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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 February 2010 to 29 June 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
deviations to Study Plan but not to guideline
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
idem above
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Di-tert-butyl polysulphides (TPS 44)
- Physical state: light yellow liquid
- Analytical purity: 97.29%
- Purity test date: 27 January 2009
- Lot/batch No.: 27-jan-2009 on the analysis (27/01/2009 on the vial label)
- Expiration date of the lot/batch: January 2012
- Storage conditions of test material: at room temperature in a well-ventilated place.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Breeder: Charles River Laboratories France, l'Arbresle, France
- Age at study initiation: on the first day of treatment, the animals were approximately 6 weeks old
- Weight at study initiation: the males had a mean body weight of 198 g (range: 181 g to 210 g) and the females had a mean body weight of 161 g
(range: 143 g to 178 g)
- Fasting period before study: no
- Housing: the animals were housed in ones or twos in Individually Ventilated Cages (IVC) (polysulfone 900 cm2, Tecniplast) containing sawdust. The cages were placed in numerical order on the racks
- Diet (e.g. ad libitum): free access to SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: the animals were acclimated to the study conditions for a period of 7 days before the beginning of the treatment period.
A larger number of animals than necessary were acclimated to permit the selection and/or replacement of individuals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 50 +/- 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00).

IN-LIFE DATES: From: 11 February 2010 To: 29 June 2010.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
aqueous solution at 0.5%
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was administered as a suspension in the vehicle. The test item was mixed with the required
quantity of vehicle (under magnetic stirring until the obtention of a satisfactory homogenization) in order to achieve the concentrations of 6.6, 20
and 60 mg/mL.
The dosage forms were prepared daily by the CIT Pharmacy and delivered to the study room in brown flasks.

VEHICLE
- Concentration in vehicle: 6.6, 20 and 60 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- nature of analytical method: GC-FID analytial method
- test item concentrations: the test item concentration in samples of each control and test item dosage form prepared for use in weeks 1, 3 and 4 was measured. Deviations from the nominal concentration were within -3.1 to +11.4%, except on day 1 for groups 2 and 3 (+65.1% and -22.0%, resp.).
- homogeneity: in another study, duplicate samples were taken from three levels of the container (top, middle and bottom) on the day of preparation, at concetrations covering those used in present study.
- stability: no assessed, dosage form prepared extemporaneously each day of treatment.
Duration of treatment / exposure:
4 weeks followed by a 2-week treatment-free period.
Frequency of treatment:
Daily.
Doses / concentrationsopen allclose all
Dose / conc.:
33 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
For 33 and 100 mg/kg/day dose-levels: 5 males and 5 females per dose
For 300 mg/kg/day dose-level: 10 males and 10 females (incl. 5 rats/sex allocated to recovery period).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, based on the results of a 14 day preliminary toxicity study by the oral route in rats
CIT/Study No. 36319 TSR). The test item was administered to three groups of three male and three female Sprague-Dawley rats at dose-levels of 100, 300 or 1000 mg/kg/day under a dosage-volume of 5 mL/kg. A control group was given the vehicle under the same experimental conditions.
Animals given 1000 mg/kg/day were prematurely sacrificed on day 10 due to the spreading of the smell of the test item, which persisted despite the
actions taken (treatment under a specific air flow safety hood, housing in individually ventilated cages, etc). In this group, ptyalism was constantly
observed in 4/6 animals. Lower body weight was observed when compared to controls (-9 and -7% on day 4 in males and females respectively;
-10% in both sexes on day 7), together with lower food consumption (in males and females, respectively: -14 and -17% from day 4 to day 6; -34 and
-25% from day 7 to day 9). At pathology, enlarged, dark-colored spleen and whitish/yellowish deposit in the forestomach were observed in all
animals.
At 300 mg/kg/day, slightly lower body weight (-5% in comparison with controls) and enlarged spleen were observed.

- Rationale for animal assignment (if not random): computerized randomization procedure

- Post-exposure recovery period: not done in satellite groups but in main, high-dose and control groups, for 5 rats/sex/group, for 2 weeks without treatment..
Positive control:
Not applicable.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day during the acclimation period and at least twice a day during the treatment and treatment-free periods, including
weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the beginning of the treatment and treatment-free periods and the once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule for examinations: once before group allocation, on the first day of treatment, then once a week until the end of the study.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

HAEMATOLOGY+CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and treatment-free periods.

