Registration Dossier

Administrative data

Description of key information

The repeated toxicity of polysulfides, di-tert-butyl was evaluated following daily oral administration (gavage) to rats in OECD 407 and 421 studies. The dose-related changes induced among hematology parameters were indicative of a slight and regenerative anemia and correlated at pathology with extramedullary hemopoiesis in the spleen. At dose levels >= 150 mg/kg/day, the increased incidence and/or severity of the changes observed at pathology and among hematology parameters, was considered to be adverse. Based on the weight of evidence, the No Observed Adverse Effect Level (NOAEL) was established at 100 mg/kg/day.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guideline; adequate coherence between data, comments and conclusions.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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: males were 10 weeks old and females were 9 weeks old
- Weight at study initiation: males: mean body weight of 391 g (range: 357 g to 416 g); females: mean body weight of 215 g (range: 191 g to 246 g)
- Housing: the animals were housed individually, except during pairing and with the litter after parturition, in Individual Ventilated Cages (IVC)
(polysulfone 900 cm2, Tecniplast) containing sawdust.
- 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: 6 days

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

IN-LIFE DATES: From: 28 April 2010 To: 20 June 2010
Route of administration:
oral: gavage
Vehicle:
other: CMC (carboxymethyl cellulose) (0.5% aqueous CMC)
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
and until the obtention of satisfactory homogenization

VEHICLE
- Concentration in vehicle: 5, 15 and 30 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:
The GC-FID analytical method for the determination of Di-tert-butyl polysulfides (TPS 44) in dosage form samples was provided by the Sponsor
and this method was validated at CIT prior to dosage form analysis.

The dosage forms were within ± 10% of nominal values except for the 25 mg/kg/day dosage form on day 3 (within +-10% at other samplings).
Duration of treatment / exposure:
The dosage forms were administered daily according to the following schedule:
in the males:
- 2 weeks before pairing,
- during the pairing period (2 weeks),
- until sacrifice (at least 5 weeks in total).

in the females:
- 2 weeks before pairing,
- during the mating period (2 weeks),
- during gestation,
- during lactation until day 4 post-partum inclusive,
- until sacrifice for non-pregnant females.

Day 1 corresponds to the first day of the treatment period.
Actual durations: 40 days (males), 40-53 days (females).
Frequency of treatment:
Once daily.
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected following the results of a previous 4-week toxicity study in the rat (CIT/Study No. 36320 TSR). In this study, lower body weight gains were recorded in males and females treated at 300 mg/kg/day and in females treated at 100 mg/kg/day but there were no effects on food consumption. Regenerative anemia was observed in males and females treated at 300 mg/kg/day and in males treated at 100 mg/kg/day and some blood biochemistry parameters were affected, primarily in males treated at 300 mg/kg/day. Due to the minimal effects observed at 100 mg/kg/day in the 4-week study, the high dose-level for the present study is slightly higher in order to ensure treatment-related effects.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day.

BODY WEIGHT: Yes
- Time schedule for examinations: males: first day of treatment (day 1), then once a week until sacrifice. females: first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 post-coitum and days 1 and 5 post-partum.

FOOD CONSUMPTION:
- Food consumption for each animal determined: male: once a week, over a 7 day period, from the first day of treatment until the start of the
pairing period. female: once a week, over a 7 day period, from the first day of treatment until the start of pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 post-coitum and during lactation for interval days 1-5 post-partum.
During the pairing period, the food consumption was measured for neither males nor females.

WATER CONSUMPTION: No

LABORATORY INVESTIGATIONS ON PARENTAL ANIMALS
- Blood collection:
Prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours.
2- Hematology
The following parameters were determined from all males on the day of sacrifice: Erythrocytes (RBC), Hemoglobin (HB), Mean cell volume (MCV), Packed cell volume (PCV), Mean cell hemoglobin concentration (MCHC), Mean cell hemoglobin (MCH), Thrombocytes (PLT), Leucocytes (WBC), Differential white cell count with cell morphology, reticulocyte count and Prothrombin time (PT).
- Blood biochemistry
The following parameters were determined from all males on the day of sacrifice: Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (I.PHOS), Glucose (GLUC), Urea (UREA), Creatinine (CREAT), Total bilirubin (TOT.BIL.), Total proteins (PROT), Albumin (ALB), Albumin/globulin ratio (A/G), Total cholesterol (CHOL), Triglycerides (TRIG), Alkaline phosphatase (ALP), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT)
Sacrifice and pathology:
SACRIFICE
- Male, female animals: All surviving animals on completion of the treatment period

GROSS NECROPSY
- Gross necropsy consisted of external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the
thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table below were prepared for microscopic examination and weighed, respectively.
Statistics:
Data are compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).

PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01) .
Clinical signs:
no effects observed
Description (incidence and severity):
All clinical signs were those that are regularly observed in laboratory rats and no dose-relationship was apparent.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Male U27597 (75 mg/kg/day) was sacrificed on day 17 of dosing (after mating) because of aggressive behavior. This was considered to be unrelated to the test item.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
(Table 1)
Both males and females treated at 150 mg/kg/day gained less body weight than the other groups during the first week of dosing (-29% for males
and -32% for females when compared with controls, not statistically significant). In the second week of dosing, the males gained slightly more
weight than the controls while females continued to gain less weight. These initial lower body weight gains were recouped during the rest of the
study and had no impact on the final body weights.
There were no effects of treatment with the test item at 25 or 75 mg/kg/day.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males treated at 150 mg/kg/day consumed a statistically significantly lower quantity of food during the first week of treatment when compare
with the controls (28 g/animal/day vs. 32 g/animal/day, p < 0.01). Mean food consumption was similar to that of the controls during the second
week of treatment. There were no effects on mean female food consumption at this dose level.
There were no effects of treatment with the test item at 25 or 75 mg/kg/day.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
(Table 2)
Lower red blood cell parameters (erythrocyte count, hemoglobin level, and hematocrit) were observed at all dose-levels. Higher mean cell volume and mean cell hemoglobin level were observed at 75 and 150 mg/kg/day. These changes were associated with higher reticulocyte count (2.05-fold at 75 mg/kg/day and 2.4-fold at 150 mg/kg/day) and were indicative of regenerative anemia, which has been shown to be reversible at the dose level of 300 mg/kg/day in a concurrent 28-day oral toxicity study with a 2-week treatment-free period (CIT study no. 36320 TSR).
In addition, mean platelet count was increased at 150 mg/kg/day when compared with the control mean value and was considered to be related to the anemia.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
(Table 3)
At 150 mg/kg/day, calcium concentration was statistically significantly increased (+3.7%), associated with statistically significantly increased protein (+7%) and albumin (+11%) concentration, and chloride (-2%) and glucose (-14%) concentrations were statistically significantly decreased. Changes in calcium, protein, albumin, and glucose levels are consistent with the reversible changes observed in the 28-day toxicity study at the dose level of 300 mg/kg/day.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
(Table 4)
The mean absolute and relative weights of the liver were greater in males given 75 or 150 mg/kg/day reaching a statistically significant value (p<0.01). These changes were considered to be due to treatment.
The mean absolute and relative weights of the spleen were statistically significant greater in both sexes at 75 and 150 mg/kg/day. These changes were considered to be due to treatment.
The mean absolute and relative weights of the kidneys were statistically significant greater in males at 75 or 150 mg/kg/day (p<0.01). In females given 75 or 150 mg/kg/day, the mean relative weights were statistically significantly greater (p<0.05)..
Other changes in the mean organ weights, including the statistically significant greater weight of epididymides in males given 75 mg/kg/day were considered to be without any relationship with the test item.
At the end of the treatment-free period of the 28-day toxicity study, 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.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Enlarged spleen in 0/4 and 2/9 male/female animals, out of 10 per sex, at 75 and 150 mg/kg/day, resp.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
(Tables 5 to 7)
Males and females treated at 150 mg/kg/day were examined, microscopic non adverse changes were seen in the liver an spleen of males and females and in the kidneys of males.
In the liver, centrilobular hypertrophy, extending to midzonal area was seen in males and females given 150 mg/kg/day with a greater severity in males than in females. This was associated with hemopoiesis and brown pigment in Kupffer cells (see incidence table below). In males, periportal microvacuolation was seen with a greater incidence in treated animals than in controls. In the absence of associated degenerative changes, these changes were considered to be non adverse.

