2-thiazolidinylidenecyanamide

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to OECD guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

427-720-1

EC Name:

-

Cas Number:

26364-65-8

Molecular formula:

C4H5N3S

IUPAC Name:

[(1,3-thiazolidin-2-ylidene)amino]formonitrile

Details on test material:

name: Cyanamide, (4,5-dihydroxy-2-thiazolyl)-
molecular formula: C4 H5 N3 S
molecular weight: 127.2
physical state: solid
analytical purity: 96.7 %

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH
- Age at study initiation: about six weeks for male and as about 7 weeks for female rats
- Weight at study initiation: 154-158 g (males), 134-139 g (females)
- Housing: polycarbonate cages (5 animals per cage, separated by sex).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): approx. 55 ± 5%
- Air changes (per hr): approx. 15 - 20 times per hour
- Photoperiod (hrs dark / hrs light): 12 hours, illumination from 6 a.m. to 6 p.m.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The formulation of test substance was done using a magnetic mixer. Directly before (<1 hour) and during the administration the applied formulations were well mixed by stirring on a magnetic mixer. An additional mixing occurred by pumping the syringe for several times.

VEHICLE
- Concentration in vehicle: 0.5 % aqueous Carboxymethyl cellulose (CMC)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical data verify that the test material was homogeneously distributed within the concentration range of 0.15 wt.% to 50 wt.% (Table A1 + study no. T8061092). Under current sample preparation and handling conditions comparable to those in the actual study the chemical stability was assured for a period of at least 4 days.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Once per day

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
140 mg/kg
Basis:
other: nominal in vehicle

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg
Basis:
other: nominal in vehicle

Doses / concentrations 3

Remarks:

Doses / Concentrations:
15 mg/kg
Basis:
other: nominal in vehicle

No. of animals per sex per dose:

5 male and 5 female each.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosages were selected on the basis of the results of an earlier tolerability study, in which a dose of 100 mg/kg body weight was fed to female rats for 7 days. At the end of this study, a slight body weight retardation was observed, and no clinical signs and no mortalities occurred.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The experimental animals were inspected at least twice a day (once daily at
weekends and on public holidays). Any clinical signs (findings) and abnormalities
were recorded. Body surfaces and orifices, posture, general behavior, breathing and
excretory products were assessed.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The experimental animals were inspected at least twice a day (once daily at
weekends and on public holidays). Any clinical signs (findings) and abnormalities
were recorded. Body surfaces and orifices, posture, general behavior, breathing and
excretory products were assessed.


BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of the individual experimental animals were determined before the
beginning of the study and daily thereafter up to scheduled necropsy.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
The feed intake of each individual rat was determined ones a week.


OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes

Blood samples for determination of glucose taken in the morning from a caudal vein of fasted, non-anaesthetized animals. The samples were deproteinized with perchloric acid.
The blood for the other clinical pathology tests was withdrawn from animals under deep diethyl ether anesthesia by cardiac puncture at the time of necropsies. The samples for the hematological determinations were collected in tubes coated with EDTA (anticoagulant). The samples for biochemical test were heparinized (plasma) and untreated (serum).

The following hematological parameters were determined in peripheral blood:
differential blood count, morphology of erythrocytes, erythrocytes, hemoglobin, hematocrit, leucocyte count, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, platelet count, thromboplastin time, Hepato quick.


CLINICAL CHEMISTRY: Yes

The blood for the other clinical pathology tests was withdrawn from animals under deep diethyl ether anesthesia by cardiac puncture at the time of necropsies. The samples for the hematological determinations were collected in tubes coated with EDTA (anticoagulant). The samples for biochemical test were heparinized (plasma) and untreated (serum).

The following parameters were determined:
Enzyme activities in plasma: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, glutamate dehydrogenase.
Substrates/Electrolytes: albumin, protein (total), bilirubin (total), cholesterol, creatinine, glucose, triglycerides, urea, calcium, chloride, potassium, sodium, phosphorus.


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: Yes
weeks 1 , 2 3 and 4.
Before starting of treatment, and during week 1, 2 and 3 the examinations were conducted individually on all animals within one day, starting with the male groups.
The FOB at week 4 was performed at two consecutive days testing males on the first day and females on the second day.
The following observations/examinations were performed:
home cage observation: posture, piloerection, gait abnormalities, involuntary motor movements, vocalization
observations during handling: ease of removing, reaction to being handled, muscle tone, palpebral closure, pupil size, lacrimation, salivation, stains
open field observations: piloerection, respiratory abnormalities, posture, involuntary motor clonic, involuntary motor tonic, stereotypy, bizarre behavior, gait abnormalities, arousal, rearing, defecation and urination
The following manipulative tests were additionally determined in week 4:
pupil response, approach response, touch response, auditory response, tail pinch response, grip strength.

