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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted prior to GLP similar to OECD guideline 408 on the registered substance itself. The method is to be considered scientifically reasonable with minor deficiencies in documentation and performance, e.g. dosing intervals were chosen as 5-fold instead of 2-4-fold. Also, the original report is in German, which does however not affect the assessment of the study as the assessor is a native speaker.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report Date:
1980

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
not applicable
GLP compliance:
no
Remarks:
test conducted prior to GLP implementation
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Name of test material (as cited in study report): N-methyl-2-thion-thiazolidin
- Substance type: pure substance
- Lot/batch No.: 29 x 0570

Test animals

Species:
rat
Strain:
other: SPF, Wistar-TNO W.74
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Züchter Winkelmann, Borchem
- Age at study initiation: 35-42 days
- Weight at study initiation: 50-60 g
- Housing: single in Makrolon cages Type II with dust-free wood granules
- Diet (e.g. ad libitum): Altromin R-powder feed (Altromin, Lage) ad libitum
- Water (e.g. ad libitum): tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Altromin R-powder feed (Altromin, Lage)
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
3 months
Frequency of treatment:
continuously
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 500, 2500 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
15
Control animals:
yes, plain diet
Positive control:
none used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: after 1 and 3 months in 5 animals/sex/dose

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- 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: No data

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 data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 1 and 3 months
- Anaesthetic used for blood collection: Yes (diethylether)
- Animals fasted: No data
- How many animals: 5 animals/sex/dose
- Parameters erythrocyte and leukocyte count, thrombocyte count, hemoglobin, hematocrit, differential blood count, MCH, MCV, and thromboplastin time were examined.

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood: after 1 and 3 months
- Animals fasted: No data
- How many animals: 5 animals/sex/dose
- The following parameters were examined:
- Enzymes: Alkaline Phosphatase (ALP), Glutamate-Oxalacetate-Transaminase (GOT), Glutamate-Pyruvate-Transaminase (GPT), Glutamate-Dehydrogenase (GlDH)
- Substrates: Creatinine, urea, glucose, cholesterol, bilirubin, and total plasma protein

URINALYSIS: Yes
- Time schedule for collection of urine: 16h collection period after oral gavage of ca. 8 ml water
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- The following parameters were examined:
- semiquantitatively: glucose, blood, protein, pH value, ketobodies, bilirubin, urobilinogen, and microscopic sediment examination after centrifugation of urine
- quantitatively: protein according to Richterich (1968)

NEUROBEHAVIOURAL EXAMINATION: No data

OTHER:
Thyroid function:
- Protein-bound iodine (PBI)
- Rectal determination of body temperature
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsy of rats which died during experimental phase:
Rats were necropsied and examined macroskopically. All dissected organs and possible changes were fixed in Bouins fixans and after ca. 30 h in 70% ethanol.
- Necropsy of rats which were sacrificed at the end of the experimental phase:
All surviving animals were anesthized with diethylether and killed via exsanguination. After necropsy animals were examined macroscopically. The weights of thyroid, thymus, heart, lung, liver, spleen, kidneys, adrenal glands, testicles resp. ovaries were determined.
Of each five male and female animals heart, lung, liver, spleen, kidneys, pituitary gland, thyroid, adrenal glands, testicles, epididymis, prostate, seminal vesicle, ovaries, uterus, salivary glands, pancreas, esophagus, stomach, intestines, lymph nodes, thymus, bladder, brain, eyes, aorta, trachea, skeletal muscles incl. nervus ischiadicus, bones and bone marrow were fixed in Bouins fixans and after 30h in 70% ethanol, parts of the liver of these animals were fixed in Formol-Ca.
The organs to bei weighted as well as brain, pituitary gland and testicles resp. ovaries were fixed in 10% buffered formaldehyde solution.

HISTOPATHOLOGY: Yes
5 µm paraplast section were prepared from the fixed organs resp. organ samples and stained with hemalaun-eosin. Additional sections of the kidneys were stained via PAS reaction. 15 µm frozen sections of formol-fixed liver were stained with Oil Red O for fat determiantion. Bones were decalcified with sodium EDTA. Histological examinations were made on each five male and female animals of the control and high dose group.
Statistics:
Arithmetic means, standard deviations, upper and lower confidence limits with the confidence niveau of 1-α = 95% and 1-α = 99%. Values of the test and control groups were compared with the test for significance (U-Test) according to Mann, Whitney and Wilcoxon on the significance niveau α = 5% and α = 1% using the computer IBM-370/145. Establishing the random list was done using "Subprogram Randu" of the "Scientific Subroutine Package" by IBM on the computer IBM-370.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Appearance and behaviour were not influenced in male and female rats at dosages up to 2500 ppm. 2 males of the high dose group died during the course of the study.
Mortality:
mortality observed, treatment-related
Description (incidence):
Appearance and behaviour were not influenced in male and female rats at dosages up to 2500 ppm. 2 males of the high dose group died during the course of the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Growth was not influenced in the 100 ppm group. In the 500 and 2500 ppm group food consumption and body weight gain were diminished significantly (p<0.01) during the whole study duration.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was not influenced in the 100 ppm group. In the 500 and 2500 ppm group food consumption and body weight gain were diminished significantly (p<0.01) during the whole study duration. A repellent effect of the test item cannot be excluded.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Blood was not damaged by the test item in the groups up to 500 ppm. An intermittent influence on the leucocytes was noticed after one month in the 2500 ppm group, which could not be observed after 3 months.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment-related adverse effects to the liver or kidneys in dosages up to 500 ppm. An intermittent increase of the enzymes GOT and GPT, a decrease in plasma protein and persistent elevated urea concentration was noted in the 2500 ppm group.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
There was no indication on kidney damages in the dosage groups up to 500 ppm. At the 2500 ppm group elevated urea concentrations were noted.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Necropsies and histopathological examinations gave no indications on treatment-related effects on any organs in groups up to 2500 ppm.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsies and histopathological examinations gave no indications on treatment-related effects on any organs in groups up to 2500 ppm.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Necropsies and histopathological examinations gave no indications on treatment-related effects on any organs in groups up to 2500 ppm.
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No differences between control and dosage groups were observed regarding appearance and behaviour. During the course of the study two males of the 2500 ppm group died.


