Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Key study. Two-generation lifetime feeding study (no TG, no GLP, study well documented, acceptable for assessment). No statistically significant or dose-related incidence of tumours was observed in any groups. Based on the available data, the test item was not carcinogenic to rats.

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
Study period:
1974
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: Two-generation lifetime feeding study
- Short description of test conditions: 50 male and 50 female weanling Sprague-Dawley rats were administered the test item at different doses in their diet. After 3 months on test, F0 rats were bred and 50 pups of each sex, from every group, were weaned onto the parental diets, which both generations received for their lifetime.
GLP compliance:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, Canadian Breeding Farms, Montréal, Québec, Canada.
- Age at study initiation: 32 days
- Housing: The animals were housed individually, except during mating, in stainless-steel cages with screened floors. Cages were steam disinfected every 2 weeks and the "pull-papers" under the cages were changed daily.
- Diet: the basal diet was Master Laboratory Cubes (Master Feeds, Toronto, Ontario, Canada) to which 4% (w/w) corn oil had been added after the cubes were ground. Weighed amounts of the appropriate feed were distributed to the animals on a weekly basis in stainless steel feed cups.
- Water: drinking water was supplied by the Municipality of Ottawa, ad libitum, provided in a clear glass bottle, with a rubber stopper and stainless-steel nipple, which had been previously sterilized. The water bottles were changed 3 times/week.

DETAILS OF FOOD AND WATER QUALITY: Four lots of feed cubes were analyzed for the following micronutrients: calcium (1.7%), phosphorus (0.99 - 1.0%), copper (9.1 - 10 mg/lb), cobalt (2.5 - 2.8 mg/lb), manganese (64 - 68 mg/lb), zinc (30 - 31 mg/lb), iron (431 - 499 mg/lb). Analysis of several batches of feed during the study identified background levels of heavy metals, an average of 20 ppb of nitrosamines, trace amounts of 10 halogenated hydrocarbons (12 ppb total content), but no detectable amounts of aflatoxin or aflatoxin-like compounds. The classification of the drinking water hardness was 'very good' (< 4.7 grains CaCO3/US gal).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1°C
- Humidity (%): 50-55%
- Photoperiod (hrs dark / hrs light): 12 h light/dark cycle
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): the appropriate amount of test chemical was premixed with 500g of ground rat chow. This concentrate premix was added to an appropriate amount of basal diet plus corn oil and mixed thoroughly on the day prior to use.
- Storage temperature of food: room temperature, protected from light.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
F0: 142 weeks; F1: 127 weeks.
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
2.5 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
plus 1% NH4CI in drinking watter, which was added to prevent the formation of alkaline urine (associated with the production of bladder calculi and bladder tumors in a previous study).
No. of animals per sex per dose:
50
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the doses were selected to be equivalent to the levels resulting from the dietary additions of Remsen-Falberg-produced saccharin used in previous studies (WARF, FDA).
- Rationale for animal assignment: Random.
- Schedule: After 3 months on test, the F0 rats were mated on a one-to-one basis; all litters were culled to 8 pups (4 males and 4 females) 4 days post partum in a random manner. The pups were weaned onto their parents' diet, and 50 males and 50 females from each group were randomly selected to constitute the second generation (F1). Thus, the pups were exposed to the test item in utero, during lactation via the dam's milk, and for the remainder of their life. The two generations remained on test for 127 (F1) and 142(F0) weeks.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE: A limited water balance study was undertaken with the male and female F1 generation animals when they were 20 months of age. These consisted of determining the volume of water consumed and the volume of urine during a 24h period, by each of 10 males and 10 females from the following groups: control, 250 mg/kg o-TS and 250 mg/kg o-TS + 1% NH4Cl in water

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 0.1 - 0.125 ml were obtained from the tail aorta at 0, 4.5, 21, 44.5, 57, 65, 69, 96 and 98 weeks for F0; and at 4.5, 18.5, 32.5 and 45.5 weeks of age.
- Anaesthetic used for blood collection: Yes (ether)
- How many animals: 10 per group
- Parameters checked: erythrocyte enumeration (RBC), hematocrit (HTC), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentratoin (MCHC), total leukocyte enumeration (WBC), differential leukocyte count including absolute values, and occasionally hematopoietic organs (bone marrow, lymph node, and spleen imprints) were examined in cases where hematologic involvement was observed in moribund animals.

