Registration Dossier

Toxicological information

Carcinogenicity

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Description of key information

Oral exposure to ethylene thiourea (ETU) caused tumors in two rodent species (rats and mice) and at two different tissue sites.
Dietary exposure of adults animals to ETU caused thyroid-gland cancer (follicular-cell carcinoma) in mice and rats of both sexes, liver cancer (hepatocellular carcinoma) in mice of both sexes, and benign pituitary-gland tumors (adenoma of the pars distalis) in mice of both sexes.
ETU is reasonably anticipated to be a human carcinogen based on sufficient evidence of carcinogenicity from studies in experimental animals.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records
Reference
Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Study : carcinogenicity of ETU with or without exposure.
Group of rats were exposed to ETU by feed.
7 treaded groups and one control group (0:0).
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Kingston, NY)
- Age at study initiation: 5-6 weeks old
- Weight at study initiation: no data
- Fasting period before study:
- Housing: one per polycarbonate cage during the pregnancy, then five per cage
- Diet (e.g. ad libitum): Purina Certified Rodent Chow, ad libitum
- Water (e.g. ad libitum): City of Columbus water, ad libitum
- Acclimation period: 7 days (minimum)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21/23°C
- Humidity (%): 40-60%
- Air changes (per hr): 15/hr
- Photoperiod (hrs dark / hrs light): fluorescent light on 12 hr/day
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The experimental design of the carcinogenicity studies consisted of groups of rats receiving perinatal exposure (F0) and/or adult exposure (F1) to varying concentrations (ppm) of ETU in diet as follow : (0:0), (0:83), (0:250), (90:0), (90:83), (90:250), (30:83), (9:25).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dietary formulations were prepared weekly by mixing appropriate amounts of ethylene thiourea and feed and were analyzed at least every 2 months. The formulations were stored in plastic bags for no longer than 2 weeks, and stability studies showed no decreases in the concentrations of ETU in the dark at room temperature. The formulated dieu were prepared within ±10% of the target concentrations approximately 95% of the time.
Duration of treatment / exposure:
Females F0 : one week before breeding, during pregnancy and lactation.
Treated pup F1 : 2 years
Frequency of treatment:
Daily (in feed)
Remarks:
Doses / Concentrations:
Adult exposure only: 83 and 250 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Perinatal/adult exposures: 9/25, 30/83, 90/83 and 90/250 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
Perinatal exposure only: 0/90 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
Pups : 50-60 animals / sex / dose (x:x)
Control animals:
yes
Details on study design:
Femelle rats were exposed at 0, 9, 30, or 90 ppm in feed for 1 week before breeding. After breeding to previously unexposed male rats, all females were housed singly and were continued on their previous diet. Exposure continued throughout pregnancy and lactation. On postpartum Day 4, litters were culled to a maximum of eight. Weaning occurred on Day 28 postpartum and exposure continued at the concentrations given to the respective dams until the pups were 8 weeks of age. Pups were separated alter weaning by sex and litter mates were cohoused. At approximalely 8 weeks of age, the pups were separated into groups of 60 males and 60 females to receive the adult (F1) dietary concentrations (0, 25, 83, or 250 ppm) for 2 years. Animals were housed five per cage and feed and water were available ad libitum. Cages were rot rotated during these studies.
Positive control:
no
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, twice per day
DETAILED CLINICAL OBSERVATIONS: Yes, twice per day
BODY WEIGHT: Yes, once per week for the first 13 weeks of the study and one per month thereafter.
Mean body weight were calculed for each group.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no data
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: after 9 months after necropsy
- Animals fasted: No data
- How many animals: 10M/10F
- Parameters examined : serum T3, T4 and TSH
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
Each animal was necropsied and the adrenal gland, brain, heart, kidney, liver, lung, pituitary gland, testis, prostate, uterus, ovary, thyroid gland, and thymus were weighed. Complete histologic examinations were performed on all rats.
Necropsy was performed on animals found dead or moribund and those surviving to the end of the 2-year studies. During necropsy, all organs and tissues were examined for grossly visible lesions.
Complete histopathological examinations were performed on all rats, on all control, that died before the end of the study.
Other examinations:
Serum T3, T4, and TSH were measured from 10 rats of each sex.
Statistics:
The experimental design of the carcinogenicity study was comptex and it was intended to evaluate both perinatal and postnatal effects. The effect of adult only exposure to ETU was analyzed by comparison ofgroups 0: 0 (F0:F1), 0:83, and 0:250 (rats). To determine perinatal effects, supplemental analyses were carrierd out in addition to the usual comparison of exposed groups to controls. Specifically, for a fixed adult (F1) exposure concentration, the effect of varying perinatal (F0) exposure was evaluated.
The majority of tumors in this study were considered to be incidental to the cause of death or not rapidly lethal. Thus, the primary statistical method used was a logistic regression analysis, which assumed that the diagnosed tumors were discovered as the result of death from an unrelated cause and thus did rot affect the risk of death. In addition to logistic regression, alternative methods of statistical analysis were used. These include the life table test, appropriate for rapidly lethal tumors, and the Fisher exact test and the Cochran-Armitage trend test, procedures based on the overall proportion of tumor-bearing animals.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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:
no effects observed
Gross pathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
Generally, there were no clinical signs related to ETU administration.
Survival of rats receiving adult exposure only was similar to those of controls. However, survival of male rats in the 90:250 ppm dose group was significantly decreased.