URINALYSIS: No.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the treatment period.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
A complete macroscopic post-mortem examination was performed on all study animals. This included examination of the external surfaces, all
orifices, the cranial cavity, the external surfaces of the brain, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and
the neck with its associated organs and tissues.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- all tissues listed in the Tissue Procedure Table for control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- all macroscopic lesions, kidneys, liver, spleen, bone marrow (sternum) and forestomach from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period,
- kidneys, liver, spleen, bone marrow (sternum) and forestomach from the control and high-dose animals sacrificed at the end of the treatment-free period.

ORGAN WEIGHTS: see table below

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was observed in 9/10 males and 9/10 females given 300 mg/kg/day and was the only test item-related clinical sign.
Mortality:
no mortality observed
Description (incidence):
No unscheduled deaths occurred during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
(Table 1)
At 300 mg/kg/day: lower body weight gain was observed at the end of the treatment period in both sexes (-14% in males; -25 % in females, when compared with controls). Even though the final body weight was still lower when compared to controls, higher body weight gain was observed in previously test item-treated animals during the treatment-free period.
At 100 mg/kg/day, lower body weight gain was observed in females during the treatment period (-10% when compared with controls).


Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
(Tables 2 and 3)
At 300 mg/kg/day: Lower red blood cell parameters (including erythrocyte count, hemoglobin level, mean cell hemoglobin concentration and packed cell volume) were observed, together with higher mean cell volume and mean cell hemoglobin level. These changes were associated with higher reticulocyte count (up to 3.6-fold in males and 5.2-fold in females) and were indicative of regenerative anemia. Slightly higher neutrophil counts were observed. At the end of the treatment-free period, normalization of the above parameters was observed but higher hemoglobin level and packed cell volume were observed in both sexes when compared to controls.
At 100 mg/kg/day, similar changes as in high-dose animals were observed, especially in males but with a lower severity.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
(Table 4)
At 300 mg/kg/day: higher potassium (+31%) and calcium (+5%) concentrations, higher protein and albumin levels (+11%) and lower phosphatase alkaline activity (-36%) were observed in males. A trend towards the same variations was observed in females, but to a lesser extent. These changes were not observed at the end of the treatment-free period.
At 100 mg/kg/day, similar changes as in high-dose animals were observed, especially in males but with a lower severity.

Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
(Tables 5 and 6)
At 300 mg/kg/day: Increased kidney, liver and spleen weights were noted at the end of the treatment period and correlated with macroscopic enlargement of these organs. At the end of the treatment-free period, kidney, liver and spleen weight increases were recorded in males and/or females previously given 300 mg/kg/day but with a lower severity when compared to those recorded at the end of treatment period, thus indicating partial reversibility.
At 100 mg/kg/day, increased kidney, liver and spleen weights were recorded at the end of the treatment period and correlated with macroscopic enlargement of the kidney and liver.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
See organ weight section.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(Tables 7 to 10)
. At the end of the treatment period
Treatment-related changes were seen in kidneys, liver, spleen, bone marrow and forestomach.
Kidneys (Table 7)
Slight to moderate hyaline droplets were seen in males given 33, 100 or 300 mg/kg/day test item (see table below). This correlated with increased kidney weights and gross finding (enlargement of kidneys). This could correlate with increased potassium concentration in males at 300 mg/kg/day. These densely eosinophilic globules were located in the cytoplasm of tubular cells of outer cortex and were considered to be consistent with alpha2µ-globulin overload. This is an adaptative change specific of male rats that is not relevant in man (Histopathology of preclinical studies, P. Greaves, 2000, Elsevier Science B.V.)
Minimal brown pigment was seen in the cortical tubular cells of two males given 300 mg/kg/day.
Slightly increased incidence and severity of tubular basophilia was recorded in males given 33 or 100 mg/kg/day and females given 300 mg/kg/day, although poorly dose-related in males. The relationship to test item was considered to be equivocal.
In the absence of degeneration of tubular cells and as the hyaline droplets are specific of male rats, these findings were considered not to be adverse.

Liver (Table 8)
Minimal to marked centrilobular to midzonal hepatocellular hypertrophy was recorded in males and females given 100 or 300 mg/kg/day, together with minimal erythroid hemopoiesis from 33 mg/kg/day and brown pigment in Kupffer cells at 300 mg/kg/day (see table below). The hepatocellular hypertrophy correlated with increased liver weights and gross findings (i.e. mainly enlargement of liver).
These findings could be considered as an adaptation to a metabolic over-load and a regenerative anemia and were considered not to be adverse in the absence of degenerative changes.

