Di-tert-dodecyl disulphide

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 April 2016 - 22 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

03 October 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Di-tert-dodecyl disulphide

EC Number:

248-468-7

EC Name:

Di-tert-dodecyl disulphide

Cas Number:

27458-90-8

Molecular formula:

C22H46S2 to C24H50S2

IUPAC Name:

2-ethyl-5,6,6-trimethyl-1-[(3,5,6,6-tetramethylheptyl)disulfanyl]heptane; 2-ethyl-5,6,6-trimethyl-1-[(5,6,6-trimethylheptyl)disulfanyl]heptane; 3-ethyl-1-[(2-ethyl-5,6,6-trimethylheptyl)disulfanyl]-5,6,6-trimethylheptane

Test material form:

other: light yellow liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: at the beginning of the treatment period, the animals were 6 weeks old
- Mean body weight at study initiation: The males had a mean body weight of 255 g (range: 238 g to 273 g) and the females had a mean body weight of 196 g (range: 178 g to 213 g)
- Fasting period before study: no
- Housing: the animals were group housed in fives by sex and group in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for 8 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 11 May 2016 to 22 June 2016.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Emulsion in the vehicle.

VEHICLE
- Justification for use and choice of vehicle: suitable formulation in corn oil
- Concentration in vehicle: 10, 50 and 200 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:

Type of method: Gas Chromatography with Mass Spectrometry detection (GC/MS)
On Weeks 2, 3 and 4.
A sample was taken from control and test item dose formulations and analyzed using the validated method.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 animals per sex for groups 1 and 4 (doses 0 and 1000 mg/kg/day)
5 animals per sex for groups 2 and 3 (doses 50 and 250 m/kg/day)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor, on the basis of the results of a previous 2 week toxicity study performed in the same species. In this study, changes were limited to slightly lower body weight gain in females given 1000 mg/kg/day over the second week of the study and to slightly higher spleen weights in males given 1000 mg/kg/day (up to +22%) for which a relationship to the test item treatment was not excluded.
Consequently, the dose-levels of 50, 250 and 1000 mg/kg/day were selected for the present study.
- Rationale for animal assignment: computerized randomization procedure.
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

no (not required)

Examinations

Observations and examinations performed and frequency:

MORTALITY/MORBIDITY: Yes
- Time schedule: each animal was checked for mortality and morbidity 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.

CLINICAL OBSERVATIONS: Yes
- Time schedule: each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed on all the animals once before the beginning of the treatment period and then at least once a week until the end of the study.

BODY WEIGHT: Yes
- Time schedule: the body weight of each animal was recorded once before the beginning of the treatment period, on the first day of treatment and then at least once a week until the end of the study.

FOOD CONSUMPTION: Yes
- Time schedule: the quantity of food consumed by the animals in each cage was recorded once a week during the study. Food consumption was calculated per animal and per day.

MONITORING OF ESTROUS CYCLE: Yes
- Time schedule: the estrous cycle stage was determined for each female sacrificed at the end of the treatment period daily for 4 consecutive days.
As no relevant changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.

NEUROBEHAVIOURAL EXAMINATION: Yes
Functional Observation Battery (FOB)
All principal animals were evaluated once in Week 4 before the daily treatment.

Motor activity
For each principal animal, motor activity was measured once by automated infra-red sensor equipment over a 60 minute period.

HAEMATOLOGY: Yes
Peripheral blood:
The parameters were determined for all animals sacrificed at the end of the treatment period.
In view of the findings observed at the end of the treatment period, these examinations were carried out at the end of the treatment-free period in females only.

Bone marrow:
Two bone marrow smears were prepared from the femoral bone (at necropsy) of each animal sacrificed on completion of the treatment and treatment-free periods.

CLINICAL CHEMISTRY: Yes
Blood biochemistry
The parameters were determined for all animals sacrificed at the end of the treatment period.
As no relevant changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.

Thyroid hormones
An additional blood sample was taken into K3-EDTA tubes from all animals sacrificed at the end of the treatment and treatment-free periods.
The levels of the thyroid hormones and thyroid stimulating hormone were determined from each animal sacrificed at the end of the treatment and treatment-free periods.

URINALYSIS: Yes
The parameters were determined for all animals sacrificed at the end of the treatment period.
As no relevant changes were observed at the end of the treatment period, these examinations were not carried out at the end of the free-treatment period.

