Imidazolidine-2-thione

Endpoint summary

Description of key information

Short-term toxicity studies demonstrated that the thyroid gland is the most affected organ. Reduced T3 and T4 secretion and increased TSH (thyroid-stimulating hormone) secretion are found in rats. Ethylene thiourea may induce ultrastructural changes in the proximal tubuli of the rat kidneys. From these short-term studies, it can be concluded that the no-observed-adverse-effect level is of the order of 25 mg/kg diet (equivalent to 1.7 mg/kg body weight) and between 0.01 and 0.04 mg/l by inhalation.

Two key studies are selected for this endpoint (for oral route). Freudenthal study showed the smallest NOAEL (1.7 mg/kg bw/d) and it was a subchronic study (90 days of exposure).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

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

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

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)

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.

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.

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.

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

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

Critical effects observed:

not specified

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.

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1.7 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Freundenthal and Graham studies are reliable with a klimisch score of 2.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb-Mar 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

there are no satellite group

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: SPF-Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFM Kleintierfarm, Madoerin AG, CH 4414 Fuellinsdorf / Switzerland
- Age at study initiation: 9 weeks
- Weight at study initiation: 227.5-261.5g (males), 165.8-193.6g (females)
- Fasting period before study: no data
- Housing: in group of 5 in Makrolon type-4 cages with wire mesh tops
- Diet (e.g. ad libitum): pelleted standard Kliba, ad libitum
- Water (e.g. ad libitum): community tap water from Geneva, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-2°C
- Humidity (%):40-70%
- Air changes (per hr):10-15
- Photoperiod (hrs dark / hrs light):12/12

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: % particules <3µm are 92.7+/-1.7 (group 2), 90.4+/-1.2 (group 3) and 83.8 +/-6.3 (group 4).

Details on inhalation exposure:

The animals were confined separately in makrolon tubes which were positioned radially around the exposure chamber.
The system is unique by comparaison with conventional nose-only exposure systems in that it insures a uniform aerosol distribition at all chamber levels, provides a constant stream of "fresh" aerosol to each animal, and precludes rebreathing from the exhaled air.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Solutions were analyzed by gas chromatography according to a method provided by the sponsor.
Analytical determinations were performed once weekly for each dose-group.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day, 5 days/week (excluding week-ends)

Remarks:

Doses / Concentrations:
0, 0.01, 0.04 and 0.2 mg/l air
Basis:
other: target concentration

Remarks:

Doses / Concentrations:
0.011, 0.043 and 0.197 mg/l air
Basis:
analytical conc.

No. of animals per sex per dose:

5 animals/ sex/ dose

Control animals:

yes

Details on study design:

- Dose selection rationale: based upon a preliminary range finding study carried out at RCC Geneva.
- Rationale for selecting satellite groups: no recovery period
- Post-exposure recovery period in satellite groups: no recovery period

Positive control:

no

Observations and examinations performed and frequency:

Clinical signs: yes, once daily
Mortality:yes, twice daily
Body weight: yes, once weekly
Food consumption: yes , weekly
Water consumption: yes, weekly
Ophthalmoscopic examination: yes at the end of study
Haematology: yes at the end of study
Biochemistry: yes at the end of study
Urinanalysis: no

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
Weighted organs: yes
Macroscopic examined: thyroid, pituitary and mandibular glands...
Microscopic: according to OECD guideline

Other examinations:

no data

Statistics:

The following statistical methods were used to analyze the following data : body weight, food consumption, water consumption, organ weights and clinicat laboratory data. If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups for each sex.
-The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
-Univariate one-way analysis of variance to assess the level of signifcance of intergroup differences.
-For the overall spontaneous mortality data, the Fisher's exact test for 2 x 2 tables was applied.
Group means with standard deviations were calculated for continuous data; for discrete data (scores), medians were calculated.
Individual values, means, standard deviations and statistics were rounded off before printing. For exemple, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

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 examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No deaths occured during the treatment.
No clinical signs were noted in control animals nor in animals of the low (0.01 mg/l) or medium (0.04 mg/l) dose-groups. In the high-dose group (0.20 mg/l), alopecia started to develop in both sexes during the second week of treatment. The incidence and severity of this alteration increased until the end of the study, particularly in the males. During test-weeks 3 and 4, hunched posture was observed in both sexes. Other clinical signs included : tachypnea (females only, test-week 4), salivation (males only, test-week 2) and reddish sore spot on the tail (males only, test-week 4).

BODY WEIGHT AND WEIGHT GAIN
At the start of treatment, mean body weights ranged between 236.7 - 250.0 g in the males and between 173.2 - 187.4 g in the females. The treatment had no influence on body weights or body weight gains in animals exposed to 0.01 mg/l (group 2) or 0.04 mg/l (group 3) of the test-article; at the high concentration (0.20 mg/l, group 4), mean body weights were reduced below control values, with statistically significant differences from test-week 2 in the males and on test-week 4 in the females. In both sexes, body weight gains of animals of group 4 became increasingly smaller than in controls, and on test-week 4, body weight gains were reduced by approximately one third (males) and one half (females) when compared to non-treated animals.

