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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 Jan - 14 Feb 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000
Reference Type:
other: Amendment
Title:
Unnamed
Year:
2000
Report date:
2000
Reference Type:
other: Amendment
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 2008
Deviations:
yes
Remarks:
The study was conducted to investigate the mechanism of thyroidal changes after treatment with the test material. Therefore, the examinations were limited. For details please refer to "Principles of method if other than guideline".
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted 1995
Deviations:
yes
Remarks:
The focus was set on the mechanism of test substance-related thyroidal changes.
Principles of method if other than guideline:
The study period was 3 weeks instead of 4, animals were housed individually instead of in groups, and were checked for morbidity and moribundity only once per day. Sensory reactivity was not assessed and no haematology was performed. Regarding clinical chemistry, only T3, T4, TSH, UDP-GT-levels and activity and plasma hormone levels were assessed. Organ weights were recorded for liver, uterus and the left thyroidal lobe only and histopathology was performed only for thyroid.
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:
Common strain 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 (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Weight at study initiation: mean of male groups: 173-178 g, mean of female groups: 131-135 g
- Fasting period before study: not applicable
- Housing: individually under conventional conditions in type IIa polycarbonate cages on low-dust wood granulate (S sniff Spezialdiäten GmbH, Soest, Germany), during acclimatization rats were kept in groups (about 5 animals per type III cage, separated by sex)
- Diet: fixed-formula standard diet (Altromin® 1321 powder supplied by Altromin GmbH, 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 checked for contaminations.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): approx. 55
- Air changes (per hr): 15.20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
peanut oil
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): fixed-formula standard diet (Altromin® 1321 powder supplied by Altromin GmbH, Lage, Germany)
- Storage temperature of food: room temperature

VEHICLE
- Justification for use and choice of vehicle: peanut oil was mixed in the feed to prevent dust-formation
- Amount of vehicle in feed: 1%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Data on homogeneity and stability of the test substance in the administration vehicle covering the concentration range used were obtained. It was found that the test substance was stable in the diet for 8 days.
Duration of treatment / exposure:
21 days
Frequency of treatment:
continuously via the diet
Doses / concentrationsopen allclose all
Dose / conc.:
25 ppm
Remarks:
corresponding to 2.6 and 3.1 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
100 ppm
Remarks:
corresponding to 9.0 and 12.3 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
400 ppm
Remarks:
corresponding to 36.9 and 44.6 mg/kg bw/day actual dose ingested for males and females, respectively
Dose / conc.:
1 600 ppm
Remarks:
corresponding to 145.1 and 190.8 mg/kg bw/day actual dose ingested for males and females, respectively
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The same doses had been administered in a subchronic feeding study in rats. In the present study, particular emphasis was given to the effects in thyroids and on thyroid hormones.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: Body surfaces and orifices, posture, general behavior, breathing and excretory products

BODY WEIGHT: Yes
- Time schedule for examinations: Before beginning of the study and weekly thereafter

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

HAEMATOLOGY: No

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Days -2 through 14 (retroorbital plexus) and Day 21 and 22 (cardiac puncture)
- Anaesthetic used for blood collection: Yes (deep diethylether anesthesia)
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked: Triiodothyronine (T3), thyroxine (total) (T4), thyroxine binding capacity (TBC) and thyroid stimulating hormone (TSH) levels and protein content (from heparinized plasma), UDP-glucuronyl transferase (UDP-GT, from liver samples at time of necropsy)

PLASMA/SERUM HORMONES/LIPIDS: Yes, see above

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
Sacrifice of surviving animals was performed by exsanguination under deep ether anesthesia,

GROSS PATHOLOGY: Yes
Organ weights were recorded for liver, uterus and the left thyroidal lobe.


HISTOPATHOLOGY: Yes
tissues collected: liver, thyroid, uterus and pinnae were fixed in aqueous 10% formalin solution, tissues investigated: thyroid
Optional endpoint(s):
Optional endpoints: No
Other examinations:
None
Statistics:
The quantitative results for individual animals were used to calculate group means, median 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.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The only effect observed was bright feces of one female (high dose) after 2 and 3 weeks. This finding was considered incidental.
Mortality:
no mortality observed
Description (incidence):
not applicable
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- 1600 ppm: decreased transient body weight (males, Week 1 and 2, -10.5 and -8.4%) and decreased body weight in females (throughout the study, -12.5 to -13.0%), reduced body weight gains in males (-14.9%) and females (-43.5%), all compared to controls.