BODY WEIGHT AND WEIGHT GAIN
Body weights were generally similar among exposed and control rats evaluated at 9 months.
Mean body weights of rats receiving adult only exposure to ETU were similar to those of the 0:0 ppm controls throughout the study. Mean body weights of females were not affected by perinatal exposure, but those of males receiving 90:250 ppm ETU were 18% lower than the 0:250 ppm group and 20% lower than the 0:0 ppm group at the end of the study. Body weights of males receiving lower F0:F1 concentrations of ETU were generally similar to controls (Table 3).

FOOD CONSUMPTION AND COMPOUND INTAKE
There were no differences in food consumption between groups of exposed rats and the 0:0 control throughout the study, except for a decrease in that of the 90:250 ppm males during the last month of exposure.

CLINICAL CHEMISTRY
At 9 months : In general, serum T4 levels were decreased in exposed rats and thyrotropin levels were increased; T3 levels were variable, but decreased in nome exposure groups (Table 2).
Effects of adult only exposure (2 years): Serum T4 levels were decreased in most of the dose groups while TSH levels were increased several fold in the top dose group.

ORGAN WEIGHTS
Organ weights were generally similar among exposed and control rats evaluated at 9 months. Thyroid weights were marginally increased in males and females in the 0:250 and 90:250 ppm groups but the increases were not statistically significant.

HISTOPATHOLOGY: NON-NEOPLASTIC
At 9 months : Thyroid follicular cell hyperplasia was observed in most groups of exposed male and female rats (Table 2). The severity increased in a dose-related manner from minimal in the lower dose group to moderate in the higher dose groups.
Effects of adult only exposure (2 years): The nonneoplastic effects of adult only exposure were determined by comparison of the incidences of lesions in the 0:0, 0:83, and 0:250 ppm groups. The principal toxic effects of ETU in rats were in the thyroid gland (Table 4). The incidences of follicular cell hyperplasia were markedly increased in both exposure groups, relative to controls, and occurred in 60-90% of the exposed rats.
Effects of perinatal only exposure (2 years) : The incidences of male and female rats with follicular cell hyperplasia were marginally but significantly increased in the 90:0 ppm group. Whether these increases are chemically related is uncertain, but similar effects in both sexes support the association with dietary exposure.
Effects of combined perinatal/adult exposure (2 years) : Combined perinatal exposure of 30 or 90 ppm ETU with adult exposure of 83 or 250 ppm was associated with increased incidences of nonneoplastic lesions of the thyroid gland, and incidences were similar to those of adult only exposure.
The incidence of follicular cell hyperplasia in the 90:83 ppm males (47/50), but not in the females, was significantly greater than that in the 0:83 ppm (30/46) group.