In the spleen congestion and hemopoiesis were seen with a greater severity in treated animals than in controls as showed in the following text table. These changes were associated with a greater severity of pigmented macrophages (consistent with hemosiderin).
Decreased marginal zone was observed in treated animals only
Increased congestion and hemopoiesis correlated with the greater weight of the spleen and enlargement seen at necropsy. Hemopoiesis correlated with changes in hematological parameters seen clinically

In kidneys of males given 150 mg/kg/day, hyaline droplets were seen in the epithelial cells of tubules with a greater incidence and severity than in controls. This was associated with increased incidence and severity of tubular basophilia and tubular dilatation.
Accumulation of hyaline droplets correlated with the greater weight of kidney noted at necropsy in males. This change is consistent with accumulation of a specific male protein called a2µ-globulin.

In the kidneys, males had increased incidence of hyaline droplets in epithelial cells of the tubules, consistent with the increased kidney weight, and increased incidence and severity of tubular basophilia and dilatation. These are considered to be related to accumulation of the male rat specific protein, a2µ-globulin.This overload was associated with tubular basophilia and in two animals with eosinophilic casts (minimal). As this change is specific to male rats, this was considered not to be adverse and not relevant in man (Histopathology of preclinical studies, P. Greaves, 2000 Elsevier Science B.V.; Handbook of Toxicologic Pathology, Haschek W. M and Rousseaux C.G, 1991, Academic Press, Inc. )

Careful examination of testes and ovaries did not show any treatment-related changes in animals given 150 mg/kg/day.
Dose descriptor:
NOAEL
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Dose descriptor:
LOAEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
haematopoietic
Organ:
blood
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Mean body weights and body weight gains (in g)

Sex

Male

Female

Dose-level (mg/kg/day)

0

25

75

150

0

25

75

150

Pre-mating

 

 

 

 

 

 

 

 

Day 1

387

393

393

392

215

215

213

216

Day 15

457

461

459

453

252

249

243

242

Day 36

501

505

518

504

/

/

/

/

Days 1-8

+41

+38

+40

+29

+19

+19

+15

+13

Days 1-15

+70

+67

+67

+61

+37

+34

+30

+26

Days 1-36

+113

+111

+126

+112

/

/

/

/

/: not applicable,p.c.:post-coitum,p.p.:post-partum.

Table 2: Relevant hematology parameters

Dose-level (mg/kg/day)

0

25

75

150

Red blood cell count (T/L)

9.01

8.17**

7.62**

7.13**

 

 (-9%)

(-15%)

(-21%)

Hemoglobin (g/dL)

15.6

14.4**

13.9**

13.2**

 

(-8%) 

(-11%)

(-15%)

Hematocrit (L/L)

0.47

0.43**

0.43**

0.42**

 

 (-9%)

(-9%)

(-11%)

Mean cell volume (fL)

51.8

53.0

56.2**

58.6**

 

 (+2%)

(+8%)

(+13%)

Mean cell hemoglobin (pg)

17.3

17.6

18.2*

18.5**

 

(+2%) 

(+5%)

(+7%)

Platelets (G/L)

1303

1212

1383

1513*

 

 (-7%)

(+6%)

(+16%)

Reticulocytes (%)

2.21

2.69

4.55**

5.33**

 

 (+22%)

(x2.1)

(x2.4)

White blood cell count (G/L)

11.99

12.90

16.91*(+41%)

15.16

 

 (+8%)

(+26%)

Neutrophils (G/L)

1.81

2.20

2.85*

2.47

 

 (+22%)

(+57%)

(+36%)

Statistically significant *: p<0.05, **: p<0.01.

In brackets, differences from controls.

Table 3: Relevant blood biochemistry parameters 

Dose-level (mg/kg/day)

0

25

75

150

Chloride (mmol/L)

102.9

102.3

101.8

100.7**

 

 

(-1%)

(-1%)

(-2%)

Calcium (mmol/L)

2.64

2.60

2.67

2.74**

 

 

(-2%)

(+1%)

(+4%)

Glucose (mmol/L)

7.14

7.36

6.49

6.16*

 

 

(+3%)

(-9%)

(-14%)

Protein (g/L)

65

65

66

70**

 

 

(0)

(+2%)

(+8%)

Albumin (g/L)

36

36

38

40**

 

 

(0)

(+6%)

(+11%)

Albumin/globulin ratio

1.27

1.28

1.32

1.35

 

 

(+1%)

(+4%)

(+6%)

Statistically significant *: p<0.05, **: p<0.01.

