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Diss Factsheets

Administrative data

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Sep - 2 Nov 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1997
Report date:
1997

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day 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 specied for toxicological studies and 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: 7 - 8 weeks (males), 14 weeks or older (females)
- Weight at study initiation: 222 - 256 g (males), 208 - 235 g (females)
- Fasting period before study: not applicable
- Housing: individually in polycarbonate cages type IIA with low-dust wood shavings, during acclimation in groups of 3 or 5 in polycarbonate cages type III
- Diet: Altromin 1324 Diet for Rats and Mice (Altromin GmbH and Co. KG in Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

DETAILS OF FOOD AND WATER QUALITY: feed and water were regularly analyzed for contaminations

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2.0
- Humidity (%): approx. 55 ± 5
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Remarks:
only moistened by tap water
Details on exposure:
TEST SITE
- Area of exposure: 5.5 x 5.5 cm = 30.25 cm²
- % coverage: over 10%
- Type of wrap if used: gauze layer ("Hansapor steril" patch), secured in place using ,,Peha-Haft" cohesive stretch tape (8 x 23 cm), at weekends, a ,"Fermoflex®" dressing was used additionally
- Time intervals for shavings or clippings: one day before first day of treatment and twice weekly afterwards

REMOVAL OF TEST SUBSTANCE
- Washing: with soap and water
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the amount was weighed out onto the gauze layer ("Hansapor steril" patch)
- Constant volume or concentration used: no
- For solids, paste formed: no, the powder was only moistened with tap water


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (mobility of the rats was impaired by a ,,Lomir Biomedical Inc." rat jacket)
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Analytical verification was not performed because the substance was applied undiluted and only moistened with water immediately before application.
Duration of treatment / exposure:
4 weeks treatment, 2 weeks recovery
Frequency of treatment:
First three weeks: 5 days/week, 4th week: 7 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
nominal dose applied to males and females of the main group, corresponding to 4.4-6.0 mg/cm²
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
nominal dose applied to males and females of the main group, corresponding to 4.1-5.5 mg/cm²
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
nominal dose applied to males and females of the main group, corresponding to 6.7-9.5 mg/cm²
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
nominal dose applied to males and females of the recovery group, corresponding to 7.0-9.4 mg/cm²
No. of animals per sex per dose:
5 (main and recovery groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses were based on a previously conducted two-week dose range-finding study in male and female rats. At 1000 mg/kg bw/day, slightly lower feed consumption in Week 1 (males and females), decreased lymphocyte counts and increased counts of segmented neutrophils (females), increased cholesterol (males) and decreased triglyceride levels (females), increased liver weight (males) and lower thymus weights (males and females), and spleens of dark-red color (females) were observed. On the application area neither erythema nor edema of the skin were observed.
- Rationale for selecting satellite groups: reversibility of the observations should be assessed
- Post-exposure recovery period in satellite groups: 2 weeks
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, (once daily at weekends and on public holidays)
- Cage side observations: Mortality, moribund state, body surfaces and orifices, posture, general behavior, breathing and excretory products, including irritation at the dose site

DETAILED CLINICAL OBSERVATIONS: No data

DERMAL IRRITATION: Yes (scoring according to Draize, including swelling)
- Time schedule for examinations: daily before treatment

BODY WEIGHT: Yes
- Time schedule for examinations: weekly, starting on Day 0 before before the first application

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 23/24 (main groups) and 41/42 (recovery groups) for glucose, on day of necropsy (Day 28 for main groups, Day 43 and 44 for recovery groups) for all other parameters
- Anesthetic used for blood collection: Yes (diethyl ether anesthesia, for all parameters except glucose) / No (glucose)
- Animals fasted: Yes (for glucose determination, blood taken from the distal vessels of the tail prior to necropsy) /No (everything else, blood taken by heart puncture at necropsy)
- How many animals: all animals
- Parameters checked: Erythrocyte count, hemoglobin, hematocrit, hepato quick, leucocyte count, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume erythrocytes (MCV), thrombocytes (THROMBO) and differential blood count (eosinophils, lymphocytes, monocytes, normal RBC, segmented neutrophils).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: see above
- Animals fasted: No
- How many animals: all animals
- Parameters checked: alanine aminotransferase (ALAT), albumin, alkaline phosphatase (APh), aspartate aminotransferase (ASAT), bilirubin total, cholesterol, creatinine, calcium, chloride, gamma-glutamyltransferase (GGT), glutamate dehydrogenase (GLDH), potassium, sodium, inorganic phosphorus, protein, triglycerides, urea and glucose.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:
Surviving animals were sacrificed by exsanguination under deep ether anesthesia.

GROSS PATHOLOGY: Yes
The following organs were weighed: adrenal glands, brain, heart, kidneys, liver, lung, spleen, testes and thymus.

HISTOPATHOLOGY: Yes
The following organs were immersion-fixed in Davidson's solution: Adrenal glands, brain (cerebrum, cerebellum, pons/medulla), heart, kidneys, liver, lung, skin (treated and untreated), spleen, testes, thymus, thyroid gland with parathyroid gland, physical identifier (tattooed ears), and all organs or tissues with macroscopic findings.
One liver lobe and lungs (fixed by instillation of 4% aqueous formaldehyde solution) of all rats were immersion-fixed in 4% aqueous formaldehyde solution.

