Imidazolidine-2-thione

Repeated dose toxicity: oral

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: Comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

publication

Title:

Dietary subacute toxicity of ethylenethiourea in laboratory rat.

Author:

Freudenthal RI, Kerchner G, Persing R and Baron RL

Year:

1977

Bibliographic source:

J. Environ. Pathol. Toxicol. 1: 147-161

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

No hematology and urine analysis, limited blood chemistry and histopathological examinations

GLP compliance:

not specified

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Imidazolidine-2-thione

EC Number:

202-506-9

EC Name:

Imidazolidine-2-thione

Cas Number:

96-45-7

Molecular formula:

C3H6N2S

IUPAC Name:

imidazolidine-2-thione

Details on test material:

ETU was obtained from Aldrich Chemical Company (Milwaukee, WI).
The ETU used in this study was analyzed by mass spectrometry and nuclear magnetic resonance spectrometry.
Purity = 96.8%

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories (Wilmington, MA)
- Age at study initiation: 8/9 weeks
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individually
- Diet (e.g. ad libitum): Ralston Purina Company (Richmond, Indiana)
- Water (e.g. ad libitum):no data
- Acclimation period:no data

ENVIRONMENTAL CONDITIONS : no data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

corn oil

Details on oral exposure:

The final corn oil concentration in the bledded feed was 1%.
The test diets were prepared fresh weekly.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

30, 60 and 90 days

Frequency of treatment:

daily (in feed)

Doses / concentrations

Remarks:

Doses / Concentrations:
1, 5, 25, 125 or 625 ppm (corresponding approximately to 0.07, 0.3, 1.7, 8 and 42 mg/kg body weight/day).
Basis:
nominal in diet

No. of animals per sex per dose:

Treated groups : 20 animals / sex / dose / duration of exposure
Negative control group : 24 animals / sex / duration of exposure
Positive control groups : 20 animals / sex / duration of exposure

Control animals:

yes, concurrent vehicle

Details on study design:

no

Positive control:

Yes, 2 groups: Propylthiouracil (PTU, 125 ppm) and amitrole (50 ppm).
These dietary levels of PTU and amitrole were previously shown to produce significant antithyroid effects.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (mortality), weekly
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes weekly
FOOD CONSUMPTION : Yes weekly
OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: No data
CLINICAL CHEMISTRY: Yes : serum T-3, T-4, TBG, and TSH concentrations
URINALYSIS: No data
NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

The tissues removed during necropsy were heart, spleen, kidney, ovary (paired), testicle (paired), liver, pituitary, thyroid (paired), adrenal (paired), and brain. The thyroid gland and liver were examined microscopically in all study groups.

Other examinations:

At 30-day intervals, 10 rats of each sex from each test group were sacrificed and serum T-3, T-4, TBG, and TSH concentrations were measured. These rats were also used for necropsy. The remaining 10 rats of each sex per group were used for thyroid 125I uptake studies.

Statistics:

yes, See tables.

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):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
Mortality was observed in this study only at the highest ETU dose level. Fourteen of the 40 rats on the 625 ppm ETU 60-day study died between days 40 and 60 of the study. Only one rat f rom the 625 ppm 90-day group died. There was no mortality noted in this study attributable to the presence of ETU in the diet at dietary levels of 125 ppm and below.
The rats which received 625 ppm ETU showed clinical signs of poisoning by the eighth day on study. Excessive salivation, loss of hair or failure to replace normal hair loss, rough and bristly hair coat, and scaly skin texture were observed. The rats receiving 125, 25, 5, 1 and 0 ppm ETU, 50 ppm amitrole, or 125 ppm PTU showed no adverse clinical effects.