In brackets, differences from controls.

Conclusions:
In conclusion, the test item, Di-tert-butyl polysulfides (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 regenerative anemia and correlated at pathology with extramedullary hemopoiesis in the spleen. At 150 mg/kg/day, the increased incidence and/or severity of the changes observed at pathology and among hematology parameters, was considered to be adverse. At 25 or 75 mg/kg/day, none of these changes were considered to be adverse, taking into consideration their limited amplitude, the absence of associated degenerative changes and/or the reversibility observed at the dose level of 300 mg/kg/day in a concurrent 28-day oral toxicity study.
Executive summary:

In a reproduction/developmental screening test performed according to OECD 421 guideline and GLP, three groups of 10 male and 10 female Sprague-Dawley rats received the test item, Di-tert-butyl polysulfides (TPS 44), daily, by oral (gavage) administration, 2 weeks before mating and through mating and, for the females, through gestation until day 4 post-partum.The dose-levels were 25, 75 or 150 mg/kg/day. Another group of 10 males and 10 females received the vehicle, 0.5% aqueous carboxymethylcellulose in purified water, alone, under the same experimental conditions and acted as a control group. The dosing volume was 5 mL/kg/day. Clinical signs and mortality were checked at least once daily during the treatment period. Body weight and food consumption were recorded weekly until mating. Blood samples were taken from all males at the end of the treatment period for analysis of hematology and blood biochemistry parameters. The males were sacrificed after completion of the mating period. The body, epididymides, testes, liver, kidneys and spleen were weighed and a complete macroscopic post-mortem examination was performed. A microscopic examination was performed on the epididymides, testes, liver, kidneys and spleen of the control and high-dose groups and on all macroscopic lesions of all males. The dams were sacrificed on day 5 post-partum. The body, liver, kidneys and spleen were weighed and a complete macroscopic examination was performed. A microscopic examination was performed on the ovaries, liver, kidneys and spleen of the control and high-dose groups and on all macroscopic lesions of all females.

There were no test item treatment-related unscheduled mortalities and clinical signs. Both males and females treated at 150 mg/kg/day gained less body weight during the first week of treatment, associated with statistically significantly lower mean food consumption of the males, but this was recouped during the rest of the study and had no effect on final mean body weights.

There were no effects at 25 or 75 mg/kg/day. Lower red blood cell parameters (erythrocyte count, hemoglobin level, and hematocrit) were observed at all dose-levels. Higher mean cell volume and mean cell hemoglobin level were observed at 75 and 150 mg/kg/day. These changes were associated with higher reticulocyte count (2.05-fold at 75 mg/kg/day and 2.4-fold at 150 mg/kg/day) and were indicative of regenerative anemia, which has been shown to be reversible at the dose level of 300 mg/kg/day in a concurrent 28-day oral toxicity study with a 2-week treatment-free period (Petitpretz, 2010). At 150 mg/kg/day, calcium concentration was statistically significantly increased (+3.7%), associated with statistically significantly increased protein (+7%) and albumin (+11%) concentration, and chloride (-2%) and glucose (-14%) concentrations were statistically significantly decreased. Changes in calcium, protein, albumin, and glucose levels are consistent with the reversible changes observed in the 28-day toxicity study at the dose level of 300 mg/kg/day. Mean spleen weights were statistically significantly increased in males and females treated at 75 or 150 mg/kg/day. In addition, mean liver and kidney weights were statistically significantly increased in males treated at 75 or 150 mg/kg/day. At the end of the treatment-free period of the 28-day toxicity study, 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. Enlarged spleen was observed in males and females treated at 150 mg/kg/day (2/10 and 9/10, respectively) and also in 4/10 females treated at 75 mg/kg/day. Males and females treated at 150 mg/kg/day were examined microscopic non adverse changes were seen in the liver an spleen of males and females and in the kidneys of males. In the liver, centrilobular hypertrophy was observed (males being more affected than females) and was associated with hemopoiesis and brown pigment in Kuppfer cells. Males also had periportal microvacuolation. In the spleen, increased severity of hemopoiesis, pigmented macrophages and congestion were observed in males and females, hemopoiesis and congestion correlating with the hematological findings (anemia) and the increased spleen weight. Males and females both had increased incidences of decreased marginal zone. Six females treated at 75 mg/kg/day were examined (two because they were not pregnant and four because they had enlarged spleen) and all had hemopoiesis. In the kidneys, males had increased incidence of hyaline droplets in epithelial cells of the tubules, consistent with the increased kidney weight, and increased incidence and severity of tubular basophilia and dilatation. These are considered to be related to accumulation of the male rat-specific protein,a2-µ-globulin. All these findings are consistent with the microscopic findings of the 28-day toxicity study, which were partial reversibility at the end of the 2-week treatment-free period.