FOOD CONSUMPTION and WATER CONSUMPTION
In both sexes, mean food consumption values of animals of group 4 (0.20 mg/l) were generally lower than in the controls (approximately 5 g/animal/day) at test-week 4). Water consumption was comparable in all groups throughout the study.

OPHTHALMOSCOPIC EXAMINATION
Increased pupillary reflex was noted in one male rat of group 4. One female of group 2 and one female of group 2 had an irregular corneal surface.

HAEMATOLOGY
The only major difference noted between the control and the treated animals was the reticulocyte count which, in the high-dose group (0.20 mg/l), was reduced by about half in both sexes. Hematocrit values of the high-dose group of males were slightly reduced below contrai levels.

CLINICAL CHEMISTRY
Differences between control and treated animals were generally small; in the high-dose group of males (0.20 mg/l), calcium was slightly reduced below contrai levels. Potassium was slightly increased in all dose-groups of males. In the highdose-group of females (0.20 mg/l), a small increase in urea, bilirubin and phosphorus was noted.
Concerning the parameters indicative of the thyroid function, a dose-related decrease in T4 values was noted in both sexes. Differences were statistically significant in the males of group 4 (0.20 mg/l) and in the females of groups 3 (0.04 mg/l) and 4 (0.20 mg/l). In the high-dose group (0.20 mg/l), mean T4 values were reduced by approximately one third (males) and one half (females) when compared to control animals. No differences were noted between the T3 values of control and treated animals.

ORGAN WEIGHTS
No differences in the absolute weight of the lungs, liver, and kidneys were found between control and treated groups. In the males of group 4 (0.20 mg/l), absolute adrenals and testes weights were reduced when compared to controls, whereas no differences were seen in relation to body weight. In the females, the weight of the adrenals was similar in all groups, in both absolute and relative values.

GROSS PATHOLOGY
All rats of bath sexes treated at a dosage of 0.20 mg/l (group 4) displayed areas of hair loss (alopecia) at necropsy.
Other recorded necropsy findings were :
-mandibular lymph-node : enlarged (males # 2, control group, # 6, group 2).
-stomach : nodule, gray-white, hard, in the forestomach/granular mucosa junction (male #2 control group).
-liver (right lateral lobe) : focus, 4x2 mm, tan caler (male # 20, group 4).
-uterus (bath horns) : distended, d = 6 mm (female # 1, control group).

HISTOPATHOLOGY: NON-NEOPLASTIC
Thyroid gland : Morphometric analysis of the thyroid gland revealed no significant change in the follicular diameter or follicular area as a result of the treatment. In group 4 (0.20 mg/l), an increase in the mean follicular epithelial height was observed in both sexes, and in group 3 (0.04 mg/l) in the females only here was no effect at 0.01 mg/l. Additional changes characterized by colloid depletion colloid agglomeration, diffuse hyperplasia, folliculoneogenesis and increased vascularity were noted in both sexes at the dose of 0.20 mg/l (group 4). Colloid depletion was observed in one female at the dose of 0.04 mg/l (group 3).
Pituitary gland: Hypertrophy of pituitary basophils was noted in rats of both sexes at the dose of 0.20 mg/l (group 4).
Mandibular salivary gland: Hypertrophy of the acinar epitelium and decreased eosinophilic staining of the striated ducts was noted in rats of both sexes in group 4 (0.20 mg/l).
Skin: All rats of both sexes of group 4 (0.20 mg/l) displayed areas of hair loss at necropsy. Microscopic examination revealed hyperkeratosis in soma sections.
A variety of spontaneous findings was recorded, which were consistent with the spectrum of findings normally encountered in young rats kept under laboratory conditions and which showed no effect of treatment on their character, severity or distribution.

Dose descriptor:

NOEL

Effect level:

0.01 mg/L air

Sex:

male/female

Basis for effect level:

other: No effects were observed at this dose.

Dose descriptor:

LOEL

Effect level:

0.04 mg/L air

Basis for effect level:

other: At this dose, microscopic findings in thyroid gland, decrease in plasma levels of T4 in females

Critical effects observed:

not specified

Conclusions:

The inhalation of Ethylenethiourea (6 hours/day, 5 days/week, for a total of 20 exposures) was found to produce changes in the thyroid gland in both male and female rats at the concentration of 0,20 mg/l air, and in the females, at 0.04 mg/l air. Based opon these results, the NOEL of ETU by inhalation was 0.01 mg/l air.

Executive summary:

Ethylenethiourea (ETU) was administered to SPF-Wistar rats (5 males and 5 females per group) by inhalation over a 28 -day period, 5 days/week, 6 hours/day, for a total of 20 exposures. The target concentrations were 0, 0.01, 0.04 and 0.20 mg/l air (corresponding to analytical concentrations of 0, 0.011, 0.043 and 0.197 mg/l air, respectively). A control group was exposed to filtered air using the same exposure system and schedule.

