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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Feb - Mar 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
methodological deficiencies (MMAD exceeds the currently recommended particle size, no BALF analysis or determination of lung burden was conducted, food and water consumption were not measured)
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
Version / remarks:
adopted 1981
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
adopted 1984
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
428-040-8
EC Name:
-
Cas Number:
138261-41-3
Molecular formula:
C9H10ClN5O2
IUPAC Name:
2-chloro-5-{[2-(nitroimino)imidazolidin-1-yl]methyl}pyridine

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelmann Experimental Animal Breeders, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 2-3 months
- Weight at study initiation: 160 - 200 g
- Fasting period before study: not applicable
- Housing: in groups of five in Type III Makrolon® cages with type S 8/15 low-dust wood shavings (Ssniff Spezialdiäten GmbH, Soest, Germany)
- Diet: Altromin® 1324 Maintenance Diet for Rats and Mice (Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least one week

DETAILS OF FOOD AND WATER QUALITY: Tap water met drinking water standards (Drinking Water Statute of May 22, 1986; Bundesgesetzblatt Part I, page 760), feet was regularly checked for contaminants, spot checked and analyzed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approx. 5o
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
head only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 2.37 - <= 5.7 other: µm (for details please refer to "Any information on materials and methods")
Geometric standard deviation (GSD):
1.96
Remarks on MMAD:
Results of particle size analysis are provided under "Any information on materials and methods".

Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: PVC inhalation chamber, 30 cm diameter, 28 cm height (volume approx. 20 liters)
- Method of holding animals in test chamber: Plexiglass tubes (closed, tail of the rat was outside the tube)
- Source of air and method of conditioning air: compressed air was produced with two Model SB 270/15/350D Boge compressors in parallel. Type A 110 compressed air dryer mounted behind the compressors were used for conditioning. The regulated operating pressure of the compressors was 8-10 bars (800 - 1000 kPa)
- System of generating particulates/aerosols: Wright Dust Feeder (5 and 30 mg/m³ air) and a RBG brush-type generator (180 mg/m³ air, only on the first day because of a defect thereafter) or an Exactomat 4200 (180 mg/m³ air, starting on the second day of exposure).
- Temperature, humidity, pressure in air chamber (for details on temperature and humidity please refer to "Additional information on Materials and Methods"):
- Air flow rate: continously monitored, 20-30 L/min
- Air change rate: 30 air exchanges per hour
- Method of particle size determination: aerodynamic particle sizer with a laser velocimeter (TSI APS 3300) for 5 mg/m³, 30 and 180 mg/m³: APS 3300 instrument in conjunction with two dilution stages (TSI Model 3200)
- Treatment of exhaust air: The exhaust air was treated by passing it through a cotton
wool aerosol filter. The cotton wool filters were destroyed by incineration.

TEST ATMOSPHERE
- Brief description of analytical method used: Leybold - Heraeus measuring system, temperature and humidity were determined at 10 min intervals (automatically), the data was compared to appropriate reference data. The sensors were located in the inhalation chamber cover.
- Samples taken from breathing zone: yes

VEHICLE:
- Dust is most appropriate in accordance with potential exposure route for humans. An inhalation toxicity study using an aerosol was considered inadequate because of the low solubility of the active ingredient in nontoxic vehicle substances (maximum technically producible concentration: 69 mg/m³ air).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gravimetric filter analysis (Sartorius SM 11106 cellulose acetate filter, pore size 0.45 µm). 100, 50 and 30 liters of test atmosphere were sampled in breathing zone for 5, 30 and 180 mg/m³ air, respectively (rate: 4 L/min). If possible, 3 air samples were taken, one at the beginning of the test, one in the middle of the test and one near the end. All concentrations stated below refer to mg test substance (95.2% purity) per m³ air. Recalculation to a 100% active ingredient basis was not performed.
Duration of treatment / exposure:
28 days, 6h per exposure
Frequency of treatment:
daily, 5 times per week
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/m³ air (nominal)
Remarks:
corresponds to 5.5 mg/m³ air as analytical concentration
Dose / conc.:
30 mg/m³ air (nominal)
Remarks:
corresponds to 30.5 mg/m³ air as analytical concentration
Dose / conc.:
180 mg/m³ air (nominal)
Remarks:
corresponds to 191.2 mg/m³ air as analytical concentration
No. of animals per sex per dose:
10
Control animals:
yes
Details on study design:
- Dose selection rationale: Acute inhalation toxicity studies summarised in the technical dossier under 7.2.2 "Acute toxicity: inhalation" served as range-finding studies (M-027586-01-1). LC50 values > 0.069 mg/L air and > 5.3 mg/L air were derived for aerosols and dust, respectively. Regarding orientative subacute inhalation (dust) with 20, 109 and 505 mg/m³ air, the NOEL was 20 mg/m³ air and the LC 50 > 505 mg/³. From 109 mg/m³ air, body weight was reduced slightly and liver enzyme activity was increased. Other changes were not found. Therefore, 5, 30 and 180 mg/m³ air were set as concentrations for this study.