CLINICAL CHEMISTRY: No data

URINALYSIS: Yes
- Time schedule for collection of urine: at 6 months intervals, at 9 AM and 2 PM (groups: for control, 250 mg/kg o-TS and 250 mg/kg o-TS + 1% NH4Cl in water)
- Metabolism cages used for collection of urine: No
- Parameters: pH, turbidity, Bililabstix determination (for presence of glucose, ketones, bilirubin, protein and blood), presence of sediment or exfoliated epithelial cells (microscopical examination), presence of parasites (Trichosomoides crassicauda).

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (all animals). Animals were anesthetized with ether and exsanguinated from the abdominal aorta.
- If the urinary bladder contained urine, it was drained with a syringe. If not, physiological saline (0.2 ml) was injected into the bladder and a sample was drawn out, to examine for bladder parasites, sediment and bladder epithelial cells. The bladder was then filled with up to 0.2 ml of 10% neutral buffered formalin prior to being placed in a container of buffered formaling along with all of the other organs, tissues, and tumors. On the next working day, all organs and tumours were opened, examined grossly, and prepared for histological examination.

HISTOPATHOLOGY: Yes. Representative portions from all organs and all grossly abnormal areas of dermal, supportive or skeletal tissues were embedded in paraffin and sectioned at a thickness of 4 µm, or directly by cryostat when appropriate. The tissues were stained with hemalum-phloxine-saffron (HPS, unless a special stain was indicated). In addition, slides of the bladder tumors were reviewed by a panel of pathologists not associated with the design/conduct of the study.
Statistics:
Unless otherwise noted, the observed results for each test diet were compared to those for the control diet separately for each sex and time of observation, limiting the overall significance to 5% for each sex and time of observation, using the Bonferroni inequality. t-tests were used for continuous data and Fisher's test for quantal data.
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
The F0 generation animals were kept on test for 142 weeks, at which time less than 3 % of the initial animals were alive. The F1 portion of the study was terminated after 127 weeks; when approximately 20 % of the initial animals were still alive. The time-to-death was normally distributed in the F0 generation and was not affected by treatment. In the F1 generation, no significant increase in the probability of dying by time t as a result of exposure to o -TSA was observed for either sex.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The growth curves for the F0 and F1 males and females receiving o-TSA at 250 mg/kg or 250 mg/kg with 1% NH4Cl in the drinking water were significantly different (p < 0.05) from control animals.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Animals consuming o-TSA at 250 mg/kg or 250 mg/kg with 1% NH4Cl in the drinking water consumed less feed.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
For the seven hematological parameters (number of erythrocytes, hematocrit, hemoglobin, mean corpuscular volume, total number of leukocytes, neutrophils and lymphocytes), there were no patterns suggesting an effect due to treatment, sex, generation, or variable measured.
Clinical biochemistry findings:
not examined
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistical analysis of urinary pH values for male rats during the 18th month on test revealed that only the urine from the group of males receiving o-TSA at 250 mg/kg with NH4Cl in the drinking water significantly different when compared to males in other treatment groups; it was more acidic. The effect of dietary treatment upon urinary pH values of female rats was not as extensively studied except for control where no statistically significant differences were observed.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
At necropsy, the most common lesions found for both F0 and F1 animals were pituitary and subcutaneous tumors. Grossly visible bladder stones were observed in 3 animals during the study. Microscopic examination revealed the following: epithelial hyperplasia in an F0 control animals, diffuse papillomatosis involving most of the bladder wall in an F0 animal receiving 2.5 mg/kg o-TS, no pathological changes in an F1 animal receiving 2.5 mg/kg o-TS. 3 F0 females from the control, 2.5 mg/kg o-TS and 250 mg/kg o-TS groups had grossly visible kidney stones, but none had urinary bladder tumors.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
See table 2 below.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant increased in the incidence of tumours (see table below). The bladder tumors were found at the apex or funds of the bladder, except for one F1 female from the 2.5 mg/kg group which had a benign papilloma arising from the base of the bladder. They arose from a narrow pedicle which measured approximately 1 mm in diameter and 2 mm in length. The benign tumors were primarily soft, papillary, edematous, masses which were not particularly hyperemic or hemorrhagic. The benign papillomata in F0 animals consisted primarily of somewhat edematous, vascular, stroma covered by a layer of transitional epithelium that appeared normal while those in F1 animals were covered by a thick hyperplastic epithelium.
The animals were free of urinary bladder parasites. There were no treatment-related effects associated with longevity. Urinary bladder tumours, all of which were benign, were observed in one male each of the 0, 2.5 and 250 mg/kg b.w. group and in one female of the 2.5 mg/kg b.w. group in the first generation, and 2 females of the 2.5 mg/kg b.w. group in the second generation. However, the incidence was neither statistically significant nor dose-related. No other dose-related tumours were observed in any groups.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No significant differences (p > 0.05) from the control group were observed for any of the reproductive parameters. However, there was a statistically significant decrease
in the litter size of the 250 mg o-TS/kg group. When the average pup body weight at 4 days postpartum was adjusted for litter size, there was a significantly lower average body weight at 4 days postpartum in both groups receiving 250 mg/kg o-TS. No other treatment-related effect was evident.
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
no