All animals survived to the end of the exposure period. No clinical signs were noted in animals of groups 2 (0.01 mg/l) or 3 (0.04 mg/l). In group 4 (0.20 mg/l), alopecia (starting on test-week 2) and hunched posture (test-weeks 3 and 4) were the major clinical signs. No treatment-related abnormalities in ophtalmoscopic examinations were observed. At the end of the 28 -day exposure period, mean body weights of animals of group 4 (.0.20 mg/l) were reduced below control values by approximately 30g in the females and 45g in the males. In this group, mean food consumption was reduced by approximately 5g/animal/d when compared to controls.

Concerning the hematology investigations, an approximately 50% decrease in reticulocyte count was observed (both sexes) in group 4 (0.20 mg/l). The clinical biochemistry showed a dose-related decrease in thyroxine (T4) ion both sexes. In group 4 (0.20 mg/l), mean T4 values were decreased by approximately 30% (males) and 50% (females), whereas triiodothyronine (T3) values remained unchanged.

Skin areas of alopecia considered to be treatment-related were seen in alla rats of group 4(0.20mg/l). no other macroscopic findings of significance were diagnosed in these rats. Pathomorphologic lesions (histopathology) considered to be treatment-related were noted in the thyroid gland of animals of group 3 (0.04 mg/l) and 4 (0.20 mg/l). They considered of an increase of follicular epithelial height, colloid depletion, colloid agglomeration, diffuse hyperplasia, folliculoneogenesis, and increased vascularity. these changes were observed in male and female rats of group 4 and in female rats of group 3. Other treatment related changes noted in male and female rats of group 4 were hypertrophy of basophils (pituitary gland), hypertrophy of the acinar epithelium and decreased eosinophilic staining of striated ducts (mandibular salivary gland), and a hyperkeratosis of skin. Histologically, no effects of treatment were observed in group 2 (0.01 mg/l).

No treatment-related changes in organ weight or organ weight ratios were observed.

The treatment with ETU was considered to induce a number of changes and to reveal some target organs. Changes were suggestive of a thyrotrophic character of ETU : decrease in plasma levels of T4 (at 0.04 mg/l in females, and at 0.20 mg/l in both sexes), changes of the thyroid gland (increase of follicular epithelial height, colloid depletion, colloid agglomeration, diffuse hyperplasia, folliculoneogenesis and inscreased vascularity).

Other changes attributed to the treatment observed in rats of both sexes dosed at 0.20 mg/l included : a moderate decrease in body weight, histological changes in the pituitary gland and mandibular salivary gland; decrease in reticulocyte count.

Based opon these results, the NOEL of ETU by inhalation lies between 0.01 and 0.04 mg/l air according to the autors.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

10 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Duchosal study is a reliable study with a klimsich score of 2.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb-Mar 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

there are no satellite group

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: SPF-Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: KFM Kleintierfarm, Madoerin AG, CH 4414 Fuellinsdorf / Switzerland
- Age at study initiation: 9 weeks
- Weight at study initiation: 227.5-261.5g (males), 165.8-193.6g (females)
- Fasting period before study: no data
- Housing: in group of 5 in Makrolon type-4 cages with wire mesh tops
- Diet (e.g. ad libitum): pelleted standard Kliba, ad libitum
- Water (e.g. ad libitum): community tap water from Geneva, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22+/-2°C
- Humidity (%):40-70%
- Air changes (per hr):10-15
- Photoperiod (hrs dark / hrs light):12/12

Route of administration:

inhalation

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: % particules <3µm are 92.7+/-1.7 (group 2), 90.4+/-1.2 (group 3) and 83.8 +/-6.3 (group 4).

Details on inhalation exposure:

The animals were confined separately in makrolon tubes which were positioned radially around the exposure chamber.
The system is unique by comparaison with conventional nose-only exposure systems in that it insures a uniform aerosol distribition at all chamber levels, provides a constant stream of "fresh" aerosol to each animal, and precludes rebreathing from the exhaled air.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Solutions were analyzed by gas chromatography according to a method provided by the sponsor.
Analytical determinations were performed once weekly for each dose-group.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours/day, 5 days/week (excluding week-ends)

Remarks:

Doses / Concentrations:
0, 0.01, 0.04 and 0.2 mg/l air
Basis:
other: target concentration

Remarks:

Doses / Concentrations:
0.011, 0.043 and 0.197 mg/l air
Basis:
analytical conc.

No. of animals per sex per dose:

5 animals/ sex/ dose

Control animals:

yes

Details on study design:

- Dose selection rationale: based upon a preliminary range finding study carried out at RCC Geneva.
- Rationale for selecting satellite groups: no recovery period
- Post-exposure recovery period in satellite groups: no recovery period

Positive control:

no

Observations and examinations performed and frequency:

Clinical signs: yes, once daily
Mortality:yes, twice daily
Body weight: yes, once weekly
Food consumption: yes , weekly
Water consumption: yes, weekly
Ophthalmoscopic examination: yes at the end of study
Haematology: yes at the end of study
Biochemistry: yes at the end of study
Urinanalysis: no

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
Weighted organs: yes
Macroscopic examined: thyroid, pituitary and mandibular glands...
Microscopic: according to OECD guideline

Other examinations:

no data

Statistics:

