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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:
4 Apr - 4 Jun 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
adopted 2018
Deviations:
yes
Remarks:
Blood was taken from non-fasted animals for glucose determination, no BALF analysis or determination of lung burden was conducted, humidity in the exposure chamber was lower than recommended
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study
Version / remarks:
adopted 1981
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin
EC Number:
601-147-9
Cas Number:
111988-49-9
Molecular formula:
C10H9ClN4S
IUPAC Name:
3-(2-chlor-5-pyridyl-methyl)-cyanimino-1,3-thiazolidin

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Commonly used for toxicology studies, recommended by the guideline.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 2-3 months
- Weight at study initiation: 243 - 246 g (group means, males), 166 - 171 g (group means, females)
- Fasting period before study: no
- Housing: individually in in conventional Makrolon® Type II cages with low-dust wood granulate (type S 8/15, Ssniff, Soest/Westfalen, Germany)
- Diet: standard fixed-formula diet (Altromin® 1324 pellets maintenance diet for rats and mice, Altromin GmbH, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: approximately 10 days

DETAILS OF FOOD AND WATER QUALITY: food and water were checked for contaminations regularly

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approximately 50
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 5 May To: 4 June 1997

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
2.9 µm
Geometric standard deviation (GSD):
1.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Plexiglas exposure retainers, designed that the rats tail remained outside to prevent hyperthermia.
- Method of holding animals in test chamber: Plexiglas exposure retainers are designed to hold the rat in place for exposure.
- System of generating particulates/aerosols: The test substance was aerosolized using a Wright-Dust-Feeder (BGI Inc., Waltham, MA, USA). For powder dispersion, conditioned compressed air (30 liters of air/min) was used. A cyclone prevented larger particles (>10 µm) from entering into the exposure chamber.
- Temperature, humidity in air chamber: 21 - 22 °C, 11 - 18%
- Inlet Air flow: 30 L/min
- Air change rate: 30 L/min x 60 min/(3.8 L) = 474 L
- Method of particle size determination: The particle-size distribution was analyzed using a BERNER-TYPE AERAS low pressure critical orifice cascade impactor (Hauke, Gmunden, Austria).
- Treatment of exhaust air: The exhaust air was purified via cotton-wool and HEPA filters which were disposed.

TEST ATMOSPHERE
- Brief description of analytical method used: The test-substance concentration was determined by gravimetric analysis (filter: cellulose-acetate filter, Sartorius, Göttingen, Germany; balance: MettlerAE 100). Samples for particle size analysis were also taken from the breathing-zone.
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
As mentioned previously, the concentrations were determined by gravimetric analysis; The number of samples taken was sufficient to characterize the test atmosphere and was adjusted to accommodate sampling duration and/or the need to confirm specific concentration values. It was intended to collect at least three samples/exposure, after the equilibrium concentration was reached. The analytical concentrations were reported as mg of test item/m³ air.
The amount of particles below 3 μm was 54.5 (2 mg/m³), 51.6 (20 mg/m³) and 53.1% (200 mg/m³).
Duration of treatment / exposure:
28 days, 6 h/day
Frequency of treatment:
5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
2 mg/m³ air (nominal)
Remarks:
gravimetric concentration in air was 2.0 mg/m³
Dose / conc.:
20 mg/m³ air (nominal)
Remarks:
gravimetric concentration in air was 18.2 mg/m³
Dose / conc.:
200 mg/m³ air (nominal)
Remarks:
nominal concentration in air, had to be reduced to 100 mg/m³ in the second week, gravimetric concentration in air was 143.4 mg/m³ (mean over entire testing period)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The concentrations were selected based on the results of an acute inhalation study and a one-week pilot study, both conducted on rats. In the acute toxicity study, rats were exposed to the test substance at concentrations of 80, 481, 1523, and 2535 mg/m³ air. The NOAEL was found to be 80 mg/m³ air for males and females and the LC50 > 2535 mg/m³ for males and approximately 1223 mg/m³ in females.
In the pilot study, rats were exposed to the test substance at 1.97, 19 or 205 mg/m³ air. Only minor influences on the respiratory tract were found up to the highest concentration tested. At 205 mg/m³ air, liver weight was increased transiently and induced hepatic enzyme induction was found. At 19 mg/m³, grip strength was increased transiently and an increased incidence of darken spleens was noticed in the females of this group. All findings were reversible within 2 weeks. 19 mg/m³ was therefore seen as the lowest effect level. Based on these results, the concentrations for the present study were set at approximately 2, 20 and 200 mg/m³ air. 2 mg/m³ was expected to be non-toxic while 20 and 200 mg/m³ air should induce toxic effects. However, during the course of the study, the highest concentration of 200 mg/m3 needed to be reduced to 100 mg/m3 air to prevent undue distress of animals or even mortality.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (once on weekends and holidays)
- Cage side observations: changes in the skin and hair-coat, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, and sensori- as well as somatomotor activity and behavior pattern. Particular attention was directed to tremors, convulsions, salivation, diarrhea, lethargy, somnolence and prostration.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Parameters checked: visual placing response and grip strength on wire mesh, abdominal muscle tone, corneal and pupillary reflexes, pinnal reflex, righting reflex, tail-pinch response, startle reflex with respect to behavioral changes stimulated by sounds (finger snapping) and touch (back).