Summarized data can be found in Attachment 1 in the attached background material.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- 1600 ppm: reduced feed consumption and daily feed intake compared to controls during Week 1 for both sexes

Not treatment related:
Decreased daily feed intake for the 100 and 400 ppm male rats compared to controls was not considered biologically relevant because of a lack of dose response.

Summarized data for the 21-day interval can be found in Attachment 2 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:
not examined
Description (incidence and severity):
not applicable
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Thyroid parameters
- 1600 ppm: significant reduction in T3 levels on Day 2 in male and female rats, significant reduction in total T4 levels on Days 2 and 7 in male rats and on Day 2 in female rats, increase in the level of TSH in males on Day 14 and in females on Days 7 and 22, all compared to controls.

As can be expected, there was variation within the observations made in the control groups at different times. Hormone levels underlie a wide intra-individual variation. For relative changes, the pre-treatment data of each treatment group was used instead of the control group as reference. Therefore, analysis of covariance has been made. Treatment-related results were

- 400 ppm: increased thyroid stimulating hormone (TSH) in males from Day 7-21 (not statistically significant but in dose-dependency with results seen at 1600 ppm) compared to controls.
- 1600 ppm: reductions in T3 and T4 on Day 2 for males (-21.2 and -32.0%, respectively) and females (-30.9 and -34.4%, respectively) compared to controls, reduction of T4 continued for males throughout the study (-11.31 to -38.88%) and for females at Day 7 and 22 (-21.8 and -41.6%, respectively) compared to controls, increased TSH throughout the study in males and females, statistically significant at Day 14 in males (532.8%) and Day 7 (127%) and Day 22 (247.7%) in females compared to controls.

Summarized data can be found in Attachment 3.

UDP levels
- 400 ppm: increased UDP-GT levels in males (+98.2%) and females (+64.1%) compared to controls.
- 1600 ppm: increased UDP-GT levels in males (+252%%) and females (+221%%) compared to controls.

Summarized data can be found in Attachment 4. Reference values for total protein and thyroid parameters are presented in Attachment 5.

Further findings not considered to be treatment-related:
Changes in plasma protein levels were statistically significant compared to controls at some instances (females at 1600 ppm, males at all dose levels). However, these were not dose-dependent and close to control values and were therefore not considered treatment-related.
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):
- 400 ppm: increased absolute liver weight in males compared to controls (+14.2%)
-1600 ppm: increased absolute (+32.2% in males, +22.9% in females) and relative (+40.6% in males, +39.4% in females) liver weight compared to controls

Further findings not considered to be treatment-related:
The decreased thyroid weights in females (absolute at > 400 ppm, relative at 400 ppm) compared to controls were not regarded as treatment-related, as there was no dose response for the relative thyroid weight.

Summarized data can be found in Attachment 6.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- 1600 ppm: increased liver (10/10 males, 9/10 females), marked lobular pattern (7/10 females)

Summarized data is presented in Attachment 7.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- 400 ppm: minimal to slight hypertrophy of the follicular epithelium compared to controls (males)
- 1600 ppm: minimal to slight hypertrophy of the follicular epithelium compared to controls (males/females)

Summarized data can be found in Attachment 8 (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
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at this dose level.
Remarks on result:
other: corresponding to 9.0 mg/kg bw/day for males and 12.3 mg/kg bw/day for females
Key result
Dose descriptor:
LOAEL
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Remarks on result:
other: corresponding to 36.9 mg/kg bw/day for males and 44.6 mg/kg bw/day for females

Target system / organ toxicity

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

Applicant's summary and conclusion

Conclusions:
The present study was conducted according to the OECD guideline 407 (dated 1995), however with the focus being set on the mechanism of thyroidal changes related to the test substance; the study conduct was GLP compliant. It was shown that the test substance induces the hepatic enzyme UDP-glucuronyl transferase (UDP-GT). In general, induction of UDP-GT leads to increased T4 glucuronide formation and excretion of T4, resulting in decreased T3 and T4 levels, which was also found in this study. TSH levels increased to compensate for decreased T4 and T3 levels and led to a continuous activation of the thyroid as already seen by hypertrophy of the follicular epithelium of the thyroid. Moreover, changes in the liver and on body weight and feed consumption were observed. It was concluded that the activation of UDP-GT was seen as a primary effect of the test substance and the consequent changes in hormone level and thyroid histopathology secondary effects due to activation of the hypothalamic-pituitary-thyroidal (HPT) axis. A dietary concentration of 100 ppm (equivalent to 9.0 and 12.3 mg/kg body weight per day, for males and females, respectively) was determined as a clear no-observed-effect level.