The following statistical methods were used to analyze the following data : body weight, food consumption, water consumption, organ weights and clinicat laboratory data. If the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between the treated groups and the control groups for each sex.
-The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
-Univariate one-way analysis of variance to assess the level of signifcance of intergroup differences.
-For the overall spontaneous mortality data, the Fisher's exact test for 2 x 2 tables was applied.
Group means with standard deviations were calculated for continuous data; for discrete data (scores), medians were calculated.
Individual values, means, standard deviations and statistics were rounded off before printing. For exemple, test statistics were calculated on the basis of exact values for means and pooled variances and then rounded off to two decimal places. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

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 examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No deaths occured during the treatment.
No clinical signs were noted in control animals nor in animals of the low (0.01 mg/l) or medium (0.04 mg/l) dose-groups. In the high-dose group (0.20 mg/l), alopecia started to develop in both sexes during the second week of treatment. The incidence and severity of this alteration increased until the end of the study, particularly in the males. During test-weeks 3 and 4, hunched posture was observed in both sexes. Other clinical signs included : tachypnea (females only, test-week 4), salivation (males only, test-week 2) and reddish sore spot on the tail (males only, test-week 4).

BODY WEIGHT AND WEIGHT GAIN
At the start of treatment, mean body weights ranged between 236.7 - 250.0 g in the males and between 173.2 - 187.4 g in the females. The treatment had no influence on body weights or body weight gains in animals exposed to 0.01 mg/l (group 2) or 0.04 mg/l (group 3) of the test-article; at the high concentration (0.20 mg/l, group 4), mean body weights were reduced below control values, with statistically significant differences from test-week 2 in the males and on test-week 4 in the females. In both sexes, body weight gains of animals of group 4 became increasingly smaller than in controls, and on test-week 4, body weight gains were reduced by approximately one third (males) and one half (females) when compared to non-treated animals.

FOOD CONSUMPTION and WATER CONSUMPTION
In both sexes, mean food consumption values of animals of group 4 (0.20 mg/l) were generally lower than in the controls (approximately 5 g/animal/day) at test-week 4). Water consumption was comparable in all groups throughout the study.

OPHTHALMOSCOPIC EXAMINATION
Increased pupillary reflex was noted in one male rat of group 4. One female of group 2 and one female of group 2 had an irregular corneal surface.

HAEMATOLOGY
The only major difference noted between the control and the treated animals was the reticulocyte count which, in the high-dose group (0.20 mg/l), was reduced by about half in both sexes. Hematocrit values of the high-dose group of males were slightly reduced below contrai levels.

CLINICAL CHEMISTRY
Differences between control and treated animals were generally small; in the high-dose group of males (0.20 mg/l), calcium was slightly reduced below contrai levels. Potassium was slightly increased in all dose-groups of males. In the highdose-group of females (0.20 mg/l), a small increase in urea, bilirubin and phosphorus was noted.
Concerning the parameters indicative of the thyroid function, a dose-related decrease in T4 values was noted in both sexes. Differences were statistically significant in the males of group 4 (0.20 mg/l) and in the females of groups 3 (0.04 mg/l) and 4 (0.20 mg/l). In the high-dose group (0.20 mg/l), mean T4 values were reduced by approximately one third (males) and one half (females) when compared to control animals. No differences were noted between the T3 values of control and treated animals.

ORGAN WEIGHTS
No differences in the absolute weight of the lungs, liver, and kidneys were found between control and treated groups. In the males of group 4 (0.20 mg/l), absolute adrenals and testes weights were reduced when compared to controls, whereas no differences were seen in relation to body weight. In the females, the weight of the adrenals was similar in all groups, in both absolute and relative values.

GROSS PATHOLOGY
All rats of bath sexes treated at a dosage of 0.20 mg/l (group 4) displayed areas of hair loss (alopecia) at necropsy.
Other recorded necropsy findings were :
-mandibular lymph-node : enlarged (males # 2, control group, # 6, group 2).
-stomach : nodule, gray-white, hard, in the forestomach/granular mucosa junction (male #2 control group).
-liver (right lateral lobe) : focus, 4x2 mm, tan caler (male # 20, group 4).
-uterus (bath horns) : distended, d = 6 mm (female # 1, control group).

HISTOPATHOLOGY: NON-NEOPLASTIC
Thyroid gland : Morphometric analysis of the thyroid gland revealed no significant change in the follicular diameter or follicular area as a result of the treatment. In group 4 (0.20 mg/l), an increase in the mean follicular epithelial height was observed in both sexes, and in group 3 (0.04 mg/l) in the females only here was no effect at 0.01 mg/l. Additional changes characterized by colloid depletion colloid agglomeration, diffuse hyperplasia, folliculoneogenesis and increased vascularity were noted in both sexes at the dose of 0.20 mg/l (group 4). Colloid depletion was observed in one female at the dose of 0.04 mg/l (group 3).
Pituitary gland: Hypertrophy of pituitary basophils was noted in rats of both sexes at the dose of 0.20 mg/l (group 4).
Mandibular salivary gland: Hypertrophy of the acinar epitelium and decreased eosinophilic staining of the striated ducts was noted in rats of both sexes in group 4 (0.20 mg/l).
Skin: All rats of both sexes of group 4 (0.20 mg/l) displayed areas of hair loss at necropsy. Microscopic examination revealed hyperkeratosis in soma sections.
A variety of spontaneous findings was recorded, which were consistent with the spectrum of findings normally encountered in young rats kept under laboratory conditions and which showed no effect of treatment on their character, severity or distribution.

