose (nasal cavity), Ovaries, Pharynx, Pancreas, Parathyroid glands, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testes (modified Davidson’s), Thymus, Thyroid glands, Tongue, Trachea, Ureter, Urethra, Urinary bladder, Uterus. From the liver, one slice each of the Lobus dexter medialis and the Lobus sinster lateralis was fixed in Carnoy’s solution and embedded in paraplast.
- Histological assessment: Fixation was followed by histotechnical processing and examination by light microscopy and assessment of findings. The extent of the examination was according to test guideline OECD TG 412. Following the initial examination, a peer review of the findings in respiratory organs (nose,
larynx, trachea and lung) was performed. Results presented in this report reflect the consensus opinion of the study pathologist and peer reviewer.
Statistics:
Dependent on parameter: DUNNETT's test (two-sided), KRUSKAL-WALLIS test (two-sided), WILCOXON-test (two-sided)
Details on results:
MORTALITY: 10 male and 10 female animals exposed to the high concentration (test group 4) died during the exposure period or were sacrificed unscheduled.

CLINICAL OBSERVATIONS: During the pre-exposure period the animals showed no clinical signs and findings different from normal. During the exposure period the animals of the control group, low concentration (3 mg/m³) and low intermediate concentration (10 mg/m³) showed no clinical signs and findings different from normal. During the exposure period the animals of the high intermediate concentration (30 mg/m³) showed following abnormal clinical signs: intermittent respiration, respiration sounds, red encrusted nose, protruding eyeballs and yellow discoloration of the fur. During the exposure period the animals of the high concentration (100/50 mg/m³) showed following abnormal clinical signs up to study day 6 or 7: gasping, intermittent respiration, respiration sounds, red encrusted nose, hypothermia, poor general state, yellow discolored fur.

BODY WEIGHT DATA: The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0. The body weight change of the male animals of the high concentration (100/50 mg/m³) was statistically significantly lower compared to the controls from study day 0 to day 4. No differences in body weight change were observed in males of low (3 mg/m³), low intermediate (10 mg/m³) and high intermediate concentration (30 mg/m³) and females of low intermediate, high intermediate and high dose group. The body weight change was significant increased in females treated with 3 mg/m³ on study day 14 to 21 and 28 compared to controls. These findings are regarded as incidental rather than treatment related because of missing dose dependency.

FOOD CONSUMPTION: A slight reduction of food consumption was observed in males after first exposure in all dose groups. No treatment related effect was observed in females.

OPHTHALMOLOGY: Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group without any concentration-response relationship. In females of the high intermediate group (30 mg/m³) pronounced eyeballs were observed in 5 of 10 animals on study day 26. This finding was not detected in females before exposure and is therefore regarded as treatment related.

CLINICAL PATHOLOGY:
- Hematology: No treatment-related changes among hematological parameters were measured. In females of test group 1 (3 mg/m3), hemoglobin and hematocrit values were lower and relative reticulocyte levels were higher compared to controls. The means of all mentioned parameters were not altered dose-dependently and therefore these changes were regarded as incidental and not treatment-related.
- Clinical chemistry: No treatment-related changes among clinical chemistry parameters were measured.