BODY WEIGHT AND WEIGHT GAIN
Growth, as evidenced by weekly recording of body weight data, was normal at dietary levels of 125 ppm and below. Only those rats which received 625 ppm ETU or 125 ppm PTU test diets showed a marked decrease in body weight gain. No difference in body weight gain was observed for those rats which received ETU at 125, 25, 5 or 1 ppm, or amitrole, as compared to the control group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Ingestion of ETU was calculated and found to range from 11.7 to 15.2 mg/day in female rats to 13.0 to 17.3 mg/day in male rats.

CLINICAL CHEMISTRY (tables 3, 4, 5)
Altered thyroid function can result from the action of a chemical on iodine uptake and incorporation, synthesis or release of thyroid hormones, synthesis or release of TSH, or inactivation of receptors activated by either TSH (at the level of the thyroid) or by thyroid hormone (at the pituitary level).
Consistent with the observable clinical changes, several biochemical changes were noted.
Rats ingesting ETU at 625 ppm showed a significant decrease in iodine uptake. The percent T-3 bound to TBG was lower than normal at the 30-and 90-day evaluations. Serum concentrations of T-3 and T-4 were also very substantially lower than the levels obtained for the control rats. The serum TSH concentration for the 625 ppm rats, measured only at the time of the 30-day evaluation, was increased to more than twice the serum TSH concentration observed for the control rats. Significant differences in free T-4 were also found as determined by the FTI.
The rate of iodine uptake, the percentage of T-3 bound to TBG, and the serum T-3 concentration in the rats receiving 125 ppm ETU were not significantly different f rom the control group. However, a statistically significant decrease in total serum T-4 concentration and in free serum T-4 measured at the 30-, 60- and 90-day evaluations was observed. The serum TSH concentration in the 125 ppm ETU rats at 30 days was approximately three times the concentration measured in the control rats.
With one exception, there were no significant measurable differences in iodine uptake, T-3 binding to TBG, or serum T-3, T-4, and TSH levels in the rats receiving ETU at either 25, 5 or 1 ppm for 30, 60, or 90 days. The rats ingesting 25 ppm ETU for 60 days showed a statistically significant increase in serum T-4 concentration. This was not evident at 30 or 90 days.

Amitrole
After 30 days of amitrole ingestion, very significantly decreased serum T-3 and T-4 levels were observed. The female rats from this group also had a decreased percent of T-3 bound to TBG. Both the male and female rats had an increased serum TSH concentration. While the decreased T-4 levels were still found after 30 days of amitrole ingestion, iodine uptake appeared to have returned to normal, probably as a result of biological compensation. At 60 and 90 days, normal values for ail of the thyroid function measurements were noted.

Propylthiouracil
The rats which received PTU had decreased serum T-3 and T-4 concentrations at 30, 60, and 90 days. The serum TBG level was affected only in the 60- and 90-day test groups. Since the PTU study was added after the ETU study had started, TSH values were obtainable for the 60- and 90-day PTU test groups. Both groups had markedly elevated serum TSH levels.

ORGAN WEIGHTS
There were no significant differences in organ weights in those rats fed 25, 5, 1, and 0 ppm ETU.
The spleen-to-body-weight ratio for the rats which ingested 625 ppm ETU was significantly decreased, while the ratios for the thyroid, brain, kidneys, testicles, and pituitary were significantly higher than the corresponding control values. Increased thyroid-to-body-weight ratios were also found for the rats which received 125 ppm PTU, 50 ppm amitrole, or 125 ppm ETU.
Organ-weight-to-brain-weight ratios were also calculated. No significant differences were found between rats receiving 125, 25, 5, 1, and 0 ppm ETU. For those rats ingesting 625 ppm ETU, the 'ratio of heart, spleen, kidney, adrenals (female), and ovaries-to-brain-weight was significantly decreased, and the thyroid-to-brain-weight ratio was significantly increased compared to control values. The thyroid-to-brain-weight ratios in the rats which received 125 ppm ETU were also significantly increased in size at 30, 60, and 90 days.
An increased thyroid-to-brain-weight ratio was the only significant organ-to-brain-weight ratio change found in the 50 ppm amitrole test animals. The animals which received 125 ppm PTU for 30, 60, or 90 days showed a very significant decrease in organ-to-brain-weight ratios for heart, spleen, kidney, ovary, liver, and adrenals. These animals had a very significant increase in thyroid-to-brain-weight ratios compared to the control values.