BODY WEIGHT AND WEIGHT GAIN
Male and female rats gained weight similarly to the ones from the control. The 500 ppm and 2500 ppm animals showed during the whole study a significantly (p<0.01) decreased body weight gain.


FOOD CONSUMPTION AND COMPOUND INTAKE
Details on food consumption and compound intake can be found in table 1, “any other information on results incl. tables”. Male and female rats consumed powder feed similarly to the ones from the control. The 500 ppm and 2500 ppm animals consumed approx. 10% resp. 45% less feed than control animals.


HAEMATOLOGY
Table 2a (“any other information on results incl. tables”) shows the results (mean values) of the blood analysis, table 2b the differential blood count after 1 and 3 months of treatment.
After 1 month, all treated females and the 100 ppm males did not differ relevantly from the control animals regarding erythrocyte and leukocyte count, thrombocyte count, hemoglobin, and haematocrit, as well as MCH and MCV. The males exhibited a significantly decreased thrombocyte (500 and 2500 ppm) and leucocyte count (2500 ppm). The 100 ppm animals did not differ from control regarding differential blood count and shape of the single cell types. In the 2500 ppm animals, the amount of neutrophile granulocytes with segmented nucleus was significantly elevated, in the females of the 500 ppm group and the 2500 ppm animals the leucocyte count was significantly decreased.
After 3 months, no significant differences were observed between all treated animals and control regarding blood analysis and differential blood count.


LIVER FUNCTION
Table 3 (“any other information on results incl. tables”) shows the results of the liver function related enzymes and total plasma protein. The values of the control and the 100 ppm group do not differ significantly. After 1 month, the 2500 ppm animals showed increased values of the transaminases GOT and GPT, significantly increased was only the GOT value of the males. Additionally, significantly decreased protein plasma levels were detected in the 500 ppm and 2500 ppm groups as well as increased ALP activities in the 2500 ppm males.
After 3 months, there were no treatment-related differences regarding ALP, GOT, GPT, GLDH and Bilirubin up to 2500 ppm. Total plasma protein was decreased in the 2500 ppm females.


KIDNEY FUNCTION
Results of the urine examinations after 1 and 3 months revealed no significant differences compared to control. In none of the examined rats glucose, keto bodies or bilirubin was found in the urine. Only in male rats after 3 months there were found protein- and blood-positive results, which are however dose-independent. The content of urobilinogen and pH value of the treated animals did not differ to a relevant extent from control, examination of sediment did not show a treatment-related influence.
The mean urea- and creatinine-concentrations as well as protein content in urine can be found in Table 4 (“any other information on results incl. tables”). Urea and creatinine-concentrations were in physiological range for animals dosed up to 500 ppm after one month and in all group after 3 months. Elevated urea was detected in the 2500 ppm group (only significant in males) after 1 month. Protein content in the urine was not elevated in any of the treated groups.


CLINICAL CHEMISTRY – OTHER
Blood sugar and cholesterol were determined not to be in pathological range in dosage groups up to 2500 ppm. Table 5 (“any other information on results incl. tables”) shows these results. All (significantly) deviating values in males and females are dose-independent, all values are within the normal range for animals of this age and hence not toxicologically relevant.


THYROID FUNCTION
The results of the determination of protein-bound iodine (PBI) after 4 and 9 weeks as well as 3 months can be found in Table 6 (“any other information on results incl. tables”). Decreased PBI values were determined only in males and females after one month and males after 3 months in the 2500 ppm group. , which were only significant in males. Additionally, after 9 weeks in males of the 100 ppm group an increased PBI value was found.
Body temperatures (also Table 6) were significantly decreased after one month in males and females in the 2500 ppm group. No deviations from control were found after 3 months. After one month in males and females of the 100 and 500 ppm and after 3 months in the 100 ppm males slightly elevated body temperatures were measured.


GROSS PATHOLOGY
Necropsy of rats which died during study:
Both perished males were necropsied. In one rat there were no distinct findings, the other rat showed bleedings in stomach and mucosa in the fundus and elevated content of air and liquids in the lung.