PATHOLOGY:
- Absolute and relative weights: The change in absolute lung weights of female test group 1 (3 mg/m³) animals (110%) was regarded as incidental as there was neither a dose relationship nor a morphological correlate. None of the other mean absolute weight parameters or any of the relative weight parameters showed significant differences when compared to the control group 0.
- Gross lesions: There was a treatment-related increased incidence of discoloration or fur discolored in animals of test groups 3 (30 mg/m³) and 4 (100 mg/m³) (group 3: males 10/10, females 9/10, group 4: males 5/10, females 1/10 as opposed to none in controls). There was no histopathological correlate for this finding. A focus on the epididymis was observed only in a few treated animals, this correlated with spermatogenic granulomas histologically. As this is a finding which can also occur in control animals it was regarded as incidental. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Histopathology: Treatment related findings were noted in larynx (all levels), lungs, nasal cavity (level I and II) and trachea. Larynx: Metaplasia, squamous referring to a metaplasia of respiratory to squamous epithelium, erosion/ulcer, necrosis of the u-shaped cartilage, hyperplasia and inflammation were noted. Lung: Degeneration, (multi)focal, bronchial epithelium referring to a degeneration of bronchial epithelium characterized by loss of cilia and flattening of epithelium and an increase of bronchus associated lymphoid tissue (BALT) were noted. Nasal cavity: Squamous metaplasia of the ventral respiratory epithelium of the nasal cavity (level I and II) and degeneration of the vomeronasal organ were noted. Trachea: Metaplasia to squamous epithelium was noted both on the tip of the carina and in the ventral epithelium.
Squamous metaplasia, erosion/ulcer, necrosis of the u-shaped cartilage, hyperplasia and inflammation in the larynx were regarded as adverse and were noted in many animals of test group 2 (10 mg/m³) and 3 (30 mg/m³) and one male animal in test group 1 (3 mg/m³) (necrosis of the u-shaped cartilage). Squamous metaplasia in the nasal cavity in animals of test group 2 (10 mg/m³) and 3 (30 mg/m³) and degeneration of the vomeronasal organ in a few test group 3 (30 mg/m³) animals was considered a consequence of irritant effects of the test substance and is regarded to be adverse. Degeneration of the bronchial epithelium of the lung in animals of all test groups was likewise considered adverse as this could impair ciliary clearance from the lung while increase in BALT was considered reactive and non-adverse.
Dose descriptor:
NOAEC
Basis for effect level:
other: local irritation effect (histopathology findings in larynx, trachea and lung)
Remarks on result:
not determinable
Remarks:
no NOAEC identified
Key result
Dose descriptor:
LOAEC
Effect level:
3 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local irritation effect (histopathology findings in larynx, trachea and lung)
Key result
Dose descriptor:
NOAEC
Effect level:
30 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic effects
Key result
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
30 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 001175MCAO

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature; avoid temperature >40°C
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
- Age at study initiation: about 8 weeks
- Weight at study initiation: mean ± SD males: 263 ± 8; females: 179 ± 7
- Housing: 5 animals per cage in Polysulfon cages (H-Temp [PSU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm²)
- Diet: mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: All measurements of particle size resulted in MMADs between 2.1 and 2.7 μm with GSDs around 1.9. The calculated mass fractions of particles below 3 μm aerodynamic size ranged between 56.6 and 70.7 %. Thus the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs. The remaining fractions may have reached the upper respiratory tract and been deposited there.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 60 volume V ~ 90 L, BASF SE) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone-shaped outlets and inlets. The exposure systems were located in exhaust hoods in an air conditioned room.
- Method of holding animals in test chamber: The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol.
- Method of conditioning air: activated charcoal filtered air conditioned to about 50 ± 20 % relative humidity and 22 ± 2 °C
- System of generating particulates/aerosols: The test substance was diluted 1 part test substance + 9 parts highly deionized water (w/w = 10 % solution). 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 and mixed with conditioned dilution air and passed into the inhalation system.
- Method of particle size determination: The particle size analysis was carried out with a cascade impactor.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentrations of the inhalation atmospheres in test groups 1 - 4 were analyzed by gravimetry. Daily means were calculated based on 3 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. In these groups, the constancy of concentrations in the inhalation system in each chamber was continuously monitored using scattered light photometers. Additionally, Monoethanol amine (MEA) concentrations were determined 2 times during exposure period by gas chromatography. Formaldehyde (FA) in the test atmosphere was monitored two times during the exposure period in each test group using a Formaldehydemeter HAL-HFX105 (HAL Technology, LLC, USA)
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling for gravimetric analyses: Sampling velocity: 1.25 m/s; Flow rate of sampling: 3 L/min; Sample volumes: Test group 1: 270L; Test group 2: 90 L; Test group 3: 30 L; Test group 4: 12 / 18 L; Sampling site: immediately adjacent to the animals' noses at a separate spare port; Sampling frequency: as a rule, three samples per exposure and concentration group.
To confirm the identity of the test item during the exposure one separate filter sample per concentration group was drawn from the inhalation atmosphere and examined by IR spectrometry in addition. These samples were drawn against the end of the exposure.
Gravimetric measurement: A preweighed filter was placed into the filtration equipment. By means of a vacuum compressed air pump a defined volume of the dust/aerosol was drawn through the filter. The aerosol concentration in mg/m³ was calculated from the difference between the weight of the preweighed filter and the weight of the filter after sampling with reference to the sample volume of the inhalation atmosphere.