Motor activity assessments were conducted in the fourth exposure week following the FOB examination.
Motor activity (MA) and locomotor activity (LMA) were examined as activity for the entire 70-minute session (Summary Session MA and LMA) and activity during each 10-minute interval (Summary Interval MA and LMA). Motor activity was measured as the number of beam interruptions that occurred during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the rat relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The following exsanguinated organs of the animals sacrificed at necropsy were weighed in the unfixed state:
adrenal glands, brain, epididymides, heart, kidneys, liver, spleen, testes, thymus.

HISTOPATHOLOGY: Yes

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not examined

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

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
No clinical signs were observed in the animals at up to and including 50 mg/kg body
weight. At 140 mg/kg in females transiently piloerection occured, and in one female
feces was discoloured. No animal died throughout the entire treatment period.

BODY WEIGHT AND WEIGHT GAIN
There was no significant effect of treatment on body weights at 50 mg/kg and below.
At 140 mg/kg a slight retardation in body weight development was observed.


OPHTHALMOSCOPIC EXAMINATION
Not examined

HAEMATOLOGY
No evidence for lexicologically significant effects on the red blood cells, including
erythrocyte morphology, white blood cells, hemogram and blood coagulation were
detected at 50 mg/kg and below. At 140 mg/kg males, the clotting time and the
counts of neutrophiles were increased. The increased clotting time as well as the
isolated statistically significant values (platelet and monocyte counts) are considered
not of lexicologically significance because they are not dose-related and/or all
individual values are in the historical range.

CLINICAL CHEMISTRY
At 15 mg/kg and above in both sexes lower concentrations of triglycerides were
noted. In females all individual values are within the 2s-range of the historical control
values.
At 140 mg/kg in males the concentrations of calcium were decreased, and in females
the concentrations of total protein increased. All individual calcium values are within
the 2s-range of the historical control values. The total proteins were increased only
in one sex, the difference to the controls was minimal (4%). Thus, these differences
were considered as not toxicologically significant.
The other isolated differences to the controls recognized as statistically significant
are not considered to be of toxicological relevance either as the difference is only
minor, not dose-related, and all individual values are within the 2s-range of the
historical control values.

URINALYSIS
not examined

NEUROBEHAVIOUR
Before commencing treatment and during weeks 1, 2, and 3 (days 2, 9 and 16) a
detailed clinical observation including home cage, handling and open field behavior
was conducted using some elements of the functional observational battery (FOB) in
all animals.
In week 4 (days 22 and 23) a FOB was conducted including home cage observation,
handling and open field observations as well as investigations of pupil response,
approach, touch, auditory and tail pinch reflexes and measurement of grip strength.
The summary tables and individual results are shown in the Annex (9.13.).
With the exception that piloerection was observed in 2 of 5 female rats of the 140
mg/kg group on day 9, no other indication of compound-related effects was recorded
during the detailed clinical observations before and during the treatment for males
and for females. In the last week of treatment, on day 22, in male rats there was
apparently an increase in rearing at 140 mg/kg. On the contrary, in females of this
group, on day 23, there was a decrease in rearing.
Reflex testing and measurement of grip strength did not provide indications of
treatment related effects.
Motor activity measurements of male and female rats were made on days 22 and 23,
respectively. Mean and individual motor (MA) and locomotor (LMA) activity data are
shown in the Annex (9.14.) for both the entire session (70-minute) MA and LMA and
also thelO-minute intervals.
The activity determination over the entire 70 minute observation period failed to
reveal a significant effect on motor (MA) and locomotor activity (LMA) at 50 mg/kg
and below. At 140 mg/kg in males and females, the motor and the locomotor
activities were reduced. The degrees of reduction were about 20 to 30%, and thus
too small to be considered a clear-cut treatment-related effect but rather a discrete
trend.
The results of the 10 minute intervals indicate that treated and control groups
reacted in a similar way and that habituation (defined as a decrement in activity
during the test session) followed the same curve and had the same slope. In
particular, at 50 mg/kg and below, there were no significant differences in the first
10-minute intervals which are regarded as best indicator of increases or decreases
in activity before the animals habituate to a minimal level of activity. At 140 mg/kg, in
males and females, the motor and the locomotor activities were slightly decreased.
For males, this effect is not in agreement with the results of the FOB data (number of
rearings).