Spleen (Table 9)
Treatment-related minimal to moderate hemopoiesis was seen in males and/or females given 33, 100 or 300 mg/kg/day (see table below). This was seen in association with congestion and increased brown pigment (consistent with hemosiderin).
In addition, minimal to slight decreased marginal zone was recorded. However, in the absence of clinical change in the lymphocyte count, this finding was considered to be most probably related with congestion seen in these animals and consequently to be of no toxicological importance.
The hemopoiesis accompanied by congestion correlated with increased spleen weights and enlargement seen at macroscopic post-mortem examination.
This extra-medullary hemopoiesis correlated with increased reticulocyte and neutrophil counts observed in hematology.

Bone marrow (sternum)
Minimal to slight not adverse increased erythroid cell numbers sometimes accompanied by increased myeloid cell numbers (in males) was recorded in males and females given 100 or 300 mg/kg/day. This correlated with increased reticulocyte and neutrophil counts observed in hematology.

Forestomach (Table 10)
Minimal to marked hyperkeratosis was recorded in males and females given 100 or 300 mg/kg/day, sometimes in association with acanthosis and inflammation (see table below). The hyperkeratosis was characterized by increased thickness of stratum corneum. The acanthosis is the increase in thickness of the stratified squamous epithelium. The subacute inflammation was located in submucosa. In view of the slight to moderate severity and the low incidence of these changes, they were considered as non adverse.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effect
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
haematology
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
blood
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Table 1: Mean body weight/Mean body weight change (g)

Sex

Male

Female

Dose-level (mg/kg/day)

0

33

100

300

0

33

100

300

Treatment period

Days 1 to 8

+59

+61

+59

+51

+25

+25

+22

+17*

Days 8 to 15

+50

+50

+48

+49

+19

+20

+18

+16

Days 15 to 22

+39

+34

+36

+35

+18

+16

+13

+13

Days 22 to 28

+28

+23

+24

+16**

+12

+10

+13

+10

Days 1 to 28
% from controls

+176
-

+169
(-4)

+167
(-5)

+151*
(-14)

+73
-

+73
(0)

+66
(-10)

+55**
(-25)

Body weight on day 28
% from controls

375
-

367
(-2)

363
(-3)

350
(-7)

233
-

235
(+1)

228
(-2)

214*
(-8)

Treatment-free period

Days 28 to 42
% from controls

+44
-

-

-

+51
(+16)

+22
-

-

-

+24
(+9)

Body weight on day 42
% from controls

423
-

-

-

398
(-6)

257
-

-

-

232*
(-10)

Statistically significant from controls: *: p<0.05; **: p<0.01.

-: not applicable.

 

Table 2: Haematology (end of treatment)

Sex

Male

Female

Dose-level
(mg/kg/day)

0

33

100

300

0

33

100

300

RBC (T/L)

8.39

-

8.21

(-2)

7.16**

(-15)

6.13**

(-27)

7.80

-

7.18

(-8)

6.72

(-14)

5.55**

(-29)

Hb (g/dL)

15.9

-

15.0*

(-6)

13.8**

(-13)

13.2**

(-17)

15.0

-

13.6

(-9)

13.7

(-9)

12.1**

(-19)

PCV (L/L)

0.48

-

0.45

(-6)

0.43**

(-10)

0.42**

(-13)

0.43

-

0.40

(-7)

0.41

(-5)

0.38**

(-12)

MCV (fL)

56.7

-

55.3

(-2)

59.8

(+5)

68.8**

(+21)

55.2

-

56.3

(+2)

60.4*

(+9)

68.7**

(+24)

MCH (pg)

18.9

-

18.3

(-3)

19.4

(+3)

21.5**

(+14)

19.2

-

18.9

(-2)

20.4

(+6)

21.7*

(+13)

MCHC (g/dL)

33.4

-

33.0

(-1)

32.4*

(-3)

31.3**

(-6)

34.8

-

33.6

(-3)

33.8

(-3)

31.6**

(-9)

RTC (%)

2.67

-

3.39

(x1.3)

6.02*

(x2.3)

9.56**

(x3.6)

2.50

-

4.39

(x1.8)

5.00

(x2.0)

13.10**

(x5.2)

N (G/L)

1.48

-

1.71

(+16)

2.74**

(+85)

2.51**

(+70)

0.68

-

1.04

(+53)

1.24

(+82)

1.18

(+74)

Statistically significant from controls: *p<0.05; **p<0.01,

RBC: Red Blood Cells; Hb: Hemoglobin; PCV: Packed Cell Volume; MCV: Mean Cell Volume; MCH: Mean Cell Hemoglobin; MCHC: Mean Cell Hemoglobin Concentration; RTC: Reticulocytes; N: Neutrophils.