HISTOPATHOLOGY: NEOPLASTIC
At 9 months : Follicular cell adenomas were found in the thyroid glands of three males and one female receiving 90:250 ppm.
Effects of adult only exposure (2 years): The nonneoplastic effects of adult only exposure were determined by comparison of the incidences of lesions in the 0:0, 0:83, and 0:250 ppm groups. The principal toxic effects of ETU in rats were in the thyroid gland (Table 4). Adenomas were marginally increased in the 0:83 ppm groups, but were seen in 46-56% of rats in the 0:250 ppm groups. Follicular cell carcinomas were increased only at the top concentration (0:250 ppm and 90:250 groups) and were more frequent in males than in females. Thus, male rats were more sensitive to the effects of ETU than were female rats. Male rats receiving 83 or 250 ppm (F1) had higher incidences of follicular cell neoplasms than females at these concentrations, and male rats at 250 ppm had higher incidences of carcinoma than females. Most of the affected rats at 0:250 ppm had bilateral or multiple thyroid neoplasms. Focal follicular cell hyperplasia, adenoma, and carcinoma constituted a morphologic continuum. Adenomas generally were well-circumscribed nodular masses which compressed adjacent normal tissue, and consisted of follicle-like structures, papillary projections, and solid nests of well-differentiated epithelium; cystic areas were often present. Follicular cell carcinomas displayed increased architectural disorganization and greater cellular pleomorphism and atypia than adenomas. Some carcinomas invaded the adjacent parenchyma and/or esophagus and trachea, and two metastasized to the lungs.
Effects of perinatal only exposure (2 years) : Comparison of the 90:0 ppm groups with the 0:0 ppm controls showed that perinatal only exposure had no effect on the incidences of neoplasms in the thyroid gland or any other organ (Table 4).
Effects of combined perinatal/adult exposure (2 years) : Combined perinatal exposure of 30 or 90 ppm ETU with adult exposure of 83 or 250 ppm was associated with increased incidences of nonneoplastic lesions and neoplasms of the thyroid gland, and incidences were similar to those of adult only exposure. Comparison of the 90:250 ppm with the 0:250 ppm group showed statistically significant increases in the incidences of follicular cell adenoma, carcinoma, and adenoma or carcinoma combined in male rats and carcinoma and adenoma or carcinoma combined in female rats associated with perinatal exposure at 90 ppm (Table 4).
Some groups receiving both perinatal and adult exposure showed statistically significant increases, relative to the 0:0 ppm controls, in neoplasms of the Zymbals gland and of the hematopoietic system. There also were slight increases in rare renal tubular cell neoplasms that were not statistically significant. Neoplasms of the Zymbal's gland were marginally increased in rats at 90:250 ppm (males - 0:0, 1/50; 90:250, 5/50; females - 0:0, 1/50; 90:250, 4/50). Mononuclear cell leukemia occurred with a significant trend in groups of male and female rats receiving perinatal exposure of 90 ppm and increasing adult concentrations (90:0, 90:83, and 90:250 ppm), and for female rats without perinatal exposure (0:0, 0:83, and 0:250 ppm). The incidences of leukemia in the 90:83 ppm males and 90:250 ppm males and females were statistically significant relative to the respective 0:0 group. Renal tubular cell adenomas occurred in exposed male rats at low incidence, but not in controls.
Relevance of carcinogenic effects / potential:
Significant increases in the incidences of thyroid follicular-cell tumours were observed in males at 83 and 250 ppm of diet, with or without perinatal exposure, and in females at 250 ppm of diet, with or without perinatal exposure when compared with their respective control groups. Marginally significant (p<0.05) increases in the incidence of Zymbal gland tumours were observed in males (5/50) at 90:250 ppm of diet and in only 1/50 control males.  The incidences of tumours were generally similar with and without  perinatal exposure, except that the incidence of thyroid tumours were  higher with perinatal exposure at the higher concentration.
Dose descriptor:
LOAEL
Remarks:
adult exposure only
Effect level:
83 ppm
Sex:
male
Basis for effect level:
other: Thyroid follicular adenoma or carcinoma. ca.4.15 mg/kg/day (83 ppm x 0.05 (assumed rat food consumption per body weight))
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
NOAEL
Remarks:
adult exposure only
Effect level:
83 ppm
Sex:
female
Basis for effect level:
other: Thyroid follicular adenoma or carcinoma.
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
NOAEL
Remarks:
perinatal and adult exposures
Effect level:
other: 9/25 ppm
Sex:
male
Basis for effect level:
other: Thyroid follicular adenoma or carcinoma.
Remarks on result:
other: Effect type: carcinogenicity (migrated information)
Dose descriptor:
NOAEL
Remarks:
Perinatal and adult exposures
Effect level:
other: 90/83 ppm
Sex:
female
Basis for effect level:
other: Thyroid follicular adenoma or carcinoma.
Remarks on result:
other: Effect type: carcinogenicity (migrated information)

TABLE 2: Incidences of Thyroid Lesions and Hormonal Changes in Rats at the 9-Month Interim
Evaluation in the Carcinogenicity Study of Ethylene Thiourea
a