In conclusion, di-tert-butyl polysulfides (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 regenerative anemia and correlated at pathology with extramedullary hemopoiesis in the spleen. At 150 mg/kg/day, the increased incidence and/or severity of the changes observed at pathology and among hematology parameters, was considered to be adverse. At 25 or 75 mg/kg/day, none of these changes were considered to be adverse, taking into consideration their limited amplitude, the absence of associated degenerative changes and/or the reversibility observed at the dose level of 300 mg/kg/day in a concurrent 28-day oral toxicity study. 

Therefore, based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 75 mg/kg/day based on the significant regenerative anemia observed at 150 mg/kg/day.

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
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
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.
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.
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.
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
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.
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
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

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.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Experimental exposure time per week (hours/week):
7
Species:
rat
System:
haematopoietic
Organ:
blood
spleen

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD 407 study

The potential toxicity of polysulfides, di-tert-butyl was evaluated following daily oral administration (gavage) to rats for 4 weeks (Petitpretz, 2010). On completion of the treatment period, designated animals were held for a 2-week treatment-free (recovery) period in order to evaluate the reversibility of any findings. The study was performed according to OECD guideline no. 407 and in compliance with the principles of Good Laboratory Practice Regulations. One group of ten male and ten female Sprague-Dawley rats received polysulfides, di-tert-butyl daily by gavage at dose-level of 300 mg/kg/day for 4 weeks. Two other groups of five males and five females received polysulfides, di-tert-butyl 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. At the end of the treatment period, the animals were killed, except for the first five animals per sex from the control and high-dose groups, which were kept for a 2-week recovery period. 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 recovery period, the animals were sacrificed and submitted for a full macroscopic post-mortem examination. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on designated tissues from control and high-dose groups and on all macroscopic lesions from low- and intermediate-dose animals.

At 300 mg/kg/day, the only treatment-related clinical sign was ptyalism, which was 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 treated animals during the recovery 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 recovery 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 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 recovery 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 recovery 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. Microscopic non adverse changes were seen in the kidneys of males, male rat-specific hyaline dropletsconsistent with a2µ-globulin overload, a mechanism not relevant to humansandbrown pigment in cortex, in the liver of males and females,hepatocellular hypertrophy considered as an adaptation to a metabolic over-load andbrown pigment in Kupffer cells and hemopoiesis considered as an adaptation to the regenerative anemia, in the spleen of males and females, hemopoiesis, brown pigment and congestion, in the bone marrow of males and females, increased erythroid and/or myeloid cell numbers and forestomach of males and females, hyperkeratosis, acanthosis and inflammation. At the end of the recovery 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 in the kidneys of males (male rat-specific 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 polysulfides, di-tert-butyl 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. Polysulfides, di-tert-butyl 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 with extramedullary hemopoiesis in the spleen and increased erythroid cells in the bone marrow and hemopoiesis in Kupffer cells in the liver. 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.

OECD 421 study

In a reproduction/developmental screening test performed according to OECD 421 guideline and GLP (Allen, 2010), three groups of 10 male and 10 female Sprague-Dawley rats received polysulfides, ditert-butyl, daily, by oral (gavage) administration, 2 weeks before mating and through mating and, for the females, through gestation until day 4 post-partum.The dose-levels were 25, 75 or 150 mg/kg/day. Another group of 10 males and 10 females received the vehicle, 0.5% aqueous carboxymethylcellulose in purified water, alone, under the same experimental conditions and acted as a control group. The dosing volume was 5 mL/kg/day. Clinical signs and mortality were checked at least once daily during the treatment period. Body weight and food consumption were recorded weekly until mating. Blood samples were taken from all males at the end of the treatment period for analysis of hematology and blood biochemistry parameters. The males were sacrificed after completion of the mating period. The body, epididymides, testes, liver, kidneys and spleen were weighed and a complete macroscopic post-mortem examination was performed. A microscopic examination was performed on the epididymides, testes, liver, kidneys and spleen of the control and high-dose groups and on all macroscopic lesions of all males. The dams were sacrificed on day 5 post-partum. The body, liver, kidneys and spleen were weighed and a complete macroscopic examination was performed. A microscopic examination was performed on the ovaries, liver, kidneys and spleen of the control and high-dose groups and on all macroscopic lesions of all females.