Real time monitoring of constancy of concentrations: Scattered light photometers (VisGuard (Sigrist) in test groups 1-4 were used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. The measurements were recorded using line recorders.

Sampling for gas chromatography of Monoethanol amine: To trap the test substance aerosol, a filter was switched between the inhalation chamber and the sampling probe. Sampling velocity: 1.25 m/s; Flow rate of sampling: 3 L/min; Sample volumes: 1080 L; The sample volumes were adjusted to achieve amounts of test substance in the samples within the calibration range of the analytical method; Sampling site: immediately adjacent to the animals' noses at a separate spare port; Sampling frequency: as a rule, 2 samples per concentration.
The samples were drawn through the absorption vessels connected in series, each of which was filled with Acetonitrile as absorption solvent. After the sampling, the content of the absorption vessels was eluted into a 500 mL graduated flask for individual analysis.

Determination of formaldehyde in test atmospheres: Equipment: FA concentrations in the test atmosphere was determined using a Formaldehyde-meter HALHFX105 (HAL Technology, LLC, USA); Sampling site: immediately adjacent to the animals' noses at a separate spare port; Sampling frequency: measured and recorded three times per exposure, two times during exposure period.

Duration of treatment / exposure:
6 hours per day, on 5 consecutive days per week for 4 weeks (20 exposures)
Remarks:
3, 10, 30 and 100/50 mg/m³ air. Due to severe clinical findings and premature death in the high dose group test concentration was reduced to 50 mg/m³ after two (males) or one (female) exposures and was stopped after five (females) or six (males) exposures.
Remarks:
3.0 ± 0.3, 10.2 ± 0.4, 30.2 ± 1.3, 104.8/49.2 ± 2.2 mg/m³ air
(analytical conc.)
Remarks:
6.1, 17.4, 50.6, 172.4/86.2 mg/m³ air (nominal conc.)
No. of animals per sex per dose:
10
Control animals:
yes, sham-exposed
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.

CLINICAL OBSERVATIONS
The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days.

BODY WEIGHT DATA
The body weight of the animals was determined at the start of the pre-exposure, at the start of the exposure period and then, as a rule, twice a week as well as prior to gross necropsy. Body weight change was calculated as the difference between body weight on the respective exposure day and body weight on the day of the first exposure. Group means were derived from the individual differences.

FOOD CONSUMPTION
Food consumption was determined at the start of exposure and weekly thereafter and calculated as mean food consumption in grams per animal and day. The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only two cages per sex, no statistical evaluation of food consumption is possible.

OPHTHALMOLOGY
Before the start of the exposure period (day -2) the eyes of all main group animals, and at the end of the study (day 26) the eyes of the animals of test group 0 (control group) and test group 3 (high intermediate concentration) were examined for any changes in the refracting media with an ophthalmoscope (HEINE Optotechnik, Herrsching, Germany) after administration of a mydriatic (Mydrum, Chauvin ankerpharm GmbH, Rudolstadt, Germany).