- Fasting period before blood sampling for clinical biochemistry: only for glucose
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: before exposure and after, and on exposure-free days
- parameters checked: appearance of visible mucous membranes of eyes and respiratory
tract, general state of muzzle skin and ear scoops, condition of coat, grooming activities, respiration, circulation (as far as evaluation was possible)

BODY WEIGHT: Yes
- Time schedule for examinations: prior to first exposure, then weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before first exposure and close to study termination
- Dose groups that were examined: all
- Number of animals per dose: 5 per group and sex
- parameters checked: changes in the retina, vitreous humor, lens, cornea and the outer surface of the eye (analyzed with a Heine indirect ophthalmoscope, pupil dilitation with Mydriatikum Roche® 5-10 minutes prior to examination)
And:
- Time schedule for examinations: daily
- Dose groups that were examined: all
- parameters checked: corneal and light reflexes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (not for glucose determination)
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: Hematocrit, hemoglobin, leukocytes, erythrocytes, mean corpuscular erythrocyte volume (MCEV), mean erythrocyte hemoglobin concentration (MEHC), mean erythrocyte hemoglobin (MEH), thrombocyte count, differential blood count, reticulocytes, Heinz bodies, methemoglobin, thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at time of necropsy
- Animals fasted: no
- for glucose determination: fasted, non-anesthetized animals, in the week before necropsy
- How many animals: all animals
- Parameters checked:aspartate aminotransferase (ASAT/GOT), alanine aminotransferase (ALAT/GPT), glutamate dehydrogenase (GLDH), lactate dehyrogenase (LDH), plasma cholinesterase, alkaline phosphatase, sorbitol dehydrogenase (IDH), albumin, blood sugar, urea, bilirubin, creatinine, total protein, triglyceride, cholesterol, serum protein electrophoresis, T3 (not in all animals), T4, TBC, sodium, potassium, calcium, magnesium, phosphate, chloride, for liver: Cytochrome-P450, N-demethylase, O-demethylase, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: overnight (from 8-16 h), one week before necropsy
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: sediment composition, pH, protein, glucose, blood, bilirubin, urobilinogen, ketone bodies

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: daily
- Dose groups that were examined: all animals
- Parameters checked: somatomotor system and behavioral pattern (including tremor, convulsions, hypersalivation, dyspnea, diarrhea, lethargy, sedation and coma), central nervous and autonomous symptoms

IMMUNOLOGY: No


BRONCHOALVEOLAR LAVAGE FLUID (BALF): No


LUNG BURDEN: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Organs that were weighed: brain, heart, testes, liver, lung, spleen, adrenals, kidneys, ovaries, pancreas, thyroid, thymus

HISTOPATHOLOGY: Yes
- Number of animals: all
- fixation: 10 % aqueous buffered formaldehyde solution
- staining: hemalum-eosin (HE), additional sections for glycogen and lipid staining were prepared from the liver, bone marrow smears were stained with May-Grünwald solution
- organs/tissues examined: aorta, eyes (including lid), vas deferens, epididymis (including accessory glands), brain (cerebellum and cerebum), skin (rhinarium - muzzle area and mamma area), harderian glands and extraorbital lacrimatory gland, urinary bladder (instillation fixation with Bouin's solution), heart, testes, pituitary gland, intestine (stomach, duodenum, jejunum, ileum, cecum, colon, rectum), bone (femur), bone marrow (femur and sternum), coagulating gland, head (nasopharynx, oropharynx, nasal and paranasal cavities), larynx, liver, lung (with main bronchi, instillation fixation), lymph nodes (mediastinal, cervical / mandibular, mesenteric), mamma, spleen, muscle (quadriceps femoral muscle), paratyroid glands, adrenals, sciatic nerve, kidneys with pelvis, esophagus, ovaries, pancreas, prostate, spinal cord (cervical, thoracic, lumbar), seminal vesicle, salivary glands (head), sternum, trachea, lacrimatory gland, thyroid, thymus, uterus (including tubes), vagina, tongue
Other examinations:
none
Statistics:
Where possible, means and standard deviation were calculated. For animal data, whenever possible, the upper and lower confidence limits at confidence levels of (1-a) = 95 % and (1-a) = 99 % were determined.

For body weight and laboratory tests, groups were compared with the Mann and Whitney rank test (U test) in the modification by Walter. Significance was set to be p < 0.05.

Organ weight was compared with ANOVA (variances between the groups were tested for homogeneity with Box's test). If differences occured, a pairwise post hoc (one and two-tailed) comparison of the groups is performed using the Games and Howell modification of the Tukey - Kramer-test was performed.

Statistical analysis of the urine was only performed for the pH (U-test).