There were no test item treatment-related unscheduled mortalities and clinical signs. Both males and females treated at 150 mg/kg/day gained less body weight during the first week of treatment, associated with statistically significantly lower mean food consumption of the males, but this was recouped during the rest of the study and had no effect on final mean body weights. There were no effects at 25 or 75 mg/kg/day.

Lower red blood cell parameters (erythrocyte count, hemoglobin level, and hematocrit) were observed at all dose-levels. Higher mean cell volume and mean cell hemoglobin level were observed at 75 and 150 mg/kg/day. These changes were associated with higher reticulocyte count (2.05-fold at 75 mg/kg/day and 2.4-fold at 150 mg/kg/day) and were indicative of regenerative anemia, which has been shown to be reversible at the dose level of 300 mg/kg/day in a concurrent 28-day oral toxicity study with a 2-week treatment-free period (Petitpretz, 2010). At 150 mg/kg/day, calcium concentration was statistically significantly increased (+3.7%), associated with statistically significantly increased protein (+7%) and albumin (+11%) concentration, and chloride (-2%) and glucose (-14%) concentrations were statistically significantly decreased. Changes in calcium, protein, albumin, and glucose levels are consistent with the reversible changes observed in the 28-day toxicity study at the dose level of 300 mg/kg/day. Mean spleen weights were statistically significantly increased in males and females treated at 75 or 150 mg/kg/day. In addition, mean liver and kidney weights were statistically significantly increased in males treated at 75 or 150 mg/kg/day. At the end of the treatment-free period of the 28-day toxicity study, 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. Enlarged spleen was observed in males and females treated at 150 mg/kg/day (2/10 and 9/10, respectively) and also in 4/10 females treated at 75 mg/kg/day. Males and females treated at 150 mg/kg/day were examined microscopic non adverse changes were seen in the liver an spleen of males and females and in the kidneys of males. In the liver, centrilobular hypertrophy was observed (males being more affected than females) and was associated with hemopoiesis and brown pigment in Kuppfer cells. Males also had periportal microvacuolation. In the spleen, increased severity of hemopoiesis, pigmented macrophages and congestion were observed in males and females, hemopoiesis and congestion correlating with the hematological findings (anemia) and the increased spleen weight. Males and females both had increased incidences of decreased marginal zone. Six females treated at 75 mg/kg/day were examined (two because they were not pregnant and four because they had enlarged spleen) and all had hemopoiesis. In the kidneys, males had increased incidence of hyaline droplets in epithelial cells of the tubules, consistent with the increased kidney weight, and increased incidence and severity of tubular basophilia and dilatation. These are considered to be related to accumulation of the male rat-specific protein,a2-µ-globulin. All these findings are consistent with the microscopic findings of the 28-day toxicity study, which were partial reversibility at the end of the 2-week treatment-free period.

In conclusion, polysulfides, ditert-butyl was generally well-tolerated at all dose-levels tested. The dose-related changes induced among hematology parameters were indicative of a slight and regenerative anemia and correlated at pathology with extramedullary hemopoiesis in the spleen. At 150 mg/kg/day, the increased incidence and/or severity of the changes observed at pathology and among hematology parameters, was considered to be adverse. At 25 or 75 mg/kg/day, none of these changes were considered to be adverse, taking into consideration their limited amplitude, the absence of associated degenerative changes and/or the reversibility observed at the dose level of 300 mg/kg/day in a concurrent 28-day oral toxicity study. 

Therefore, based on the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) for parental toxicity was considered to be 75 mg/kg/day based on the significant regenerative anemia observed at 150 mg/kg/d.

Justification for classification or non-classification

According to the available data and criteria of Regulation (EC) No 1272-2008, no classification is warranted for repeated dose toxicity.