CLINICAL PATHOLOGY
In the morning blood was taken from the retroorbital venous plexus from fasted animals. The animals were anaesthetized using isoflurane (Isoba, Essex GmbH Munich, Germany). The blood sampling procedure and subsequent analysis of blood and serum samples were carried out in a randomized sequence. The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results. The results of clinical pathology examinations were expressed in International System (SI) units. The following examinations were carried out in 10 animals per test group and sex:
- Hematology: The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (Advia 120 model; Bayer, Fernwald, Germany): Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes. Furthermore, blood smears were prepared and stained according to WRIGHT without being evaluated, because of non-ambiguous results of the differential blood cell counts measured by the automated instrument. Clotting tests were carried out using a ball coagulometer (AMAX destiny plus model; Trinity biotech, Lemgo, Germany).
- Clinical chemistry: An automatic analyzer (Hitachi 917; Roche, Mannheim, Germany) was used to examine the following clinicochemical parameters: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG).
Sacrifice and pathology:
- Necropsy: The animals were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The animals were necropsied and assessed by gross pathology. Due to the severe clinical findings and premature death of high concentration group animals, test group 4 (100/50 mg/m³) was terminated early and the histological examination of the full panel of organs and tissues was performed in animals of test group 3 (30 mg/m³).
- Organ weights: The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lungs, Spleen, Testes, Thymus, Thyroid glands, Ovaries, Uterus.
- Organ/tissue fixation: The following organs or tissues were fixed in 4% buffered formaldehyde solution or in modified Davidson’s solution: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain with olfactory bulb, Cecum, Colon, Duodenum, Epididymides, Esophagus, Extraorbital lacrimal gland, Eyes with optic nerve and eyelid (modified Davidson’s), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lung, Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes), Mammary gland (male + female), Nose (nasal cavity), Ovaries, Pharynx, Pancreas, Parathyroid glands, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testes (modified Davidson’s), Thymus, Thyroid glands, Tongue, Trachea, Ureter, Urethra, Urinary bladder, Uterus. From the liver, one slice each of the Lobus dexter medialis and the Lobus sinster lateralis was fixed in Carnoy’s solution and embedded in paraplast.
- Histological assessment: Fixation was followed by histotechnical processing and examination by light microscopy and assessment of findings. The extent of the examination was according to test guideline OECD TG 412. Following the initial examination, a peer review of the findings in respiratory organs (nose,
larynx, trachea and lung) was performed. Results presented in this report reflect the consensus opinion of the study pathologist and peer reviewer.
Statistics:
Dependent on parameter: DUNNETT's test (two-sided), KRUSKAL-WALLIS test (two-sided), WILCOXON-test (two-sided)
Details on results:
MORTALITY: 10 male and 10 female animals exposed to the high concentration (test group 4) died during the exposure period or were sacrificed unscheduled.

CLINICAL OBSERVATIONS: During the pre-exposure period the animals showed no clinical signs and findings different from normal. During the exposure period the animals of the control group, low concentration (3 mg/m³) and low intermediate concentration (10 mg/m³) showed no clinical signs and findings different from normal. During the exposure period the animals of the high intermediate concentration (30 mg/m³) showed following abnormal clinical signs: intermittent respiration, respiration sounds, red encrusted nose, protruding eyeballs and yellow discoloration of the fur. During the exposure period the animals of the high concentration (100/50 mg/m³) showed following abnormal clinical signs up to study day 6 or 7: gasping, intermittent respiration, respiration sounds, red encrusted nose, hypothermia, poor general state, yellow discolored fur.

BODY WEIGHT DATA: The mean body weights of the test substance exposed groups were not statistically significantly different from the control group 0. The body weight change of the male animals of the high concentration (100/50 mg/m³) was statistically significantly lower compared to the controls from study day 0 to day 4. No differences in body weight change were observed in males of low (3 mg/m³), low intermediate (10 mg/m³) and high intermediate concentration (30 mg/m³) and females of low intermediate, high intermediate and high dose group. The body weight change was significant increased in females treated with 3 mg/m³ on study day 14 to 21 and 28 compared to controls. These findings are regarded as incidental rather than treatment related because of missing dose dependency.

FOOD CONSUMPTION: A slight reduction of food consumption was observed in males after first exposure in all dose groups. No treatment related effect was observed in females.

OPHTHALMOLOGY: Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group without any concentration-response relationship. In females of the high intermediate group (30 mg/m³) pronounced eyeballs were observed in 5 of 10 animals on study day 26. This finding was not detected in females before exposure and is therefore regarded as treatment related.