Dietary ETU concentration (Fo:F1)

0:0

90:0

9:25

0:83

30:83

90:83

0:250

90:250

MALES

Histopathologic changes

Follicular cell hyperplasia

0

4 t

1

10 tt

8 tt

10 tt

10 tt

10 tt

Follicular cell adenoma

0

0

0

0

0

0

0

3

Hormonal changeb

T3

-

103

72**

87

69'*

64**

96

94

T4

-

101

81**

651*

67**

66**

64**

55**

TSH

-

105

113

123

146

154

161

157

FEMALES

Histopathologic changes

Follicular cell hyperplasia

0

0

0

5t

10 tt

10 tt

10 tt

10 tt

Follicular cell adenoma

0

0

0

0

0

0

0

1

Hormonal changeb

T3

-

111

71**

74**

71**

80**

100

78**

T4

-

100

72**

49**

45**

60**

61**

54**

TSH

-

110

156

183*

178*

245**

149

260**

aTen animals examined per group except for 90:0 males for which the number was nine.

bThe values are as percentage of the 0:0 group.

* p < 0.05 vs 0:0 group (Dunnett's test).

** p < 0.01 vs 0:0 group (Dunnett's test).

t p < 0.05 vs 0:0 group (Fisher's exact test).

tt p < 0.01 vs 0:0 group (Fisher's exact test).


TABLE 3 : Final Body Weights and Survival of Rats in the Carcinogenicity Studies of Ethylene Thiourea

DietaryETUconcentration (Fo:F1)

0:0

90:0

9:25

0:83

30:83

90:83

0:250

90:250

MALEa

Animals initially in study

50

50

50

50

50

50

50

50

Terminal number

18

15

17

15

18

14

14

4

Survival p valueb

-

1.000

0.555

0.732

0.917

0.644

0.730

0.009

Final body weights (g)

415

383

406

395

410

388

398

325•

FEMALEa

Animals initially in study

50

50

50

50

50

50

50

50

Terminal number

23

30

34

30

26

32

20

13

arvival p valueb

-

0.287

0.052

0.080

0.604

0.065

0.849

0.069

Final body weights (g)

331

330

336

331

327

332

317

326

aDay of first terminal sacrifice:738for males and740for females.

bResults of the life table pairwisecomparison with the 0:0 ppm control.

*p < 0.01vs 0:0 ppm controls (Fisher's least significant difference test).

TABLE 4 : Incidences of Thyroid Follicular Adenoma or Carcinoma in Rats Exposed to Ethylene Thiourea in Feed

ETU (Fo-F,)ppm

Male

Female

Adult exposure groups

0:0

1/49

3/50

0:83

12/46•*

7/44

0:250

37/50**

30/49**

Perinatal / adult exposure groups

9:25

3/46

1/49

30:83

14/47**

6/47

90:83

13/50**

9/47

90:250

48/50.'t

37/50*`t

Perinatal exposure groups

90:0

4/49

3/50

** p < 0.01 vs 0 :0 group (logistic regression test)

t p< 0.05 vs 0:250 ppm group (logistic regression test)


Conclusions:
There were statistically significant pair-wise increases in thyroid follicular cell adenomas at 83 and 250 ppm in males and females; and in combined adenomas/carcinomas at 83 and 250 ppm in males and at 250 ppm in females. Thyroid follicular cell carcinomas were statistically significantly increased in males and females at 250 ppm. Thyroid adenomas, carcinomas, and combined tumors had significant positive trends in males and females.
Executive summary:

This study was designed to show whether perinatal exposure, in addition to adult exposure, enhanced the sensitivity of a cancer study. There were 3 exposure groups in this study: an adult exposure group, a perinatal/adult exposure group, as well as a perinatal-only exposure group, all of 24 months duration, all of 24 months in duration, in which ethylene thiorurea (97% a.i.) was administered to F344/N rats in the diet. There were 50 rats per dose group and an additional 10 rats per dose group in an interim sacrifice group in each study. Dietary doses for rats treated only as adults were 0, 83, or 250 ppm. Another group of rats received perinatal exposure and exposure as adults as follows: dams were administered ETU for 1 week prior to breeding and during gestation. Pups were weaned at 28 days of age and exposure continued at concentrations given to the respective dams, until 8 weeks of age when doses were changed to doses which they received until the end of the 24 month study. Doses showing Fo (perinatal)/F1(adult) exposure were: 0/0, 9/25, 30/83, 90/0, 90/83, or 90/250 ppm. There were no clinical signs attributed to treatment. In the interim sacrifice at 9 months, the group which had received perinatal-only exposure (90/0 ppm) had thyroid hormone levels comparable to controls. Thyroid follicular cell hyperplasia was reported to be marginally, but significantly increased in this group. Thyroid hormone levels were similar in animals which were treated as adults only and in animalswhich also were treated in the postnatal period. Females had slightly greater depression of thyroid hormone levels than males at the interim sacrifice, but not at terminal sacrifice. For animals which received treatment as adults (adult only or perinatal/adult exposure), thyroid hormones at the interim sacrifice at 9 months were affected as follows: T4 hormone levels were decreased (45-81% of control values) in a dose related manner in males and females of all dose groups (dietary concentrations of 25, 83, or 250 ppm). T3 levels showed no dose-response related changes, and TSH was variably increased (113-260% of controls). Incidence of thyroid follicular cell hyperplasia was increased in males and females at doses of 83 and 250 ppm at the interim sacrifice; thyroid histopathology for non-neoplastic lesions was not shown for the terminal sacrifice. At terminal sacrifice, T4 was decreased and TSH was increased in males in the 83 and 250 ppm groups and in females in the 250 ppm group.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
4.15 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Both studies are reliable with a klimisch score of 2.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Cohort study

 

A list of 1929 workers at several large rubber manufacturing firms where ethylenethiourea was used and at a firm producing ethylenethiourea in England was drawn up from employment records (Smith, 1976). According to the records of the Birmingham Cancer Registry for the period 1957–71, none of the workers developed thyroid cancer. [The lack of details on methods, including the number of expected cases, makes it difficult to assess the relevance of this finding.] Although workers in the rubber industry and pesticide applicators may be exposed to ethylenethiourea, no specific mention of this compound was found in epidemiological studies of the cancer risks of these populations.

 

Studies of Cancer in Experimental Animals

Mouse: In a preliminary report of a screening study, groups of 18 male and 18 female hybrid mice of the (C57BL/6 × C3H/Anf)F1 (B6C3F1) and (C57BL/6 × AKR)F1 (B6AKF1) strains, 7 days of age, were given doses of 0 (control) or 215 mg/kg bw commercial-grade ethylenethiourea [purity not specified] daily in 0.5% gelatin in water by gavage for 3 weeks. The dose determined at 7 days of age was not adjusted for body weight. The mice were weaned at 4 weeks of age, and the chemical without vehicle was mixed into the diet at a concentration of 0 (control) or 646 mg/kg and provided ad libitum from 4 weeks to approximately 18 months. The concentration of the compound in the diet was calculated from the weight and food consumption of the 4-week-old mice to be approximately the maximum tolerated dose on a mg/kg bw basis. The same concentration was maintained throughout the duration of the study up to 82–83 weeks

of age. The incidence of ‘hepatomas’ was 14/16 (male) and 18/18 (female) in treated B6C3F1 mice and 18/18 (male) and 9/16 (female) in treated B6AKF1 mice, with incidences in the pooled control groups of 8/79 (male) and 0/87 (female) for the B6C3F1 mice and 5/90 (male) and 1/82 (female) for the B6AKF1 mice. The increases in incidences of hepatomas in male and female mice of both strains were statistically significant (p < 0.01) (Innes et al., 1969).

The carcinogenic potential of ethylenethiourea was evaluated during and after perinatal exposure (in uteroand throughout suckling) (Chhabra et al., 1992; National Toxicology Program, 1992).. Female C57BL/6 mice, 10–11 weeks of age (F0generation), were fed a diet containing 0, 33, 110 or 330 mg/kg ethylenethiourea for 1 week before breeding. After mating with previously unexposed male C3H/HeN mice, all the females were continued on the diets containing ethylenethiourea. On day 7post partum, the litters (F1generation) were standardized to a maximum of eight, weaned on day 28 and separated by sex. Up to 8 weeks of age, the litters were exposed to ethylenethiourea at the same concentrations in the diet as those given to their dams; at approximately 8 weeks of age, the pups were divided into groups of 50 animals per sex and exposed to the adult concentrations of 0, 330 and 1000 mg/kg of diet for 2 years. The F0:F1 treatments were thus 0:0, 0:330, 0:1000, 330:0, 330:330, 330:1000, 33:100 and 110:330 mg/kg of diet. The tumour incidences in the various groups are shown in Text table 1. Significant (p< 0.01) increases were found in the incidences of liver tumours in males and females at 330 mg/kg of diet, with or without perinatal exposure. Significant (p< 0.01) increases in the incidences of thyroid follicular-cell tumours were observed in females at 330 mg/kg of diet with perinatal exposure and in males and females at 1000 mg/kg of diet with or without perinatal exposure. Significant (p< 0.01) increases in the incidences of anterior pituitary tumours were observed in females at 330:330 and 330:1000 mg/kg of diet and in F1males and F1females at 0:1000 mg/kg of diet. The incidences of tumours were generally similar with and without perinatal exposure, except that the incidences of thyroid and anterior pituitary tumours in the females were higher after perinatal exposure.