Dose descriptor:

NOEL

Effect level:

0.01 mg/L air

Sex:

male/female

Basis for effect level:

other: No effects were observed at this dose.

Dose descriptor:

LOEL

Effect level:

0.04 mg/L air

Basis for effect level:

other: At this dose, microscopic findings in thyroid gland, decrease in plasma levels of T4 in females

Critical effects observed:

not specified

Conclusions:

The inhalation of Ethylenethiourea (6 hours/day, 5 days/week, for a total of 20 exposures) was found to produce changes in the thyroid gland in both male and female rats at the concentration of 0,20 mg/l air, and in the females, at 0.04 mg/l air. Based opon these results, the NOEL of ETU by inhalation was 0.01 mg/l air.

Executive summary:

Ethylenethiourea (ETU) was administered to SPF-Wistar rats (5 males and 5 females per group) by inhalation over a 28 -day period, 5 days/week, 6 hours/day, for a total of 20 exposures. The target concentrations were 0, 0.01, 0.04 and 0.20 mg/l air (corresponding to analytical concentrations of 0, 0.011, 0.043 and 0.197 mg/l air, respectively). A control group was exposed to filtered air using the same exposure system and schedule.

All animals survived to the end of the exposure period. No clinical signs were noted in animals of groups 2 (0.01 mg/l) or 3 (0.04 mg/l). In group 4 (0.20 mg/l), alopecia (starting on test-week 2) and hunched posture (test-weeks 3 and 4) were the major clinical signs. No treatment-related abnormalities in ophtalmoscopic examinations were observed. At the end of the 28 -day exposure period, mean body weights of animals of group 4 (.0.20 mg/l) were reduced below control values by approximately 30g in the females and 45g in the males. In this group, mean food consumption was reduced by approximately 5g/animal/d when compared to controls.

Concerning the hematology investigations, an approximately 50% decrease in reticulocyte count was observed (both sexes) in group 4 (0.20 mg/l). The clinical biochemistry showed a dose-related decrease in thyroxine (T4) ion both sexes. In group 4 (0.20 mg/l), mean T4 values were decreased by approximately 30% (males) and 50% (females), whereas triiodothyronine (T3) values remained unchanged.

Skin areas of alopecia considered to be treatment-related were seen in alla rats of group 4(0.20mg/l). no other macroscopic findings of significance were diagnosed in these rats. Pathomorphologic lesions (histopathology) considered to be treatment-related were noted in the thyroid gland of animals of group 3 (0.04 mg/l) and 4 (0.20 mg/l). They considered of an increase of follicular epithelial height, colloid depletion, colloid agglomeration, diffuse hyperplasia, folliculoneogenesis, and increased vascularity. these changes were observed in male and female rats of group 4 and in female rats of group 3. Other treatment related changes noted in male and female rats of group 4 were hypertrophy of basophils (pituitary gland), hypertrophy of the acinar epithelium and decreased eosinophilic staining of striated ducts (mandibular salivary gland), and a hyperkeratosis of skin. Histologically, no effects of treatment were observed in group 2 (0.01 mg/l).

No treatment-related changes in organ weight or organ weight ratios were observed.

The treatment with ETU was considered to induce a number of changes and to reveal some target organs. Changes were suggestive of a thyrotrophic character of ETU : decrease in plasma levels of T4 (at 0.04 mg/l in females, and at 0.20 mg/l in both sexes), changes of the thyroid gland (increase of follicular epithelial height, colloid depletion, colloid agglomeration, diffuse hyperplasia, folliculoneogenesis and inscreased vascularity).

Other changes attributed to the treatment observed in rats of both sexes dosed at 0.20 mg/l included : a moderate decrease in body weight, histological changes in the pituitary gland and mandibular salivary gland; decrease in reticulocyte count.

Based opon these results, the NOEL of ETU by inhalation lies between 0.01 and 0.04 mg/l air according to the autors.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

40 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Duchosal study is a reliable study with a klimsich score of 2.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Human data

A study of 49 Mexican workers who sprayed tomatoes with ethylenebisdithiocarbamate fungicides without using protective clothing or masks revealed elevated TSH levels (2.13 ± 0.15 mIU/liter; 1.61 ± 0. l9 mIU/liter in 24 unexposed controls). Levels of T4 were unaffected, however, and no symptoms of changes in thyroid function were observed, although no clinical examination was made. Exposure to ethylene thiourea was estimated by measuring the concentration of ethylene thiourea in morning urine the day after taking the blood samples used for the other analyses. The average level among the exposed subjects was 58 ± 26 ppb. All the controls and 34% of the exposed subjects had urine levels below the detection limit of 10 ppb (Steenland et al., 1977).