CLINICAL PATHOLOGY:
- Hematology: No treatment-related changes among hematological parameters were measured. In females of test group 1 (3 mg/m3), hemoglobin and hematocrit values were lower and relative reticulocyte levels were higher compared to controls. The means of all mentioned parameters were not altered dose-dependently and therefore these changes were regarded as incidental and not treatment-related.
- Clinical chemistry: No treatment-related changes among clinical chemistry parameters were measured.

PATHOLOGY:
- Absolute and relative weights: The change in absolute lung weights of female test group 1 (3 mg/m³) animals (110%) was regarded as incidental as there was neither a dose relationship nor a morphological correlate. None of the other mean absolute weight parameters or any of the relative weight parameters showed significant differences when compared to the control group 0.
- Gross lesions: There was a treatment-related increased incidence of discoloration or fur discolored in animals of test groups 3 (30 mg/m³) and 4 (100 mg/m³) (group 3: males 10/10, females 9/10, group 4: males 5/10, females 1/10 as opposed to none in controls). There was no histopathological correlate for this finding. A focus on the epididymis was observed only in a few treated animals, this correlated with spermatogenic granulomas histologically. As this is a finding which can also occur in control animals it was regarded as incidental. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
- Histopathology: Treatment related findings were noted in larynx (all levels), lungs, nasal cavity (level I and II) and trachea. Larynx: Metaplasia, squamous referring to a metaplasia of respiratory to squamous epithelium, erosion/ulcer, necrosis of the u-shaped cartilage, hyperplasia and inflammation were noted. Lung: Degeneration, (multi)focal, bronchial epithelium referring to a degeneration of bronchial epithelium characterized by loss of cilia and flattening of epithelium and an increase of bronchus associated lymphoid tissue (BALT) were noted. Nasal cavity: Squamous metaplasia of the ventral respiratory epithelium of the nasal cavity (level I and II) and degeneration of the vomeronasal organ were noted. Trachea: Metaplasia to squamous epithelium was noted both on the tip of the carina and in the ventral epithelium.
Squamous metaplasia, erosion/ulcer, necrosis of the u-shaped cartilage, hyperplasia and inflammation in the larynx were regarded as adverse and were noted in many animals of test group 2 (10 mg/m³) and 3 (30 mg/m³) and one male animal in test group 1 (3 mg/m³) (necrosis of the u-shaped cartilage). Squamous metaplasia in the nasal cavity in animals of test group 2 (10 mg/m³) and 3 (30 mg/m³) and degeneration of the vomeronasal organ in a few test group 3 (30 mg/m³) animals was considered a consequence of irritant effects of the test substance and is regarded to be adverse. Degeneration of the bronchial epithelium of the lung in animals of all test groups was likewise considered adverse as this could impair ciliary clearance from the lung while increase in BALT was considered reactive and non-adverse.
Dose descriptor:
NOAEC
Basis for effect level:
other: local irritation effect (histopathology findings in larynx, trachea and lung)
Remarks on result:
not determinable
Remarks:
no NOAEC identified
Key result
Dose descriptor:
LOAEC
Effect level:
3 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local irritation effect (histopathology findings in larynx, trachea and lung)
Key result
Dose descriptor:
NOAEC
Effect level:
30 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: systemic effects
Key result
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
3 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Peer reviewed data base
Qualifier:
according to
Guideline:
EPA OPPTS 870.3250 (Subchronic Dermal Toxicity 90 Days)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Stable in distilled water over a four-week period, in the dark at room temperature. Stable in pyrogen-free sterile water over 14 days in the dark at room temperature.
Species:
rat
Strain:
other: CD(SD)BR (VAF plus)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, England
- Age at study initiation: not stated
- Weight at study initiation: the rats were in a weight range of 230 to 276 g (males) and 197 to 234 g (females) at the start of the dosing period
Type of coverage:
semiocclusive
Vehicle:
water
Details on exposure:
- Area covered: Not reported.
- Occlusion: Application sites were covered with a semi-occlusive dressing for six hours after application of test article. Plastic Elizabethan collars were used during the exposure period.
Vehicle: Pyrogen-free sterile water for Group 1 (controls), Group 2 (5 mg/kg/day), and Group 3 (50 mg/kg/day). Group 4 (250 mg/kg/day) was treated with undiluted test article.
- Concentration in vehicle:
5 and 50 mg/kg/day group: 2.5mg/mL and 25 mg/mL, respectively.
250 mg/kg/day group: as received, 78.5%.
- Total volume applied:
0, 5, and 50 mg/kg/day group: 2 mL/kg bodyweight.
250 mg/kg/day group: 0.216 mL/kg/day.
Individual volumes were adjusted according to the most recently recorded bodyweight.
- Duration of exposure: Semi-occlusion for 6 hours. Application site was not washed between doses.
- Removal of test substance: None.
- Controls: Pyrogen-free sterile water.