 

Text table 1: Incidences of neoplasms in B6C3F1 mice exposed to ethylenethiourea in the diet with or without perinatal exposure

 

Concentration of ethylene thiourea (F0:F1) (mg/kg of diet)

Hepatocellular adenoma and carcinoma combined

Thyroid follicular cell adenoma and carcinoma combined

Anterior pituitary adenoma and carcinoma combined

 

Males

Females

Males

Females

Males

Females

Adult exposure only

0:0

20/49

4/50

1/50

0/50

0/44

11/47

0:330

32/50*

44/50**

1/49

2/50

0/42

19/49

0:1000

46/50**

48/50**

29/50**

38/50**

8/41**

26/49**

Perinatal and adult exposure

33:100

9/33

4/28

1/47

1/29

0/28

2/28

110:330

26/47

46/50**

1/47

5/50*

0/41

14/48

330:330

34/49*

46/50**

2/48

10/49**

0/45

26/47**

330:1000

47/49**

49/50**

35/49**

38/50**

4/39

24/47**

Perinatal exposure only

330:0

13/49

5/49

1/46

1/49

0/42

11/48

From Chhabraet al. (1992); National Toxicology Program (1992). Incidences are numbers of lesions observed/number of animals

*p< 0.05 versus 0:0 group (logistic regression test)

**p< 0.01 versus 0:0 group (logistic regression test)

 

Rat: Groups of 26 male and 26 female Sprague-Dawley (Crl:CD) rats, 5–6 weeks of age, were fed diets containing 175 or 350 mg/kg technical-grade ethylenethiourea (97% pure) for 18 months (Ulland et al., 1972; Weisburger et al., 1981). Five rats per dose group were killed and necropsied at 18 months, and the remaining rats at the two concentrations were continued on control diet for up to a further 6 months, for a total of up to 24 months. Thyroid (follicular or papillary) carcinomas were observed in 0/30 and 0/30 male and female controls, 2/26 and 2/26 males and females at the lower concentration and 15/26 and 6/26 males and females at the higher concentration, respectively.

 

Graham et al. (1973, 1975) studied the long-term effects on the thyroid gland of ETU ingestion. Groups of 68 male/68 female Charles River rats were fed ETU (purity not stated) in the diet at levels of 0, 5, 25, 125, 250, or 500 ppm for 2 years. Body weight and food consumption were measured weekly. At week 66, 3 male/3 female rats from each test group were fed control diet only for the remainder of the 2-year study. At 3, 11, 17 and 22 months blood samples were collected from the tail vein of 10 male and 10 female rats. At 6, 12, 18 and 24 months 10 male/10 female rats from each group were administered 5 µCi131I i.p., fasted for 24 hours, sacrificed and thyroid, heart, liver, kidneys and spleen examined for radioactive uptake. Body weights in both sexes were significantly decreased at>25 ppm initially; at>500 ppm (males) and>125 ppm (females) at 12 months; and at > 500-ppm (both sexes) for the remainder of the study. Liver to body weight ratios were significantly increased at>125 ppm through 6 months in males, but comparable to control for the remainder of the study. Relative liver weights in females were significantly increased at>125 ppm at 2 months and > 250 ppm through 18 months; no differences between control and dose groups was observed at 24 months. Thyroid to body weight ratio was significantly increased in males at > 250 ppm for 2, 6 and 18 months, and at>125 ppm in females for the first 12 months. Thyroid weights were significantly increased at > 125 ppm in males at 12 and 24 months, and at > 250 ppm in females at 18 and 24 months. Uptake of131I, expressed as counts/min/mg tissue, was significantly decreased in males at 500 ppm throughout the study. Thyroids of females fed > 125 ppm were hypofunctioning at 6 months and hyperfunctioning at 12 months. At 24 months females had a hypofunctioning thyroid at 500 ppm. There were fewer rats surviving to 24 months in the 500 ppm dose group compared to control and other dose groups. There was also a significant increase in pneumonia in high dose group rats. This may have been further complicated by obstruction of the trachea from enlarged thyroids in high dose group animals. Effects in the thyroid were evident at all doses (> 5 ppm). However, histologic data were summarized and not separated by sex. Increased vascularity and hyperplasia in the thyroid were evident at 5 ppm and increased in incidence and severity at > 25 ppm. Thyroids of treated rats were distinguishable from controls by lobulation, follicular size and uniformity, height of follicular epithelium, colloid staining, keratinization of follicles, and general size. It is possible that ETU initially reduces thyroid activity, after which compensation occurs by an increased release of TSH and that this increase in TSH stimulated thyroid weight in an attempt to overcome the blocking effect of ETU. The progression to neoplasia is believed a result of excessive pharmacological stimulation. This is supported, in part, by a lack of thyroid tumours at 1 year at 5 or 25 ppm, an increase in tumour incidence after 1 year at 125 ppm, and confirmed after 2 years in rats fed 250 and 500 ppm.