-: not applicable.

Table 3: (Haematology (recovery)

Sex

Male

Female

Dose-level (mg/kg/day)

0

300

0

300

RBC (T/L)

8.81

-

8.64

(-2)

8.09

-

7.51*

(-7)

Hb (g/dL)

15.6

-

16.7**

(+7)

14.9

-

15.7

(+5)

PCV (L/L)

0.45

-

0.50**

(+11)

0.42

-

0.45*

(+7)

MCV (fL)

51.2

-

57.7**

(+13)

52.0

-

60.0*

(+15)

MCH (pg)

17.7

-

19.3**

(+9)

18.4

-

20.9*

(+14)

MCHC (g/dL)

34.6

-

33.5**

(-3)

35.4

-

34.9

(-1)

RTC (%)

 

2.26

-

2.64

(+17)

2.09

-

1.56

(-25)

Statistically significant from controls: *p<0.05; **p<0.01,

RBC: Red Blood Cells; Hb: Hemoglobin; PCV: Packed Cell Volume; MCV: Mean Cell Volume; MCH: Mean Cell Hemoglobin; MCHC: Mean Cell Hemoglobin Concentration, RTC: Reticulocytes.

-: not applicable.

Table 4: Blood chemistry (end of treatment)

Sex

Male

Female

Dose-level
(mg/kg/day)

0

33

100

300

0

33

100

300

Potassium (mmol/L)

4.05

-

4.32

(+7)

4.22

(+4)

5.32**

(+31)

3.82

-

3.98

(+4)

4.04

(+6)

4.14

(+8)

Calcium (mmol/L)

2.67

-

2.73

(+2)

2.78*

(+4)

2.80**

(+5)

2.75

-

2.71

(-1)

2.73

(-1)

2.80

(+2)

Proteins (g/L)

63

-

63

(0)

65

(+3)

70**

(+11)

66

-

64

(-3)

64

(-3)

69

(+5)

Albumin (g/L)

38

-

37

(-3)

39

(+3)

42*

(+11)

39

-

38

(-3)

39

(0)

42

(+8)

ALP

(IU/L)

599

-

458*

(-24)

461*

(-23)

384**

(-36)

274

-

261

(-5)

297

(+8)

173

(-37)

Statistically significant from controls: *p<0.05; **p<0.01,

ALP: alkaline phosphatase activity.

Applicant's summary and conclusion

Conclusions:
Di tert butyl polysulfides (TPS 44), administered orally to male and female Sprague-Dawley rats by gavage for 4 weeks at dose-levels of 33, 100 or 300 mg/kg/day, was characterized by a NOAEL of 100 mg/kg/day and the only adverse effect was regenerative anemia and impaired weight gain.
Executive summary:

The potential toxicity of Di-tert-butyl polysulfides (TPS 44) was evaluated following daily oral administration (gavage) to rats for4 weeks. On completion of the treatment period, designated animals were held fora 2-week treatment-free period in order to evaluate the reversibility of any findings. The study was performed according to OECD(No. 407, 03 October 2008) and Commission Regulation (EC) (No. 440/2008, B.7, 30 May 2008)guidelines and in compliance with the principles of Good Laboratory Practice Regulations. One group of ten male and ten female Sprague-Dawley rats received Di-tert-butyl polysulfides (TPS 44) daily by gavage at dose-level of 300 mg/kg/day for 4 weeks. Two other groups of five males and five females received TPS 44 at 33 or 100 mg/kg/day under the same experimental conditions. A further group of ten males and ten females received the vehicle alone (0.5% carboxymethylcellulose in purified water) under the same experimental conditions, and acted as a control group.The dosage forms were administered under a constant dosage-volume of 5 mL/kg/day. At the end of the treatment period, the animals were sacrificed, except for the first five animals per sex from the control and high-dose groups, which were kept for a 2-week treatment-free period. Actual concentrations of TPS 44 in the dosage forms prepared for use in weeks 1 [days 1, 2 (groups 2 and 3 only) and 6], 3 and 4 were determined.The animals were checked daily for mortality and clinical signs. In addition, detailed clinical examinations were performed once a week. A Functional Observation Battery (FOB), including a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli,and motor activity, was performed at the end of the treatment period. Body weight was recorded once during the pre-treatment period, on the first day of treatment and then once a week until the end of the study. Food consumption was recorded once a week during the study. Hematology and blood biochemistry investigations were performed at the end of the treatment and treatment-free periods. At the end of the treatment or treatment-free period, the animals were sacrificed and submitted for a full macroscopicpost-mortemexamination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on designated tissues from control and high-dose groupsand on all macroscopic lesions from low- and intermediate-dose animals.