Histological examinations were compared with the pairwise Fisher test. Necropsy findings were compared with the pairwise Fisher test with a preliminary R x C chi square test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 180 mg/m³: significantly reduced body weight throughout the study in males (-5.8 to -8.6%) compared to controls

Summarized data can be found in Attachment 1 of the attached background material.
Food consumption and compound intake (if feeding study):
not examined
Description (incidence and severity):
not applicable
Food efficiency:
not examined
Description (incidence and severity):
not applicable
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
not applicable
Ophthalmological findings:
no effects observed
Description (incidence and severity):
not applicable
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
- 180 mg/m³: HQUICK increase in females (+10%), increased blood coagulation time and elevated total serum bilirubin (females) compared to controls

Not considered toxicologically relevant were slightly decreased thrombocyte counts (-15.1%) in males compared to control animals. Isolated significantly altered values in SEGM, erythrocytes, hemoglobin, MCH and MCHC are considered as incidental findings not indicative for hematotoxiciy as those effects appeared occasionally in one sex without dose-response.

Summarized relevant data can be found in Attachment 2 in the attached background material
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 30 mg/m³: increased ALAT (+24.7%) and APh (+20.4%) activity in females, decreased CHE activity in females (-26.1%, but not statistically significant), increased N-demethylase activity (+26.6%) in females, decreased a-globulin level in males (-7.4%) and females (-7.1%)
- 180 g/m³: increased ALAT (+70.3%), APh (+46.1%) activity in females, increased GLDH activity in males (+333.3%) and females (+731.6%), increased bilirubin (+33.3%) in females, decreased triglyceride levels in males (-48.8%) and females (-72.1%), decreased CHE activity in females (-28.2%, but not statistically significant), increased O-demthylase activity in males (+83.2%) and females (+21.5%), increased N-demthylase activity in males (+51.2%) and females (+76.5%), increased P450 content in males (+33.8%), decreased a-globulin level in males (-11.6%) and females (-8.2%)

Findings that were not considered toxicologically relevant were changes in urea content, since it is mainly dependent on food consumption and inhalation studies depend on long withdrawal from food and water. Thus, decreased plasma urea levels are a commonly occurrance. Decreased plasma protein levels in males was considered to be due to depressed hematocrit values (slight hypervolemia) determined in this sex and altered phosphorus levels in males and females were considered not biologically significant due to lack of dose-dependency.

Summarized relevant data can be found in Attachment 3, 4 and 5 in the attached background material. A summary of the results can be found in Attachment 9.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
- 180 mg/m³: significantly higher pH in females compared to controls
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
not applicable
Immunological findings:
not examined
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- 30 mg/m³: increased relative liver weight in females compared to controls (+8.8%, not statistically significant)
- 180 mg/m³: increased relative liver weight in females (+12.4%, statistically significant), slight reduction in relative heart (-5.3%) and thymus (-24.4%) weight in females, all compared to controls

Other changes in organ weights were slightly decreased relative brain and heart weights in males. But these were within the 2-sigma scattering ranges of pooled historical organ weight data for Wistar rats and therefore not considered significant.

Summarized relevant data can be found in Attachment 6 in the attached background material. The historical organ weight data can be found in Attachment 7.
Gross pathological findings:
no effects observed
Description (incidence and severity):
not applicable
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Effects that were observed were not considered treatment related.
180 mg/m³: decreased incidence of periportal round cell infiltration in males (not dose-dependent, incidence 0/2/0/5).

Further observations included a slight hyperemia of the upper respiratory tract in few animals which is most probably due to sacrifice procedure with diethyl ether.
Statistically significant changes were found in bone marrow morphology: proerythroblasts were significantly increased in all groups of treated males. In females, an increase was seen but this was not statistically significant. Moreover, the count in controls of males and females differed by a factor of 2.5 and in peripheral blood, concentration of reticulum cells was unchanged, so that this finding can be considered incidental. Segmented neutrophils were decreased in males and females in all dose groups but no change in granulopoiesis occurred in bone marrow (band neutrophils) or peripheral blood (differential blood count). Also, the amount of segmented neutrophils in the cell population is very low (0.5-1.5%), so that counting errors have a strong impact. Due to these factors, the finding is considered to be a coincidence.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable
Details on results:
General summarized results are found in Attachment 8 in the attached background material.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Effect level:
5.5 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at this dose level
Key result
Dose descriptor:
LOAEC
Effect level:
30.5 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
haematology
organ weights and organ / body weight ratios
other: effects on the liver (increased organ weight and enzyme induction)

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
30.5 mg/m³ air (analytical)
System:
hepatobiliary
Organ:
liver

Applicant's summary and conclusion

Conclusions:
The study was conducted according to GLP guidelines and according to OECD Guideline 412. Since the study was conducted in 1989, there are deviations to the current guideline as described above.

Based on the effects observed on the liver, including elevated organ weights and enzyme induction, a NOAEC of 5.5 mg/m³ and a LOAEC of 30.5 mg/m³ were derived.