 

Gak et al. (1976) studied the effects of feeding five groups of 20 male and 20 female rats [strain or stock and age not specified

with 0, 5, 17, 60, or 200 ppm ETU for 24 months. Body weight, food consumption, serum enzyme activities (e.g. glutamic pyruvic transaminase, alkaline phosphatase), hepatic enzyme activities (glutamic pyruvic transaminase, alkaline phosphatase, glucose-6-phosphate dehydrogenase), cholesterol levels, weights of thyroid and other organs, and histopathology were studied. There was a strong negative association between food consumption and bodyweight gain and dietary concentration, the decreases in food consumption and bodyweight gain being > 10% at the two higher concentrations. Hypercholesterolemia was found at dose levels of 5 ppm and above. The incidences of thyroid tumours were 0, 0, 5.9, 42.1 (p< 0.01) and 82.4% (p< 0.001) in males and 5.3, 6.3, 18.8, 22.2 and 56.3% (p< 0.001) in females at 0, 5, 17, 60 and 200 ppm ethylenethiourea, respectively.

 

Female Fischer 344 rats, 10–11 weeks of age (F0 generation), were fed a diet containing 0, 9, 30 or 90 mg/kg ethylenethiourea for 1 week before breeding (Chhabra et al., 1992; National Toxicology Program, 1992). After mating with previously unexposed male Fischer 344 rats, all the females were continued on their previous diets. On day 4 post partum, the litters (F1generation) were standardized to a maximum of eight and weaned on day 28. The pups continued to be exposed at the concentrations given to their dams until they were 8 weeks of age. The pups were separated by sex at weaning, and at approximately 8 weeks of age were divided into groups of 50 animals per sex and exposed to the adult dietary concentrations of 0, 25, 83 and 250 mg/kg for 2 years. The F0:F1treatments were thus 0:0 (control), 0:83, 0:250, 90:0, 90:83, 9:250, 30:83, and 9:25 mg/kg of diet. The incidences of thyroid tumours in the various groups are shown in Text table 2. Significant increases in the incidences of thyroid follicular-cell tumours were observed in males at 83 and 250 mg/kg of diet, with or without perinatal exposure, and in females at 250 mg/kg of diet, with or without perinatal exposure when compared with their respective control groups. Marginally significant (p< 0.05) increases in the incidence of Zymbal gland tumours were observed in males (5/50) at 90:250 mg/kg of diet and in only 1/50 control males. The incidences of tumours were generally similar with and without perinatal exposure, except that the incidences of thyroid tumours were higher with perinatal exposure at the higher concentrations.