Treatment sites were subdivided into quadrants, and application was rotated among quadrants daily.
Duration of treatment / exposure:
90 days
Frequency of treatment:
6 hours/day, 5 days/week
Remarks:
Doses / Concentrations:
0 (water), 5, 50, 250 mg/kg bw (corresp. to concentrations of ca. 2.5%, 25% and the undiluted test substance)
Basis:
nominal per unit body weight
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
Observations:
Animals were observed and the appearance of the application sites was assessed daily.
- Clinical signs: Yes, animals were observed daily for changes in condition or behavior.
- Mortality: Yes. Mortality checks were performed twice daily.
- Body weight: Yes. Body weights were recorded weekly.
- Food consumption: Yes. Food consumption was recorded weekly
- Water consumption: Not measured.
- Ophthalmoscopic examination: Yes. Ophthalmoscopic examinations were performed before the start of treatment. All high dose and control animals were reexamined during week 13 prior to blood sampling.
- Haematology: Yes. Haematology was evaluated in all animals during the final week of treatment. Haematology Parameters: packed cell volume, haemoglobin concentration, erythrocyte count, total and differential leukocyte count, coagulation test (prothrombin time, partial thromboplastin time).
- Clinical Chemisty: Yes. Blood chemistry was evaluated in all animals during the final week of treatment. Blood Chemistry Parameters evaluated were: blood urea nitrogen, glucose, alanine aminotransferase, aspartate aminotransferase, total protein, albumin, A/G ratio, sodium, potassium, calcium, chloride, inorganic phosphorus, bilirubin, and creatinine.
- Urinalysis: Yes. Urinalysis was performed on all animals during the final week of treatment. Parameters evaluated were: volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood pigments, erythrocytes, leucocytes, crystals, and casts.
Sacrifice and pathology:
- Organ Weights: Yes. Thymus, heart, liver, spleen, kidneys, adrenals, testes or ovaries, and brain were weighed.
- Gross and histopathology: Yes. All animals were examined at the end of treatment for gross and histopathology. Tissues of organs were preserved for microscopic analysis. All tissues from all control and high dose animals, as well as lungs, liver, kidneys, skin, and all gross lesions from all animals were examined microscopically.
Statistics:
Statistical analysis of body weights, organ weights, & haematological data was by analysis of variance, and where differences were significant at the 5% level, by pairwise t-tests between control and treated groups. Blood chemistry data were examined for significant differences by Kruskal-Wallis test for between group differences, and where differences were significant at the 5% level, by with Wilcox rank sum test.
Details on results:
Observations:
- Clinical signs: Other than yellow staining at the application site in the 50 and 250 mg/kg/day groups, there were no treatment-related clinical signs. Animals in all groups exhibited periorbital, perinasal, and cranial fur staining, which was attributed to effects of the Elizabethan collar.
- Mortality: No animals died during treatment
- Body weight gain: Body weight gain was not adversel affected by treatment
- Food consumption and compound intake: Food consumption was not affected by treatment.
- Ophthalmoscopic examination: No treatment-related ocular changes occurred.

- Haematology: All haematological parameters, i.e., packed cell volume, haemoglobin concentration, erythrocyte count, total leucocyte count, and leukocyte differential count, as well as prothrombin time and partial thromboplastin time, were within the normal range for the performing laboratory.