BODY WEIGHT: Yes
- Time schedule for examinations: twice per week

FOOD CONSUMPTION AND COMPOUND INTAKE: No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to first exposure and towards end of the exposure period
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at terminal sacrifice by cardiac puncture or, for glucose determination, blood was sampled next to last study week (after urine collection) from the caudal vein
- Anesthetic used for blood collection: Yes (pentobarbital), not for blood withdrawal for glucose determination
- Animals fasted: No
- How many animals: 10 animals per group and sex
- Parameters checked: Hematocrit, hemoglobin, leukocytes, erythrocytes, mean corpuscular volume, mean corpuscular hemoglobin concentration, mean corpuscular hemoglobin, thrombocyte count, reticulocytes, Heinz' bodies, leukocyte differential count (lymphocytes, granulocytes, segmented neutrophils, eosinophilic neutrophils, basophils, monocytes, plasma cells and miscellaneous abnormal cell types)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above (hematology)
- Animals fasted: No
- How many animals: 10 animals per group and sex
- Parameters checked: aspartate aminotransferase, optimized (AST), alanine aminotransferase, optimized (ALT), glutamate dehydrogenase (GLDH), lactate dehydrogenase (LDH), alkaline phosphatase (APh), y-glutamyltranspeptidase (GGT), albumin, blood glucose, urea, bilirubin, bile acids, creatinine, total protein, triglycerides, cholesterol, serum protein electrophoresis, clotting time (Hepato quick test), sodium, potassium, calcium, magnesium, phosphate, chloride

From liver samples, the following parameters were checked: N-DEM (N-Demethylase, Aminopyrin-N-demethylase), O-DEM (O-Demethylase, p-Nitroanisol-N-demethylase), cytochrome P450, triglycerides

URINALYSIS: Yes
- Time schedule for collection of urine: collected overnight during the next to last study week
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- How many animals: 10 animals per group and sex
- Parameters checked: Sediment composition, urine osmolality, specific density, pH, volume, protein, glucose, blood, bilirubin, urobilinogen, ketone bodies
Protein (Q)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: twice daily (once on weekends and holidays)
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): No

LUNG BURDEN: No
Sacrifice and pathology:
Surviving animals were sacrificed one day after the last exposure to the test substance after cardiac exsanguination using sodium pentobarbital (i.p.) for euthanasia.

GROSS PATHOLOGY: Yes

Organ weight was determined for the following organs: adrenals, brain, heart, kidneys, liver, lungs, ovaries, spleen, testes, thymus, thyroid (half), uterus

Relative organ to body and organ to brain weight was calculated.

HISTOPATHOLOGY: Yes
- Tissues collected: adrenal glands, aorta, brain (cerebrum, cerebellum, pons/medulla), epididymides, esophagus, eyes, eyelids, extraorbital lacrimal glands, femur, harderian glands, head (with nasal and paranasal cavities), heart, intestine (duodenum, jejunum, ileum, cecum, colon, rectum and remaining intestine), kidneys, larynx, liver, lymph nodes (mandibular and mesenteric), mammary gland, ovaries, oviducts, pancreas, pituitary, prostate, salivary glands, sciatic nerve, seminal vesicles (incl. coagulating glands), skeletal muscle (thigh), skin (mammary region), skin (muzzle), spinal cord (cervical, thoracic, lumbar), spleen, sternum, stomach (forestomach and glandular stomach), testes, thymus, thyroid gland with parathyroid glands, tongue, trachea, ureter, urethra, urinary bladder, uterus with uterine cervix, vagina, physical identifier (tattooed ears), and all organs or tissues with macroscopic findings. All organs not scheduled for fixation that exhibited gross changes wee also fixed if necessary.

- animals examined: for nasal cavity, larynx, trachea, lungs, lung associated lymph nodes,
heart, liver, kidneys, thyroid with parathyroid glands and esophagus all animals were examined, for all other organs control and high dose group were assessed, if differences occurred, the remaining groups were also checked.
- thickness of sections: 5 µm
embedding media: paraplast
- staining: hematoxylin and eosin (H&E), cryo-cuts obtained from the formalin-fixed livers were stained with Oil-Red-0 (ORO).
Other examinations:
Rectal temperature was assessed daily after cessation of exposure (within 30 min of cessation of exposure) using a digital thermometer.
Statistics:
All variables that are not dichotomous are described by sex, dosage group and date using appropriate measures of central tendency (mean, median) and general variability (standard deviation, in most instances also minimum, maximum).