 

Text table 2. Incidences of thyroid follicular-cell adenoma and carcinoma combined in Fischer 344 rats exposed to ethylenethiourea in the diet with or without perinatal exposure

 

Concentration of ethylenethiourea (F0:F1) (mg/kg of diet)

No. of lesions observed/number of animals

 

Males

Females

Adult exposure only

0:0

1/49

3/50

0:83

12/46*

7/44

0:250

37/50*

30/49*

Perinatal and adult exposures

9:25

3/46

1/49

30:83

14/47*

6/47

90:83

13/50*

9/47

90:250

48/50*

37/50*

Perinatal exposure only

90:0

4/49

3/50

From Chhabraet al.(1992); National Toxicology Program (1992)

*p< 0.01 versus 0:0 group (logistic regression test)

 

Hamster: Groups of 20 male and 20 female hamsters were administered ETU (purity not stated) in the diet for 20 months, at dose levels of 0, 5, 17, 60 and 200 ppm. (Strain of animals not reported.) Body weight, food consumption, selected clinical chemistry parameters and organ weights were measured. Histopathological examinations of selected tissues were performed at necropsy. SGPT, SAP and cholesterol were measured in the serum; GPT, AP and G6PDH were determined in the liver. Organs weighed included liver, thyroid, testes, kidneys and spleen. Food consumption and body weight were reduced at > 60 ppm. SAP was increased in both sexes initially, then decreased through 18 months. No effect was observed on SGPT. Cholesterol levels were significantly increased in both sexes at all doses compared to controls. Hepatic enzymes, GFT and AP, were significantly increased in both sexes at all doses. G6PDH was significantly decreased in both sexes at all dose levels. Relative thyroid weights were significantly increased at > 200 ppm in both sexes. No data were available on the histologic examination (Gak et al., 1976).

 


Justification for selection of carcinogenicity via oral route endpoint:
Two key studies are chosen to evaluate the carcinogenic potential of ETU. NTP's study is more recent and has a longer duration of exposure (2 years).

Carcinogenicity: via oral route (target organ): digestive: liver; glandular: thyroids

Justification for classification or non-classification

1/ EU Mandatory classification od ETU :

Ethylenethiourea (ETU, CAS: 96-45-7) is not classified to the Annex I of the DSD classification (19thATP, 1993) and to the Annex VI of the Regulation EC no.1272/2008 for the carcinogenicity endpoint.

2/ IARC classifications

In 1987, Ethylenethiourea was classified in Group 2B for carcinogenicity, based on inadequate evidence for carcinogenicity to humans, but sufficient evidence for carcinogenicity to animals.

In 2001, new data were available on ETU, and a new evaluation was made by IARC. According to the IARC (2001), Ethylenethiourea is not classifiable as to its carcinogenicity to humans (Group 3), based on inadequate evidence in humans but sufficient evidence in experimental animals for the carcinogenicity of ETU. The justification is “In making its evaluation, the Working Group concluded that ETU produces thyroid tumours in mice and rats by a non-genotoxic mechanism, which involves interference with the functioning of thyroid peroxidase resulting in a reduction in circulating thyroid hormone concentrations and increased secretion of TSH. Consequently, ETU would not be expected to produce cancer in humans exposed to concentrations that do not alter thyroid hormone homeostasis.”.

3/ Classification in the REACH dossier

In the 2-year carcinogenicity study (NTP 1992), ETU was administered in diet for 2 years at concentrations of 330 and 1000 ppm (42.9 and 130 mg/kg bw/d, respectively) to mice and at concentrations of 83 and 250 ppm (4.15 and 12.5 mg/kg bw/d, respectively) to rats. Based upon statistical results, the administration of ETU on mice was associated with the increased incidence of hepatocellular adenomas and carcinomas at 330 and 1000 ppm, thyroid follicular-cell adenomas and carcinomas at 1000 ppm and, anterior pituitary adenomas and carcinomas at 1000 ppm in male and in female mice. On rats, thyroid follicular-cell adenomas and carcinomas were observed at 83 ppm in males only and at 250 ppm in both sexes. Under the conditions of the bioassay, ETU was carcinogenic in mice and rats. The LOAEL for carcinogenicity are 330 ppm (42.9 mg/kg bw/d), based on liver adenomas and carcinomas observed at this dose in male and female mice, and 83 ppm (4.15 mg/kg bw/d) based on thyroid follicular-cell adenomas and carcinomsa in male rats

Moreover, ETU is reasonably anticipated to be a human carcinogen based on sufficient evidence of carcinogenicity from studies in experimental animals (Report on Carcinogens, 12th edition, 2011).

Considering the sufficient evidence of carcinogenicity in animal studies in both species (rat & mice), a classification of ETU as carcinogenic is warranted.

Self classification :

Regulation (EC) No 1272/2008 : Carc. category 2 - H351 "Suspected of causing cancer".

Directive 67/548/EEC : Carc. category 3 - R40 "Limited evidence of a carcinogenic effect".