In an English study from 1984 (Smith, 1984), thyroid function was examined in eight production workers from a plant that produced ethylene thiourea and five workers (mixers) from a factory where ethylene thiourea was used in rubber production (84). Air concentrations of ethylene thiourea ranged from 10 to 330 µg/m3in the production plant and from 120 to 160 µg/m3in the rubber factory. Thyroid function was measured as levels of T4(thyroxine), TSH (thyroid-stimulating hormone) and TBG (thyroxine-binding globulin) in serum. It was found that T4levels were lower in the mixers (geometric mean 80.5 nmol/l) than in the process workers (geometric mean 96.4 nmol/l) and an unexposed control group (geometric mean 105.7 nmol/l), but the individual values were within the range of normal reference values for T4(50 to 150 nmol/l) (53). TSH and TBG levels were normal in all the subjects except one mixer, who had an elevated TSH level.

Animal studies

Oral route

Effect on the thyroid gland

Repeated dose animal experiments indicate that in rats the organ primarily affected by exposure to ethylene thiourea is the thyroid gland.

In a ninety-day study, Freudenthalet al. (1977) gave ethylene thiourea to groups of rats at levels of 0, 1, 5, 25, 125 or 625 mg ethylene thiourea per kg feed. Rats which had been given the highest dose showed acute clinical signs of poisoning, which were not found at the other doses. At 125 mg/kg feed, substantial decreases in T3 and T4 were found, as well as a marked increase in TSH. On the other hand, for the 25 mg/kg feed group, an increase in T4 and thyroid hyperplasia were found at day 60, which had returned to normal by the ninetieth day. From this experiment it may be concluded that a NOAEL for rats is about 25 mg ethylene thiourea per kg feed (1.7 mg per kilogram body weight per day).

A 120-day feeding study of male rats was performed by Graham and Hansen (1972). Groups of rats were fed ethylene thiourea at rates of 0, 50, 100, 500 and 750 mg/kg food. A significant decrease in body weight was found in the groups given 500 and 750 mg ethylene thiourea per kg food and a significant increase in relative thyroid weight was found in the groups given 100 to 750 mg ethylene thiourea per kg food. Twenty-four hours after injection of 131I, thyroidal uptake was significantly decreased in the groups given 100 to 750 mg/kg food at different stages of the study. No difference in histological appearance could be detected between sections of the thyroid glands of rats given 50 mg ethylene thiourea/kg food and sections of the thyroids of the control animals. It can be concluded that the NOAEL in this experiment was 50 mg ethylene thiourea per kg food (approximately 5 mg per kilogram body weight per day).

In the extention of the previous study (Graham et al., 1973), both male and female Charles River rats showed decreased body weight and body-weight gain when fed diets containing 250 or 500 mg/kg ethylenethiourea for 2–12 months. The body weight of the female rats was also decreased at 125 mg/kg of diet. Iodine uptake was decreased in male rats after 12 months at 500 mg/kg of diet. Females had an initial decrease in iodine uptake at a dose of 125 or 500 mg/kg of diet at 6 months, but by 12 months the uptake had increased even on diets containing 125, 250 or 500 mg/kg.

In a 28 day study, imidazolidine-2-thione was repeatedly orally administered in doses of 0, 1, 6 and 30 mg/kg to rats of both sexes, the changes in life functions and form were monitored, and the toxicity and recovery evaluated (Hiratsuka et al., 2004). During observations on the general symptoms, abnormal fur conditions were noted in both sexes in the 30 mg/kg group. Low body weight and food consumption was noted in both sexes in the 30 mg/kg group. For the blood chemical examination, high total cholesterol values, and low values for ALP and inorganic phosphorus were noted in the males in the 30 mg/kg group, and high chlorine values for both sexes in the 30 mg/kg group. For organ weight measurements, high values for relative liver weights were noted for females in the 30 mg/kg group, high values for absolute and relative thyroid gland weights were noted for both sexes in the 30 mg/kg group, low values for absolute and relative thymus weights were noted for females in the 6 and 30 mg/kg groups. At the end of the recovery period, these changes had recovered or lessened. During the necropsy, enlargement of the thyroid gland was noted in males in the 6 mg/kg and in both sexes in the 30 mg/kg group. At the end of the recovery period, these changes had recovered or lessened. Histopathological examination confirmed hypertrophic centrilobular hepatocyte, hypertrophic basophilic cells in the pituitary gland and atrophy of the sebaceous gland in both sexes in the 30 mg/kg group. Additionally, a reduction of colloids in the thyroid gland and hypertrophy of diffuse follicular cells was noted in the males in the 6 mg/kg group and both sexes in the 30 mg/kg group. At the end of the recovery period, hypertrophic basophilic cells in the pituitary gland were noted in males in the 30 mg/kg group but the degree had lessened. The other changes had recovered. As indicated above, both sexes in groups subject to 6 mg/kg or higher experienced changes believed to be attributed to administration of the test substance. Therefore, under the conditions of this study, the no observed effect level (NOEL) for both sexes was determined to be 1 mg/kg/day for imidazolidine-2-thione.