GROSS PATHOLOGY (table 7)
Gross examinations revealed that the majority of the rats which received ETU at 125 or 625 ppm, as well as those animals which received PTU or amitrole for 30, 60, or 90 days, had enlarged red thyroids. Grossly visible patterns of centrilobular congestion were present in a few random rats fed amitrole or ETU (table

HISTOPATHOLOGY: NON-NEOPLASTIC
Thyroid hyperplasia was observed in aIl animals which received 625 and 125 ppm ETU, 125 ppm PTU, or 50 ppm amitrole in their diet for 30, 60, or 90 days.
Microscopic examination of tissues was performed on thyroid and liver. The thyroids were graded for degree of hyperplasia. Varying degrees of thyroid microfollicular hyperplasia resulted at feeding levels of ETU above 5 ppm (Table 9). Relatively mild changes were noted in animal fed 125 ppm ETU for 30 days, compared to the moderate changes observed in the rats fed 625 ppm ETU for the same time period. Marked thyroid microfollicular hyperplasia and reduced colloid formation were found in animals fed amitrole or PTU for 30 days. At 60 days, the rats receiving the three highest levels of ETU (625, 125, and 50 ppm) and the amitrole treatment group showed moderate thyroid hyperplasia as compared to the 60-day PTU group, which was more marked in its thyroid hyperplastic response and reduced colloid production. Ninety days of dietary exposure to 625 ppm ETU also resulted in microfollicular hyperplasia, but not as severe as seen with PTU in the same time period. Numerous changes ranging from adenomatous hyperplasia of follicles to what appear to be true solid adenomas were present in the thyroids in the 625 ppm ETU group. No adenomas were observed in the thyroid sections taken from the other dietary groups.

HISTOPATHOLOGY: NEOPLASTIC
No liver tumors were observed in the rats at any dosage level of ETU or in those animals which received PTU or amitrole. The majority of the livers examined microscopically had mild centrilobular changes described as follows: Portal triad areas had a smooth sheet of hepatocytes cut relatively across the nucleus and cytoplasm at about the same level giving well defined cell boundaries and cytoplasmic proportions. As the examination approached the mid-zonal area progressing into the centrilobular area or small central veins, the pattern changed to less distinct cell borders of swollen cytoplasmic material with fewer apparent nuclei. The chord pattern was less distinct, and there was a mild increase in small polyploid nuclei. The general appearance was that of increased cytoplasm, fewer nuclei, and less orderly hepatic chords. The liver changes, although not accompanied by any clinical or biochemical disorder, were present in enough detail and magnitude to suggest the possibility of future hepatic involvement.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

25 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: equivalent to 1.7 mg/kg bw/d in males and 1.9 mg/kg bw/d in females

2

Dose descriptor:

LOAEL

Effect level:

125 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: = 8 mg/kg bw/d/ thyroid toxicity