Necropsy of rats killed at the end of the study:
Necropsies of all animals gave no indications of a specific damage in all dosage groups.


ORGAN WEIGHTS
Tables 7a and 7b (“any other information on results incl. tables”) show the absolute and relative organ weights of thyroid, thymus, heart, lung, spleen, kidneys, adrenal glands and testicles resp. ovaries.
Organ weights of the 100 ppm males and females did not differ significantly from control. Deviations from control in the 500 and 2500 ppm groups compared to control were commonly decreased. Relative organ weights however were in the 2500 ppm group significantly increased, except thymus (males) and spleen, adrenal glands and ovaries (females). Furthermore, in the 500 ppm group the weights of thymus, heart, liver, spleen and kidneys were decreased, the ones of the testicles were increased.


HISTOPATHOLOGY: NON-NEOPLASTIC
Incidental finding in individual animals of various groups were: unspecific chronic pneumonia (control (2 animals), 2500 ppm (1)), Discharge of follicles in the spleen and progressed thymus involution (2500 ppm (1)), bilateral testicle atrophy (2500 ppm (1)), pituitary cyst (control (1), 2500 ppm (1)), unilateral dilatation of uterine horns (2500 ppm (2)), slight foreign body reaction in the greater omentum (2500 ppm (2)), slight phagocytosis of hematogen pigment in lymph nodes (2500 ppm (1)), unspecific inflammatory-cellular infiltration of the eye (control (1), 2500 ppm (3)) resp. traches (2500 ppm (1)).
The organs of the rats which died during the study were autolytically altered; other changes were not noted.

Effect levels

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Dose descriptor:
NOAEL
Effect level:
100 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the average food consumption and mean body weight of the animals in the mid of the study, this corresponds to 7.91 mg/kg bw/d (males) and 9.78 mg/kg bw/d (females).
Dose descriptor:
LOAEL
Effect level:
500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight; food consumption Based on the average food consumption and mean body weight of the animals in the mid of the study, this corresponds to 42.70 mg/kg bw/d (males) and 49.54 mg/kg bw/d (females).
Dose descriptor:
LOAEL
Effect level:
2 500 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: mortality; clinical chemistry; urinalysis; Based on the average food consumption and mean body weight of the animals in the mid of the study, this corresponds to 202.54 mg/kg bw/d (males) and 228.87 mg/kg bw/d (females).

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

See attachment, free text field does not allow entering the required amount of characters.

Applicant's summary and conclusion

Conclusions:
The study was conducted prior to GLP similar to OECD guideline 408 on the registered substance itself The method is to be considered scientifically reasonable with minor deficiencies in documentation and performance, e.g. dosing intervals were chosen as 5-fold instead of 2-4-fold. Hence, the results can be considered as sufficiently reliable to assess the repeated dose oral toxicity in rats.
3-methylthiazolidine-2-thione did not induce any relevant effects in male and female rats under the conditions of the test in the dosage of 100 ppm. Hence, the NOAEL can be set as 100 ppm, which corresponds based on feed intake determinations to 7.91 mg/kg bw/day in males and 9.78 mg/kg bw/day in females.
Executive summary:

In a subchronic toxicity study similar to OECD 408 3-methylthiazolidine-2-thione was administered to 15 SPF, Wistar-TNO W.74 rats/sex/dose in diet at dose levels of 0, 100, 500, 2500 ppm.

 

Appearance and behaviour were not influenced in male and female rats at dosages up to 2500 ppm. 2 males of the high dose group died during the course of the study.

Food consumption and growth were not influenced in the 100 ppm group. In the 500 and 2500 ppm group food consumption and body weight gain were diminished significantly (p<0.01) during the whole study duration. A repellent effect of the test item cannot be excluded.

Blood was not damaged by the test item in the groups up to 500 ppm. An intermittent influence on the leucocytes was noticed after one month in the 2500 ppm group, which could not be observed after 3 months.

Clinical analyses, necropsies as well as histopathological examinations revealed no indication on of treatment-related adverse effects to the liver in dosages up to 500 ppm. After one month an increase of the enzymes GOT and GPT as well as a decrease in plasma protein was noted in the 2500 ppm group. These differences compared to control could not be observed at the end of the study.

Urinalysis, clinical analyses,necropsies as well as histopathological examinations revealed no indication on kidney damages in the dosage groups up to 500 ppm. At the 2500 ppm group elevated urea concentrations were noted.

Blood sugar and cholesterol concentrations were in dosages up to 2500 ppm within the norm.

Thyroid function was not influenced by the test item in the dosage groups up to 100 ppm.

Necropsies and histopathological examinations gave no indications on treatment-related effects on any organs in groups up to 2500 ppm.

Dosages of 100 ppm of the test item were tolerated under these conditions without any effects.

 

The LOAEL is 500 ppm, based on changes in body weight and food consumption. The NOAEL is 100 ppm.

 

This subchronic toxicity study in the rat is acceptable, and satisfies scientifically sufficiently the guideline requirement for a subchronic oral study (OECD 408) in rats.