NTP (1992) and Chhabra et al. (1992) performed a thirteen-week feeding study on rats and mice of both sexes. The exposure levels of ethylene thiourea were 0, 60, 125, 250, 500 or 750 mg/kg food for rats and 0, 125, 250, 500, 1000 or 2000 mg/kg food for mice. There were no clinical signs of toxicity in the rats and all rats survived to the end of the study. Chemical-related lesions were observed in the thyroid gland, pituitary gland and liver of the exposed rats at all exposure levels. Thyroid follicular cell adenoma were found in male rats in the 250 to 750 mg/kg exposure groups, and in female rats in the 500 to 750 mg/kg groups. From this experiment it may be concluded that no tumours occurred below the level of 60 mg/kg (approximately 4.5 mg per kilogram body weight per day), which is the NOAEL for this study. No overt clinical signs of chemical toxicity were found in mice. Only diffuse thyroid follicular cell hyperplasia were found in male and female mice at concentrations of 500 mg/kg and higher. This indicates that the NOAEL for mice is 250 mg/kg food (approximately 50 mg per kilogram body weight per day). On the basis of this information, rats seems much more susceptible to the toxic effects of ethylene thiourea than mice.

Groups of 50 male and 50 female Sprague-Dawley rats were fed diets containing 0, 75, 100 or 150 ppm ethylene thiourea (purity not stated) for 7 weeks. ethylene thiourea was mixed in corn oil and added to the diet. Ten rats from each group were killed at 7 weeks and at 2, 3 and 4 weeks post-dosing on control diets in order to assess the extent of effect on the thyroid and the subsequent reversibility of these effects. Body weight, food consumption, thyroid weight, brain weight, serum T₃and T₄levels (0 and 150 ppm groups only) were measured. All animals were necropsied and thyroids examined histologically. At 7 weeks body weights decreased with increasing dose, while thyroid weights (absolute and relative) increased in both sexes. T₃levels were somewhat variable, while T₄levels were significantly decreased at 150 ppm in both sexes at 7 weeks. These effects partially reversed after 4 weeks on control diets. Histopathological findings included reduced colloid content of thyroid acini in high dose rats. Acinar epithelial cell size and height were not different from control. Two tumours were identified in the high dose male group: a follicular cell adenoma and a medullary carcinoma. The authors conclude that the relationship between the duration of exposure to ethylene thiourea and the possible reversibility of various thyroid lesions requires further study (Arnold et al., 1983).

Kurttio et al. (1986) studied the effect of administration of 100, 200 and 300 mg/l ethylene thiourea in drinking water ad libitum for twenty-eight days [corresponding to 10.6, 17.6 and 23.4 mg/kg bw per day] on the function and morphology of the thyroid glands of rats. Ethylene thiourea induced a dose-dependent inhibition of T3 (triiodo-thyronine) and T4 (thyroxine) secretion, and a ten-fold increase in the TSH (thyroid-stimulating hormone). Electron-microscopic examination showed that ethylene thiourea induced ultrastructural changes in the thyroid, namely an increased number of myelin bodies, dilatation of the rough endoplasmic reticulum and increased vacuolization in the epithelial cells of thyroid follicles. The authors estimated that these changes were associated with daily doses ranging from 10.6 to 23.4 mg ethylene thiourea per kilogram body weight per day. This means that the NOAEL is below 10.6 mg ethylene thiourea per kilogram bw per day.

From these short-term animal exposure studies it can be concluded that in rats the thyroid gland is the organ most affected by ethylene thiourea. Rats are much more susceptible to ethylene thiourea than mice. The NOAEL in rats is estimated to be 1.7 mg per kilogram body weight per day, and the LOAEL is about 8 mg per kilogram body weight per day. In mice, the NOAEL for the thyroid is estimated to be 250 ppm (mg per kilogram diet).

 

Effect on the liver

Molleret al. (1986) administered ethylene thiourea to male rats at concentrations of 1, 5, 50 an 500 mg/l in drinking water ad libitum for up to eight months. No changes in liver cell morphology were found at the concentrations up to 50 mg/l, but at 500 mg/l ethylene thiourea induced alterations in the hepatic cell morphology. These alterations included a dramatic increase in the amount of smooth endoplasmic reticulum (SER) with a concomitant reduction in rough endoplasmic reticulum (RER), and a relocation of microbodies and mitochondria to the periphery of the SER. From this experiment it can be concluded that the NOAEL for the effect of ethylene thiourea on the liver of rats is 50 mg/l ethylene thiourea in drinking water (15 mg per kilogram body weight per day).

Short-term exposure of rats indicated an NOAEL of 15 mg per kilogram body weight per day for the effect of ethylene thiourea on liver morphology.