- Clinical chemistry: Statistical differences from controls occurred as follows: 250 mg/kg/day: sodium (male) and glucose (female); 50 mg/kg/day: Sodium (female) and chloride (male); 5 mg/kg/day alanine aminotransferase (male), inorganic phosphorus (female), and chloride (female).
However, all the listed parameters were within the normal range for rats of this age and sex. None of the changes were dose-related or associated with other pathological findings. Therefore, the differences in blood chemistry from controls were not considered to be biologically significant or related to administration of the test material.

- Urinalysis
No changes in urine composition or cellularity occurred.

Sacrifice and pathology:
- Organ weights: Statistically significant increases in absolute and brain weight-related spleen weights in females from all treated groups were attributed to a slightly increased bodyweight in the affected animals. Body weight related organ weights were unaffected.
- Gross and histopathology:
Macroscopic pathology: One male from the 250 mg/kg/day group had a small area of scab formation at the application site, and two females from the same group had abnormal raised brown areas at the application site. Areas of hairloss and scab formation also occurred on untreated areas in one male and one female from the 250 mg/kg/day group, and hair loss at an untreated area occurred in one female from the 50 mg/kg/day group.
Microscopic pathology: No systemic changes showing a treatment-related distribution were observed. Animals from the 5 mg/kg/day group did not show any significant pathological changes.
Abnormal histopathological findings occurred at the application sites of all treated groups. Incidences of epidermal hyperplasia, scab formation, and ulceration of application sites were slightly higher at 250 mg/kg/day than at 50 mg/kg/day. Animals in the 5 mg/kg/day group did not show any significant pathological changes.
Key result
Dose descriptor:
NOEL
Remarks:
local effects (skin)
Effect level:
5 mg/kg bw/day
Sex:
male/female
Basis for effect level:
dermal irritation
Key result
Dose descriptor:
NOAEL
Remarks:
systemic effects
Effect level:
> 250 mg/kg bw/day (nominal)
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
LOAEL
Remarks:
systemic effects
Effect level:
> 250 mg/kg bw/day
Sex:
male/female

Table 1: Mortality:

Dose

[mg/kg/day]

Number dead /
number investigated

0

0/10

5

0/10

50

0/10

250

0/10

 

Table 2: Results of blood chemistry investigations – group mean values:

Parameter

Unit

Control

Low Dose

Medium Dose

High Dose

Dose

mg/kg/day

0

5

50

250

Blood Chemistry – Males

Alanine minotransferaseA

CalciumA

 

U/L

mmol/L

 

35

99.7

 

44*

99.8

 

30

101.2*

 

37

99.8

Blood Chemistry – Females

GlucoseA

Inorganic PhosphorusA

ChlorineA

PotassiumA

 

mg%

mg%

mmol/L

mmol/L

 

1.9

4.9

106.5

3.5

 

113

4.3*

104.0**

3.5

 

115

4.4

106.1

3.7

 

128*

4.5

108.3

3.8*

A All values were within expected laboratory ranges.

* = significantly different from controls, p<0.05.

** = significantly different from controls, p<0.01.

 

Table 3: Absolute and Bodyweight-related Spleen Weights, and Bodyweight Gains - Group Mean Values:

Parameter

Unit

Control

Low Dose

Mediu Dose

High Dose

Dose

mg/kg/day

0

5

50

500

Absolute spleen weight

Males

Females

grams

grams

0.68

0.51

0.73

0.59**

0.67

0.59*

0.78

0.59*

Brain-related spleen weight

Males

Females

grams

grams

34.08

26.35

35.76

30.62*

34.03

31.81*

38.83

30.74*

Total bodyweight gain

Males

Females

grams

grams

180

39

178

48

175

40

177

48

* = significantly different from controls, p<0.05.