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

TABLE 3. Thyroid Hormone Levels - After 30 Days On Study

ETU (ppm)	Sex	125 [I] (percent uptake)	TBG (percentT-3 bound)	T-3 (ng percent)	T-4 (µg percent)	TSH (µIU per ml)
625	M	1.2± 0.4^1	60.3± 2.6^1	57.3 ±3.7^1	0.9± 0.6^1	14.3± 0.9^1
625	F	2.1± 1.4^1	60.6± 1.8^1	58.4 ±9.9^1	1.1± 1.0^1	14.6± 1.9^1
125	M	3.6± 0.8	62.7± 1.3^1	71.1±11.8	2.6± 0.4^1	23.3± 5.9^1
125	F	4.0± 1.7	61.5± 1.3^1	104.4±16.3^1	2.1± 0.5^1	18.3± 4.0^1
25	M	2.9± 0.6	65.7± 2.3	67.1±15.9	5.6±1.1	7.3± 1.5
25	F	3.2± 1.3	63.5± 2.0	86.3 ± 14.8	3.8± 0.8	5.1± 1.3
5	M	3.6± 0.6	69.3± 6.3	79.0 ±8.1	4.7± 0.4	6.7± 1.4
5	F	3.8± 1.0	68.9± 1.3	88.1± 12.8	2.9± 0.9	4.9± 1.4
1	M	3.7± 0.7	64.5± 1.2	82.1± 13.0	5.1± 1.0	6.4± 0.8
1	F	3.0± 0.5	63.4±1.3	90.9±11.3	3.5± 1.0	4.5± 0.9
0	M	3.6± 0.9	68.0± 5.6	76.0 ± 11.8	5.0± 1.7	6.7±2.5
0	F	3.5± 0.9	66.0± 5.2	83.2 ± 16.2	3.8± 1.4	6.0±4.1
Amitrole(ppm)
50	M	3.7± 2.1	63.0± 1.5	49.±19.5^1	0.6± 0.8^1	9.3± 0.6^1
50	F	5.0± 3.5	61.4± 1.0^1	55.2 ±7.2^1	0.3± 0.3^1	8.9± 0.6^1
PTU (ppm)
125	M	2.9±1.1	67.8± 2.0	58.9±6.1^1	0.9± 0.2^1	-
125	F	3.3± 0.7	69.5± 1.6	52.0±8.0^1	0.7±0.11	-
^1Significantly different (p <0.05) from corresponding control. Student's test was used to make comparison betweenthe control and treated animals. All data reported as the mean, ± S.D.

TABLE 4. Thyroid Hormone Levels - After 60 Days On Study

ETU (ppm)	Sex	125 [I] (percent uptake)	TBG (percentT-3 bound)	T-3 (ng percent)	T-4 (µg percent)	TSH (µIU per ml)
625	M	1,9± 1.0^1	79.0±0.9	56.9±10.3^1	0.2± 0.1^1	-
625	F	2.4±1.8^1	71.8 ± 1.4	56.8± 6.9^1	0.2± 0.1^1	-
125	M	3.6 ± 1.4	66.3 ± 1.3	79.8± 28.1	2.8± 0.5^1
125	F	3.3 ± 1.0	66.3±2.1	78.5± 28.6^1	2.0± 0.5^1	-
25	M	3.2 ± 0.7	76.9 ± 1.6	86.4± 7.6	2.8± 0.5^1	-
25	F	3.7 ± 1.3	74.7 ± 1.7	126.2± 15.1	2.6± 0.5^1	-
5	M	3.5 ± 0.8	66.4 ± 1.2	85.4 ± 12.7	4.9± 0.5	-
5	F	4.0 ± 0.8	64.0 ± 1.8	118.5± 14.3	2.9± 0.9	-
1	M	2.7 ± 0.6	70.4 ± 1.2	80.3± 12.0	4.9± 0.7	-
1	F	3.2 ± 0.7	67.1 ± 1.3	93.3± 13.5	2.8± 0.8	-
0	M	4.3 ± 0.9	73.6 ± 4.9	77.3± 8.5	4.8± 0.7	5.8 ±0.4^2
0	F	3.5 ± 0.8	69.4 ± 4.3	103.8± 19.1	3.3± 0.5	6.4 ±0.9^2
Amitrole(ppm)
50	M	4.0± 0.7	74.8±   1.6	83.4±   8.9	5.9± 0.8
50	F	4.8± 0.9	69.8±   1.3	111.6± 10.7	3.5± 0.6
PTU (ppm)
125	M	3.9± 1.6	61.7± 2.6^1	46.1± 3.9^1	1.2± 0.2^1	9.8 ± 1.0^1
125	F	5.4± 1.7	62.2± 2.1^1	50.9± 9.7^1	0.8± 6.1^1	10.8 ± 1.9^1
^1Significantly different (p <0.05) from corresponding control. ^2TSH values to be used as control for PTU group.