 

Effect on the kidneys

The effect of ethylene thiourea on kidney function and morphology in rats was studied by Kurttioet al. (1991). Rats were exposed to ethylene thiourea in drinking water ad libitum for twenty-eight days at concentrations of 0, 100, 200 or 300 mg/l. No permanent effects were found on the urinary sodium or potassium excretion, urine osmolality, urine excretion of uric acid, protein or glucose. A tendency towards an increase in vasopressin urinary excretion was observed. No prominent light microscopical changes were observed in the kidney of rats; however, ethylene thiourea induced clear ultrastructural changes in the epithelium of renal proximal tubuli, as observed by electron microscopy. At the highest dose level, increases in lysosome numbers and myelin as well as vacuolization and edema were observed in the cytoplasm of the epithelial cells of proximal tubulus.Thus, the NOAEL for the effect of ethylene thiourea on the kidneys of rats is about 200 mg/l in drinking water; this equates to a dose of 17.6 mg per kilogram body weight per day.

 

Inhalation exposure

In an OECD guideline # 412 study (Duchosalet al., 1988), ethylenethiourea (ethylene thiourea) was administered to SPF-Wistar rats (5 males and 5 females per group) by inhalation over a 28 -day period, 5 days/week, 6 hours/day, for a total of 20 exposures. The target concentrations were 0, 0.01, 0.04 and 0.20 mg/l air (corresponding to analytical concentrations of 0, 0.011, 0.043 and 0.197 mg/l air, respectively). A control group was exposed to filtered air using the same exposure system and schedule.

All animals survived to the end of the exposure period. No clinical signs were noted in animals of groups 2 (0.01 mg/l) or 3 (0.04 mg/l). In group 4 (0.20 mg/l), alopecia (starting on test-week 2) and hunched posture (test-weeks 3 and 4) were the major clinical signs. No treatment-related abnormalities in ophtalmoscopic examinations were observed. At the end of the 28 -day exposure period, mean body weights of animals of group 4 (0.20 mg/l) were reduced below control values by approximately 30g in the females and 45g in the males. In this group, mean food consumption was reduced by approximately 5g/animal/d when compared to controls.

Concerning the hematology investigations, an approximately 50% decrease in reticulocyte count was observed (both sexes) in group 4 (0.20 mg/l). The clinical biochemistry showed a dose-related decrease in thyroxine (T4) ion both sexes. In group 4 (0.20 mg/l), mean T4 values were decreased by approximately 30% (males) and 50% (females), whereas triiodothyronine (T3) values remained unchanged.

Skin areas of alopecia considered to be treatment-related were seen in alla rats of group 4(0.20mg/l). no other macroscopic findings of significance were diagnosed in these rats. Pathomorphologic lesions (histopathology) considered to be treatment-related were noted in the thyroid gland of animals of group 3 (0.04 mg/l) and 4 (0.20 mg/l). They considered of an increase of follicular epithelial height, colloid depletion, colloid agglomeration, diffuse hyperplasia, folliculoneogenesis, and increased vascularity. these changes were observed in male and female rats of group 4 and in female rats of group 3. Other treatment related changes noted in male and female rats of group 4 were hypertrophy of basophils (pituitary gland), hypertrophy of the acinar epithelium and decreased eosinophilic staining of striated ducts (mandibular salivary gland), and a hyperkeratosis of skin. Histologically, no effects of treatment were observed in group 2 (0.01 mg/l).

No treatment-related changes in organ weight or organ weight ratios were observed.

The treatment with ethylene thiourea was considered to induce a number of changes and to reveal some target organs. Changes were suggestive of a thyrotrophic character of ethylene thiourea: decrease in plasma levels of T4 (at 0.04 mg/l in females, and at 0.20 mg/l in both sexes), changes of the thyroid gland (increase of follicular epithelial height, colloid depletion, colloid agglomeration, diffuse hyperplasia, folliculoneogenesis and inscreased vascularity).

Other changes attributed to the treatment observed in rats of both sexes dosed at 0.20 mg/l included: a moderate decrease in body weight, histological changes in the pituitary gland and mandibular salivary gland; decrease in reticulocyte count.

Based upon these results, the NOEL of ethylene thiourea by inhalation lies between 0.01 and 0.04 mg/l air according to the authors.

Justification for classification or non-classification

Imidazolidine-2-thione is not classified in the Annex VI of the Regulation EC n°1272/2008 for the repeated toxicity.

Regulation (EC) No 1272/2008 (Self-classification)

Ethylene thiourea should be classified as STOT RE category 1 with "thyroid" as target organ for all routes of exposure.

Justification: The LOAEL for thyroid toxicity (90 day oral study) is lower or equal than 10 mg/kg bw/d in both key studies (8 and 10 mg/kg/d), that justified that ethylenethiourea must be classified in the category 1 of CLP. Moreover, the LOAEC for thyroid toxicity (28 -day inhalation study) is below 0.06 mg/l (0.04 mg/l), and suggested a classification in the category 1 of CLP.

Directive EEC/67/548 (Self classification)

Ethylene thiourea should be classified as Xn; R48/20/22 for thyroid toxicity.

Justification: The LOAEL for thyroid toxicity (90 day oral study) is between 5 and 50 mg/kg bw/d in both key studies (8 and 10 mg/kg/d), that justified that ethylenethiourea must be classified as R48/22. Moreover, the LOAEC for thyroid toxicity (28 -day inhalation study) is between 0.025 and 0.25 mg/l (0.04 mg/l), and suggested a classification R48/20.