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Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
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 for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system 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
histopathology: non-neoplastic
organ weights and organ / body weight ratios
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.

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
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No. 440/2008, Part B.3, 30 May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
polysulfides, di-tert-butyl
IUPAC Name:
polysulfides, di-tert-butyl
Constituent 2
Chemical structure
Reference substance name:
Polysulfides, di-tert-Bu
EC Number:
273-103-3
EC Name:
Polysulfides, di-tert-Bu
Cas Number:
68937-96-2
Molecular formula:
not applicable
IUPAC Name:
Polysulfides, di-tert-Bu
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 or test system 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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% aqueous CMC
Details on 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.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: each female was placed with the same male until mating occurred or 14 days had elapsed
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 post-coitum
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
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.
Details on study schedule:
- Age at mating of the mated animals in the study: 13 weeks
Doses / concentrationsopen allclose all
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.
Positive control:
None.

Examinations

Parental animals: Observations and examinations:
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)



Oestrous cyclicity (parental animals):
The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the mating period, until the
females were mated.
The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on day 5 post-partum.
Sperm parameters (parental animals):
see post-mortem investigations. No in vivo investigation.
Litter observations:
STANDARDISATION OF LITTERS: No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross
anomalies, weight gain, clinical signs, behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
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.
Postmortem examinations (offspring):
Pups found dead and pups sacrificed on day 5 post-partum were carefully examined externally for gross external abnormalities. No tissues were preserved.
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) .
Reproductive indices:
. pre-implantation loss:
Number of corpora lutea - Number of implantation sites
_____________________________________________ x 100
Number of corpora lutea

. post-implantation loss:
Number of implantation sites - Number of live pups
_____________________________________________ x 100
Number of implantations

. mating index:
Number of mated animals
_____________________ x 100
Number of paired animals

. fertility index:
Number of pregnant female partners
_______________________________ x 100
Number of mated pairs


. gestation index:
Number of females with live born pups
________________________________ x 100
Number of pregnant females
Offspring viability indices:
. live birth index:
Number of live born pups
_____________________ x 100
Number of delivered pups

. viability index on day 5 post-partum:
Number of surviving pups on day 5 post-partum
_______________________________________ x 100
Number of live born pups

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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):
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):
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).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
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
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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, µa2-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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Description (incidence and severity):
only post-mortem examinations
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
75 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
Remarks:
parental toxicity
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
Dose descriptor:
NOEL
Remarks:
reproductive performance (mating and fertility)
Effect level:
>= 150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
While the mean pup body weights on post-natal day 1 were only minimally lower than those of the controls for both male and female pups from the group treated at 150 mg/kg/day, both sexes had lower mean body weight gains and lower mean body weights on post-natal day 5. Mean body weight gain was 29% lower for the male pups and 21% lower for the female pups and mean body weights on post-natal day 5 were -12% and -11%, respectively.
There were no effects of treatment with the test item on mean pup body weights at 25 or 75 mg/kg/day.
Sexual maturation:
not examined
Description (incidence and severity):
sex ratio unaffected
Gross pathological findings:
no effects observed

Effect levels (F1)

open allclose all
Dose descriptor:
NOEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect
Dose descriptor:
LOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Applicant's summary and conclusion

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:

The objective of this study was to evaluate the potential toxic effects of the test item, Di-tert-butyl polysulfides (TPS 44), following daily oral administration (gavage) to male and female rats from before mating, through mating and, for the females, through gestation until day 4 post-partum. This study provides information on all aspects of reproduction and development. The study was conducted as a reproduction/developmental screening test 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, purity 97.29%), 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 and then at designated intervals throughout gestation and lactation. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5 post-partum. The total litter sizes and numbers of pups of each sex were recorded after birth. Pups were observed daily for clinical signs and pup body weights were recorded on days 1 and 5 post-partum. 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. Pups were sacrificed on post-natal day 5 and were carefully examined for gross external abnormalities. Found dead and prematurely sacrificed pups were also examined for gross external abnormalities.

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. There were no effects on mating, fertility or delivery at any dose-level. There were no increases in pup mortality in the test item-treated groups and there were no relevant clinical signs or gross abnormalities in the pups.

Mean pup body weight gain at 150 mg/kg/day was lower and the mean body weight on post-natal day 5 was also lower for both sexes when compared with the controls. 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, the No Observed Effect Level (NOEL) for reproductive performance (mating and fertility) was considered to be 150 mg/kg/day, and the NOEL for toxic effects on progeny was 75 mg/kg/day based on the slightly lower body weight gain of the pups at 150 mg/kg/day.