2,2',2''-(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol

Repeated dose toxicity: inhalation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2',2''-(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol

EC Number:

225-208-0

EC Name:

2,2',2''-(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol

Cas Number:

4719-04-4

Molecular formula:

C9H21N3O3

IUPAC Name:

2,2',2''-(1,3,5-triazinane-1,3,5-triyl)triethanol

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

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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)

Doses / concentrations

Doses / concentrations 1

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.

Doses / concentrations 2

Remarks:

3.0 ± 0.3, 10.2 ± 0.4, 30.2 ± 1.3, 104.8/49.2 ± 2.2 mg/m³ air
(analytical conc.)

Doses / concentrations 3

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

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.

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)

Results and discussion

Results of examinations

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.

Effect levels

1

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

2

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)

3

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

Target system / organ toxicity

Key result

Critical effects observed:

no