- embedding media: paraplast
- Thickness: 5 µm
- Staining: hematoxylin and eosin (H&E), cryo-cuts obtained from the formalin-fixed liver lobe were stained with Oil-Red-O (ORO)

Histopathological examination was limited to the following organs/groups:
Liver (HE), thyroid gland and necropsy findings: all animal groups (including the recovery group)
Skin treated and untreated, liver (ORO): main groups (0, 100, 300, 1000 ppm, no recovery groups)
Adrenal glands, brain, heart, kidneys, lungs, spleen, testes, thymus: control and high dose group (main group only)
Statistics:
The quantitative results for individual animals were used to calculate arithmetic group means and standard deviations. The results for the groups that received the test substance were compared with those for the control group and significant differences indicated by "+" for p < 0.05 and "++" for p < 0.01. In case of numbers of values too low to calculate test statistics this is indicated by "0".

The Dunnett test was used for body weight, body weight gain, feed consumption and organ
weight data (relative organ weights subsequent to logarithmic transformation).
If possible, an ANOVA was used followed by a Dunnett's test for parametric measurements. For non-parametric data, a Kruskal-Wallis test followed by adjusted U test was performed.

Macro- and micropathological data were processed with the PATHDATA program, version 3.6. B.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No significant difference between control and treatment group was observed.
Dermal irritation:
no effects observed
Description (incidence and severity):
The skin redness and skin thickness were comparable between control and treated animals.
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant difference between control and treated animals was observed.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 1000 ppm: transient decrease of feed intake in females in the first week compared to controls

This significant transient decrease could also be a sign of stress caused by the treatment rather than the test substance so this finding is ambiguous. As the effect is transient, it is not considered toxicologically relevant. The statistically significantly increased values for feed consumption in g/kg body weight and day that was recorded for males of the recovery group are considered incidental and a result of the slightly lower body weights of these animals, as the mean feed intakes per animal and day show no abnormalities.

Summarized data can be found in Attachment 1 in the attached background material.
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:
not examined
Description (incidence and severity):
not applicable
Haematological findings:
no effects observed
Description (incidence and severity):
There were no significant differences between control and treatment groups.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant differences were observed in males of the main groups regarding cholesterol (100 and 300 mg/kg bw/day, -18.1 and -16.7%, respectively), creatinine (-13% for 100 mg/kg bw/day and -14.8% for 300 mg/kg bw/day), protein (-4.5% for 100 mg/kg bw/day and -6.3% for 300 mg/kg bw/day), albumin (-8.5% for 300 mg/kg bw/day) and glucose (-7.9% for 300 mg/kg bw/day) compared to controls. They are considered as incidental as they are not dose related and were observed only in one sex. At the end of the recovery period no toxicologically significant changes compared to controls were observed, the only statistically significant observations was slightly decreased creatine in males, considered incidental.
Endocrine findings:
not examined
Description (incidence and severity):
not applicable
Urinalysis findings:
not examined
Description (incidence and severity):
not applicable
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):
- 1000 mg/kg bw/day: increased absolute (+12.7%/+13.5%) and relative (+17.2%/+11.9%) liver weights (males/females) in the main group. This finding was reversible in the recovery group, so that this observation can therefore be seen as part of an adaptive process rather than a toxicologically relevant effect.

Summarized data can be found in Attachment 2 of the attached background material.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No significant differences between control and treatment groups were observed.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 300 mg/kg bw/day: liver hypertrophy of centrilobular hepatocytes associated with a more homogeneously structured cytoplasm (3/5 males) in the main group compared to controls. At the end of recovery period these changes were still observed in males that received 1000 mg/kg bw/day (2/5).
- 1000 mg/kg bw/day: liver hypertrophy of centrilobular hepatocytes associated with a more homogeneously structured cytoplasm (5/5 males, 3/5 females) compared to controls. At the end of the recovery period, this was still observed in males (2/5) but not in females. In the main group, hypertrophy of the follicular epithelium in thyroids (3/5 males, 2/5 females, all compared to controls) was observed. These findings were reversible for females but only partially in males, since hypertrophy of the follicular epithelium in thyroids occurred in 1/5 males of the recovery group.

The changes are considered to be toxicologically relevant.

Summarized results can be found in Attachment 3 in the attached background material.
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
not applicable
Other effects:
not examined
Description (incidence and severity):
not applicable

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects occurred at this dose level.
Key result
Dose descriptor:
NOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects occurred at this dose level.
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
LOAEL
Remarks:
systemic
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
dermal, local
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed up to this dose level.
Remarks on result:
other: Highest dose tested

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Applicant's summary and conclusion

Conclusions:
The study was conducted according to OECD guideline 410 and under GLP. Under the conditions of the test, the test substance caused liver and thyroid changes when applied dermally for four weeks in males and females. The changes in liver and thyroid were fully reversible for females but only partly reversible in males within the two-week recovery period. No dermal irritation was seen. Thus, 1000 mg/kg bw/day is regarded as the NOAEL for local skin effects and the NOAEL for systemic effects is 100 and 300 mg/kg bw for males and females, respectively.