On each occasion, chemical analysis of the dosage forms demonstrated that the actual concentrations of the test item were close to the nominal concentrations (-3.1 to +11.4%); except on day 1 for groups 2 and 3 (+65.1% and -22%, respectively). At 300 mg/kg/day, the only TPS 44-related clinical sign was ptyalism, observed on several occasions in almost all animals (9/10 males; 9/10 females). Lower body weight gain was observed at the end of the treatment period in both sexes (-14% in males; -25 % in females, when compared with controls). Even though the final body weight was still lower when compared to controls, higher body weight gain was observed in previously test item-treated animals during the treatment-free period. Food consumption was unaffected throughout the study. Lower red blood cell parameters [including erythrocyte count, hemoglobin level (-17% in males and -19% in females), mean cell hemoglobin concentration and packed cell volume] were observed, together with higher mean cell volume and mean cell hemoglobin level. These changes were associated with higher reticulocyte count (3.6-fold in males and 5.2-fold in females) and were indicative of regenerative anemia. Slightly higher neutrophil counts were observed. At the end of the treatment-free period, normalization of the above parameters was observed buthigher hemoglobin level and packed cell volume were observed in both sexes when compared to controls. At blood biochemistry, higher potassium (+31%) and calcium (+5%) concentrations, higher protein and albumin levels (+11%) and lower phosphatase alkaline activity (-36%) were observed in males. A trend towards the same variations was observed in females, but to a lesser extent. These changes were not observed at the end of the treatment-free period. Increased kidney, liver and spleen weights were noted at the end of the treatment period and correlated with macroscopic enlargement of these organs. At the end of the treatment-free period, kidney, liver and spleen weight increases were recorded in males and/or femalespreviously given 300 mg/kg/day but with a lower severity when compared to those recorded at the end of treatment period, thus indicating partial reversibility. Microscopic non adverse changes were seen inthe kidneys of males (hyaline droplets and brown pigment in cortex), liver (hepatocellular hypertrophy, brown pigment in Kupffer cells and hemopoiesis), spleen (hemopoiesis, brown pigment and congestion), bone marrow (increased erythroid and/or myeloid cell numbers) and forestomach (hyperkeratosis, acanthosis and inflammation). At the end of the treatment-free period, partial reversibility of these changes was noted. 

At 100 mg/kg/day, lower body weight gain was observed in females during the treatment period (-10% when compared with controls). At hematology investigations, similar changes as in high-dose animals were observed, but with a lower severity (hemoglobin level: -13% in males and -9% in females). Increased kidney, liver and spleen weights were recorded at the end of the treatment period and correlated with macroscopic enlargement of the kidney and liver. Microscopic non adverse changes were seen inthe kidneys of males (hyaline droplets), liver (hepatocellular hypertrophy and hemopoiesis), spleen (hemopoiesis, brown pigment and congestion), bone marrow (increased erythroid and/or myeloid cell numbers) and forestomach (hyperkeratosis and acanthosis).

 At 33 mg/kg/day, no relevant clinical changes were observed. Microscopic non adverse changes were seen in the kidneys of males (hyaline droplets), in the liver of females (hemopoiesis) and in the spleen of females (hemopoiesis and brown pigment).

The toxicity of Di-tert-butyl polysulfides (TPS 44), was evaluated in Sprague-Dawley rats following daily oral administrations (gavage) for 4 weeks at dose-levels of 33, 100 or 300 mg/kg/day. TPS 44 was generally well-tolerated at all dose-levels tested. The dose-related changes induced among hematology parameters were indicative of a slight and reversible regenerative anemia and correlated at pathology with extramedullary hemopoiesis in the spleen and increased erythroid cells in the bone marrow. These latter findings were considered to be a compensatory effect, secondary to the test-item related effect on red blood cells. At 300 mg/kg/day, the lower body weight gain observed at the end of the treatment period in both sexes, in addition to the increased incidence and/or severity of the changes observed at pathology and among hematology parameters, was considered to be adverse. At 33 or 100 mg/kg/day, none of these changes were considered to be adverse, taking into consideration their limited amplitude, the reversibility observed at the dose level of 300 mg/kg/day and/or the absence of associated degenerative changes. Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) was established at 100 mg/kg/day.