TABLE 5. Thyroid Hormone Levels - After 90 Days On Study

ETU (ppm)	Sex	125 [I] (percent uptake)	TBG (percentT-3 bound)	T-3 (ng percent)	T-4 (µg percent)	TSH (µIU per ml)
625	M	2.5± 0.8^1	62.7±2.0^1	27.9±13.3^1	1.1±0.6^1	-
625	F	3.7±1.8	62.7±0.9^1	35.2±4.3^1	1.1±0.6^1	-
125	M	2.8±0.7	65.3±1.1	86.1±15.0	2.3±0.6^1	-
125	F	3.9±1.1	64.3±1.6	105.5±16.0	1.6±0.3^1	-
25	M	3.3±0.7	68.6±1.5	79.4±12.6	3.8±1.0	-
25	F	3.4±0.9	65.6±2.3	108.7±11.6	2.9±0.7	-
5	M	3.7±0.6	71.4±0.8	76.1±13.1	5.0±1.0	-
5	F	4.2±1.1	70.1±2.2	105.2±16.6	3.0±0.7	-
1	M	3.5±0.6	65.8±1.1	68.7±9.9	4.0±1.0	-
1	F	3.2±0.9	63.1±1.4	116.7±17.6	2.5±0.7	-
0	M	3.8±0.5	69.3±2.7	72.0±21.5	4.5±0.8	5.8±0.4^2
0	F	4.1±1.0	65.2±2.9	106.8±25.0	3.3±0.8	6.4±0.9^2
Amitrole(ppm)
50	M	5.8±3.1	71.4±1.5	67.3±11.8	3.6±1.3	-
50	F	4.8±3.2	69.6±2.1	94.8±13.3	3.5±1.1	-
PTU (ppm)
125	M	4.7±1.7	59.1±1.3^1	73.2±9.9	0.6±0.2^1	9.4±1.3^1
125	F	5.6±2.5	60.4±1.7^1	69.6±9.4^1	0.4±0.2^1	10.7±2.1^1
^1Significantly different (p <0.05) from corresponding control. ^2TSH values to be used as control for PTU group.

Applicant's summary and conclusion

Conclusions:

A no effect level in rats of 25 ppm in the diet for 90 days was assumed by the authors.

Executive summary:

Ethylene thiourea (ETU) was fed to groups of rats at 0, 1, 5 125 or 625 ppm for up to 90 days. Other groups of rats received either propylthiouracil (PTU; 125 ppm) or amitrole (50 ppm) in their diets as positive controls. Only those rats which received ETU at 125 or 625 ppm and those ingesting PTU or amitrole demonstrated a measur­able toxic response. This toxicity was reflected as an alteration in thyroid function and a significant change in thyroid morphology.

Ingestion of 625 ppm ETU or 125 ppm PTU resulted in very substantial decreases in serum triiodothyronine (T-3) and thyroxine (T-4). Marked increases in serum thyroid stimulating hormone (TSH) levels were Pound in the 625 and 125 ppm ETU rats, the125 PTU rats, and the rats receiving amitrole, each time this hormone was measured.Rats which ingested 625 ppm ETU also exhibited a decrease in iodide uptake by the thyroid. While a statistically significant increase in serum T-4 and degree of thyroid hyperplasia was observed for rats ingesting 25 ppm ETU for 60 days, normal thyroid hormone levels and thyroid morphology was found in rats on 25 ppm ETU for either 30 or 90 days.

Based on biochemical and microscopic changes examined, the no-effect level for dietary ETU in this 90-day study is considered to be 25 ppm.