Table 1.Incidence of bladder tumors for rats fed diets containing o-TS:

F0 generation

F1 generation

o-TS

Benign

Malignant

Benign

Malignant

Males

Control

1

0

0

0

2.5 mg/kg

1

0

0

0

25 mg/kg

0

0

0

0

250 mg/kg

1

0

0

0

250 mg/kg + 1%NH4Cl

0

0

0

0

Females

Control

0

0

0

0

2.5 mg/kg

1

0

2

0

25 mg/kg

0

0

0

0

250 mg/kg

0

0

0

0

250 mg/kg + 1%NH4Cl

0

0

0

0

 

Table 2. Non-neoplastic lesion with significant dose response* in F0 male rats 

Dose (mg/kg)

0

2.5

25

250

250 + NH4Cl

No.of Animals

49

49

50

50

39

Lung

-Chronic respiratory disease-slight

13

17

26

20

28ab

Liver

-Peliosis

4

9

18

13

8a

Non-neoplastic lesion with significant dose response in F0 female rats

Dose (mg/kg)

0

2.5

25

250

250 + NH4Cl

No.of Animals

50

50

50

50

38

Spleen

-Dense hematosiderosis

16

12

16

26

13

Kidneys

-Pelvic subepithelial telangiectasia

1

3

2

9

2

Non-neoplastic lesion with significant dose response in F1 male rats 

Dose (mg/kg) 

0

2.5

25

250

250 + NH4Cl

 No.of Animals 

50

50

50

50

49

 Liver 

 

 

 

 

 

 -Peliosis 

10

9

5

20

14

 Spleen 

 

 

 

 

 

 -Dense hematosiderosis 

5

2

7

13

 17a 

 Pancreas 

 

 

 

 

 

 -Focal chronic pancreatitis-slight 

19

18

16

27

22

Non-neoplastic lesion with significant dose response in F1 female rats

Dose (mg/kg) 

0

2.5

25

250

250 + NH4Cl

 No.of Animals 

50

50

50

50

50

 Liver 

 

 

 

 

 

 -Centrilobular basophil chromogenesis

-

1

1

7

13a

 -Peliosis 

1

3

9

10

 6a 

 Spleen 

 

 

 

 

 

 -Dense hematosiderosis 

4

18

7

20

 25a 

*: Bartholomew's test; a: Significantly different from control (p<0.05); b: Significantly different from 250 mg o-TSA group (p<0.05)

 

Conclusions:
The test item was not carcinogenic to rats under test conditions.
Executive summary:

In a two generation lifetime feeding study (no TG, no GLP), SD rats (32 days old) were given o-TS in the diet at 0 (control), 2.5, 25, 250 mg/kg bw/day (50 animals/sex/group) or 250 mg/kg bw/day with 1% NH4Cl in the drinking water (40 males and 38 females). Rats were exposed to this chemical from 90 days before mating in the first generation (for 142 weeks) and after weaning in the second generation (for 127 weeks). The animals were free of urinary bladder parasites. There were no treatment-related effects associated with longevity. Urinary bladder tumours, all of which were benign, were observed in one male each of the 0, 2.5 and 250 mg/kg bw group and in one female of the 2.5 mg/kg bw group in the first generation, and 2 females of the 2.5 mg/kg b.w. group in the second generation. However, the incidence was neither statistically significant nor dose-related. No other dose-related tumours were observed in any groups. Based on the available data, the test item was not carcinogenic to rats under test conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
chronic
Species:
rat

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the test item is not classified for carcinogenicity according to CLP, Regulation (EC) No. 1272/2008.

Additional information