For normally distributed data with equal variances, an ANOVA followed by Dunnett's test is used. If heteroscedasticity appeared to be more likely a p value adjusted Welch test is applied. For non-parametric measurements, Kruskal-Wallis test followed by adjusted MWW tests (U tests) where appropriate. Global tests including more than two groups are performed by sex and date, i. e. each sex x date level defines a family of tests in the context of multiple comparison procedures.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At 200 mg/m³, animals showed signs of morbidity (bradypnea, labored breathing pattern, reduced motility, piloerection, ungroomed hair-coat, atony, rales, salivation, mydriasis, miosis, tremor, vocalization). Therefore, the concentration was reduced to 100 mg/m³ in Week 2, the most severe signs (labored breathing, reduced mobility, ungroomed hair) decreased after having reduced the test concentration. It was concluded that most of the signs predominantly related to respiratory distress rather than
specific central nervous effects (mydriasis, miosis, tremor) which subsided completely after reduction of concentration.

Summarized data can be found in Attachment 1 in the attached background material.
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 200 mg/m³: decreased body weight in the first week (this was reversible after reducing the concentration to 100 mg/m³)

Summarized data can be found in Attachment 2 in 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):
No differences between control and treatment groups.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Occasional findings were elevated MCHC in females of the low and middle dose group, increased neutrophils in males of the middle dose group, and decreased lymphocyte counts in the female high dose group. However, none of these were dose dependent and were therefore considered not treatment related.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
- 200 mg/m³: statistically significant increase in alkaline phosphatase (females), phosphate (males), glucose (males, females), cholesterol, bile acids (females) and calcium (females), all compared to controls

APh and phosphate were also increased in the other sex but not statistically significant. Females were mildly more susceptible than males.

Liver tissues
- 20 mg/m³: statistically significantly increased N-DEM (males) compared to controls
- 200 mg/m³: statistically significantly increased N-DEM (males, females), O-DEM (males, females), P450 (males, females), all compared to controls

In females at 2 mg/m³, N-Dem was decreased statistically significantly compared to controls. This was considered incidental.

Summarized results can be found in Attachment 3 of the attached background material.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
no effects observed
Description (incidence and severity):
No differences were observed between control and treatment groups.
Behaviour (functional findings):
not examined
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):
- 200 mg/m³: statistically significantly increased absolute (females +16.8%) and relative to body (males +12.2%, females +13.8%) liver weight, female livers were also statistically significantly increased relative to brain weight (+16.1%), all compared to controls

In males of the 2 and 200 mg/m³ groups the lung weights were statistically significantly increased, however, there was no evidence of any consistent concentration-dependence of effect. Thyroid weights were mildly increased in the middle and high dose group (absolute weight was increased 54.8% at 20 mg/m³ and 37.1% at 200 mg/m³) but this was not statistically significant.

Summarized results can be found in Attachment 4 in the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No differences between control and control groups.
Neuropathological findings:
no effects observed
Description (incidence and severity):
The assessment of reflexes did not reveal any differences between the groups. The only sign observed was that on day 2, some rats of the 200 mg/m³ group experienced a decreased tonus and light reflex (reversible).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 20 mg/m³: slight hypertrophy of hepatocytes (males)
- 200 mg/m³: slight hypertrophy of hepatocytes (males, females), hypertrophy of the thyroidal follicular epithelium (males)

Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Rectal temperature
- 200 mg/m³: decreased body temperature (34.1 °C for males and 34.5/35.3°C for females in Week 1).

Summarized data can be found in Attachment 5 in the attached background material.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEC
Remarks:
systemic
Effect level:
18.2 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: corresponding to the nominal concentration of 20 mg/m³
Key result
Dose descriptor:
LOAEC
Remarks:
systemic
Effect level:
143.44 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
histopathology: non-neoplastic
Remarks on result:
other: corresponding to the nominal concentration of 200/100 mg/m³
Key result
Dose descriptor:
NOAEC
Remarks:
respiratory system
Effect level:
>= 143.44 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect on the respiratory system was found up to the highest concentration tested.
Remarks on result:
other: corresponding to the nominal concentration of 200/100 mg/m³

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
18.2 mg/m³ air
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
The present subacute inhalation toxicity study was conducted according to the OECD guideline 412 (dated 1981) and GLP conditions. Male and female rats were exposed to the test item as aerosol at measured concentrations of 2, 18.2, and 143.4 mg/m³ air, under dynamic directed-flow nose-only exposure conditions. The aerosol was highly respirable to rats, with an average mass median aerodynamic diameter (MMAD) of 2.9 µm. Signs of hepatotoxicity were noticed at the highest tested concentration (liver enzyme induction, minimal to slight hepatocellular hypertrophy); furthermore, slight thyroid follicular cell hypertrophy also was noticed. Accordingly, the LOAEC was set at 143.4 mg/m³ whereas the NOAEC was 18.2 mg/m³.