** = significantly different from controls, p<0.01.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Study duration:
subchronic
Species:
rat

Additional information

Oral application:

A subchronic oral toxicity study in Wistar rats with administration of the test substance in drinking water for 3 months was identified as the key study for assessment of repeated dose toxicity (BASF AG, 2002). In this study, that was performed according to OECD TG 408 in compliance with GLP, the test substance was administered in drinking water at 0, 200, 1000 or 5000 ppm, equivalent to dose levels of approx. 14 / 21, 64 / 91 or 285 / 339 mg/kg bw/day in male / female rats, respectively. Treatment-related changes occurring at the highest dose level (285 / 339 mg/kg/day) included reduced food consumption in males and females, reduced water consumption in males and females, decreased bodyweight or reduced bodyweight gains for males, increased urea and urinary specific gravity in both sexes, decreased urinary volume for both sexes, and significantly increased mean absolute and relative kidney weights for females. The kidney effects may have been an adaptive response to disruption of cellular water balance arising from reduced water intake. Marginal swelling of renal tubular cells (“cloudy swelling”), would revert to normal on withdrawal of treatment. No degenerative cellular alterations indicative of tubular epithelia (such as nuclear pyknosis, karyorrhexis or cellular necrosis) or regenerative responses to tubular damage (e.g. basophilic tubuli) were apparent. The NOAEL was established at 64 mg/kg bw/day, based on reduced water consumption at this dose level but without any corroborating changes in-life or pathologically (since the test material was administered in the drinking water the reduced water consumption may have been attributable to palatability issues).

A NOEL in a similar range was determined in a supporting oral gavage study (BASF, 1970). 60 Wistar rats (30/sex) were treated for a period of 12 weeks at concentrations of 30, 100 and 300 mg/kg bw/d. A NOEL between 30 and 100 mg/kg bw/d was assumed for male and female animals.

Inhalative application:

A subacute aerosol inhalation study in rats is available for assessment (Triazine Task Force, 2011). This study was carried out according to OECD guideline 412 in compliance with GLP at liquid aerosol concentrations of 3, 10, 30 and 100/50 mg/m³ (Due to severe clinical findings and premature death in the high dose group test concentration was reduced to 50 mg/m³ after two (males) or one (female) exposures and was stopped after five (females) or six (males) exposures). In conclusion, exposure of male and female Wistar rats to liquid aerosol of the test substance caused concentration–related local irritation of the respiratory tract. The maximum tolerated dose (MTD) was clearly exceeded at the initial high concentration of 100 mg/m³ due to severe clinical findings, the concentration was lowered to 50 mg/m³ after the first exposure day for females and the second exposure day for males, respectively. The lowered high concentration led to premature death and was interrupted on study day 6 and 7, for females and males, respectively. The local irritation was manifested as gasping, intermittent respiration and respiration sound and confirmed by histological changes of nasal cavity, larynx and lung. Systemic toxicity was not observed in clinical chemistry, hematology nor in histological examinations up to 30 mg/m³. As the high concentration group was terminated, no statement concerning systemic effect on that concentration could be made. The reduced body weight gain and premature death were considered to be associated to the severe local irritation. Based on histopathology findings in larynx, trachea and lung, a NOAEC could not be established for the local irritation effect under the current study conditions. For systemic effect the NOAEC is 30 mg/m³.

Dermal application:

In a subchronic toxicity study the test substance was administered dermally at doses of 0 (water), 5, 50 or 250 mg/kg/day to 10 Sprague-Dawley rats/sex/group (cited in EPA, 2008). The doses of 5 and 50 mg/kg/day were applied diluted in water. The high dose, 250 mg/kg, was applied neat at 0.216 ml/kg. Exposure time was 6 hours/day, 5 days/week for 13 weeks. Erythema at the application sites was seen with increasing incidence and severity at 50 and 250 mg/kg/day. No effects were seen in males at the low dose but 2/10 females showed very slight erythema for 2 days and 7 days. Edema was seen on "occasion" and "isolated" at the high and mid doses. Dermal NOEL was established at 5 mg/kg/day based on skin irritation at the site of application. Systemic NOEL was >250 mg/kg/day as no systemic toxicological effects were observed.

Justification for classification or non-classification

Based on histopathology findings in larynx, trachea and lung, a NOAEC could not be established for local irritation in a subacute inhalation study with liquid aerosol exposure in rats (Triazine Task Force, 2011). In this study, the lowest dose still producing severe effects was ≤ 3mg/m³/6h/day.

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance needs to be classified as STOT RE 1 (H372) under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/ 1179.