Registration Dossier

Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
there are no satellite group
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Other name = Ethylenethiourea (ETU)
- Purity: 99.3%
-Description : white powder
- Other: based on its particle size distribution, the dust was respirable
-Batch number : T154
The test article was generated as supplied by the sponsor and generated by using an rBG 1000 aerosol generator.

Test animals

Species:
rat
Strain:
other: SPF-Wistar
Sex:
male/female
Details on test animals 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

Administration / exposure

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)
Doses / concentrationsopen allclose all
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

Examinations

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.

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 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.

Effect levels

open allclose all
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

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

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.