ORGAN WEIGHTS
At 140 mg/kg, liver weights of females were statistically significant increased
(relative).

GROSS PATHOLOGY
All gross findings are evenly distributed among all dose groups and/or are
known from control animals of previous or concurrent studies with rats
of this strain and age. Thus, they are considered to be of spontaneous
origin.

HISTOPATHOLOGY: NON-NEOPLASTIC
As shown in the histopathology report, microscopic examination revealed only
changes in lymphoid organs. These were enlarged and/or increased numbers of
germinal centers.
In the spleen, all 140 mg/kg animals revealed an increase in number and size of the
germinal centers of the lymph follicles. Increased numbers of germinal center
reaction were observed also in animals of the 50 mg/kg, and in the males of the 15
mg/kg group. However, they were no dose-related in males at 15 and 50 mg/kg.
In the mandibular lymph nodes, germinal centre reaction was observed at 140
mg/kg. The significance of this finding remains equivocal due to the greater variation
of available tissue in the specimen.
In the mesenteric lymph nodes no changes were detected.
Sperm granulomas in epididimydes were observed in 2 of 5 males at 140 mg/kg.
They are regarded of spontaneous origin, since this finding represents a lesion
relatively frequently seen in young animals.
All other histopathologic findings seen in this study are evenly distributed among all
dose groups and/or are known from previous studies to be spontaneously occurring
changes in rats of that age.

Effect levels

Dose descriptor:

NOAEL

Remarks on result:

not determinable

Remarks:

no NOAEL identified

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

All data can be taken from the attached document in Overall remarks, attachments.

Applicant's summary and conclusion

Conclusions:

Based on the findings in this study the substance is not subject to classification.

Executive summary:

The systemic tolerance of rats to 2-cyanimino-1,3-thiazolidin was examined in a subacute toxicity study involving oral administration by gavage (four-week treatment). The study methodology conformed to the OECD-Guideline for Testing of Chemicals; Section 4: Health Effects, No. 407 (updated July 27, 1995; OECD Guidelines for Testing of Chemicals, Section 4, Health Effects).

Groups of 5 male and 5 female rats of the strain HsdCpb:WU were administered 2- cyanimino-1,3-thiazolidin each day at levels of 0, 15, 50 and 140 mg/kg orally by gavage over a period of 4 weeks.

At 50 mg/kg body weight and below there was no difference between the treated and untreated animals regarding survival rate, state of health or general behavior of the animals, feed intake, body weights or body weight development, red blood cells including erythrocyte morphology, white blood cells, hemogram and blood coagulation.

At 140 mg/kg, in females transiently piloerection occured, and in one female feces was discoloured.

At 140 mg/kg the feed intake was decreased in the first week of treatment, and a slight retardation in body weight development was observed.

At 140 mg/kg, the counts of neutrophiles were increased in males.

At 15 mg/kg and above in both sexes lower concentrations of triglycerides were noted.

Relative liver weights of females treated with 140 mg/kg were statistically significant increased. However, no similar finding was apparent in males and no morphological correlate was seen in the hisopathological examination.

At 15 mg/kg and above in males and at 50 mg/kg and above in females, an increase in number and size of the germinal centers of the lymph follicles was detected in the spleen. This effect is regarded as a treatment related immunostimulation of the spleen. The significance of the germinal centre reaction observed at 140 mg/kg animals in the mandibular lymph nodes remains equivocal due to the higher physiologic variability with respect to germinal center reactions and to the greater variation of available tissue in the specimen.

The detailed clinical observations including home cage, handling and open field behavior using some elements of the functional observational battery (FOB), the observation of reflexes and the results of the grip strength measurements, and the assessment of motor and locomotor activity showed no indication of neurotoxic changes during the treatment for males and for females. The observed effects in animals at 140 mg/kg most probably reflect the described alterations of the general health status.

In conclusion, administration of 2-cyanimino-1,3-thiazolidin to Wistar rats for 4 weeks in concentrations of 15 mg/kg and higher resulted in toxic effects. Main target is the hematopoietic system.