Sacrifice and pathology:

ORGAN WEIGHTS: Yes
The body weight of each animal was recorded before euthanasia at the end of the treatment or treatment free period.

GROSS PATHOLOGY: Yes
Sacrifice
On completion of the treatment or treatment-free period, after at least 14 hours fasting, all animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and euthanized by exsanguination.

Macroscopic post-mortem examination
A complete macroscopic post-mortem examination was performed on all study animals.

HISTOPATHOLOGY: Yes
- on all tissues listed in the table below for the control and high dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- on all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.

According to kidney histopathology, immunostained kidneys from all males (groups 1 to 4) sacrificed at the end of the treatment were examined at the request of the Sponsor.
According to the microscopic results of the high-dose group male, and to some organ weight changes, a microscopic examination was performed on:
- kidneys from low- and intermediate-dose main males (groups 2 and 3) and from recovery males (groups 1 and 4),
- adrenals from recovery males (groups 1 and 4),
- liver and thyroids from recovery females (groups 1 and 4).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

No test item-related clinical signs were observed during the study.
The only clinical signs recorded during the treatment and/or treatment free period, i.e. ptyalism, exophthalmos, alopecia, chromodacryorrhea, missing digits, scabs and thinning of hair, were considered to be unrelated to the test item treatment as they were recorded both in control and test item-treated animals and/or, with no dose-relationship, and/or were isolated findings.

Mortality:

no mortality observed

Description (incidence):

No unscheduled deaths occurred during the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects on mean body weight or mean body weight gain were noted at any dose-level during the study (Table 1).
At 50 mg/kg/day, in males, the statistically significantly lower final mean body weight observed was not attributed to the test item treatment in the absence of dose-relationship.
Some instances of lower or higher mean body weight gain, statistically significantly on one occasion (p < 0.05 over the first week of the treatment free-period in males previously treated at 1000 mg/kg/day), were reported in comparison with controls over the treatment-free period. As these differences were occasional, of minimal amplitude and/or of opposite trend, and/or did not affect the mean body weight, they were considered to be of no toxicological importance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related effects on mean food consumption were noted at any dose-level during the study.
Some differences in food consumption between control and test item-treated groups over the treatment or treatment-free period were considered to be of no toxicological importance as they were not statistically significant, not dose-related and/or of opposite trends.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In females treated at 1000 mg/kg/day, statistically significantly lower mean white blood cell count was observed when compared with controls (Table 3). This was associated with statistically significantly lower mean lymphocyte count. The same trend was noted in females given 250 mg/kg/day, but to a lesser extent. Statistically significantly higher mean platelet counts were also observed in females given 1000 mg/kg/day. As at microscopic analysis, normal cellularity was observed in bone marrow and lymphoid organs and since values were closed to the mean values of the Historical Control Data (HCD) whereas most of control values were outside or close to the lower or upper upper limits of the HCD, a relationship with the test item treatment seems to be improbable and whatever to be of minor toxicological importance.
No test item treatment-related effects were observed on the hematology parameters at the end of the treatment period in males when compared to the control group.
No effects on hematology parameters of females were observed at the end of the treatment-free period. The statistically significant differences observed between control and previously test item-treated females, namely in the red blood cell parameters (red blood cell count, hemoglobin concentration and packed cell volume), were of low magnitude, within the historical control values and not observed at the end of the treatment period (Week 5).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects were observed on the blood biochemistry parameters at the end of the treatment or treatment-free period (Table 4).
The only statistically significant differences from controls (i.e. slightly higher mean chloride level in all test item-treated females, lower mean inorganic phosphorus level in females given 1000 mg/kg/day and lower mean alanine aminotransferase activity in females given 250 or 1000 mg/kg/day) were not attributed to the test item treatment as they were of slight magnitude, and/or not dose-related and/or remained within the range of the Historical Control Data.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No changes were observed in the urinary parameters at the end of the treatment period, when compared with controls.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, higher mean numbers of horizontal and rearing movements were recorded in males (+54% and +125% vs. controls, respectively) (Table 2). Since this higher activity was mainly due to the contribution of one male, did not correlate with any clinical signs, and as no such effect was observed in the females, these differences were considered to be unrelated to the test item treatment.
The other findings (e.g. grooming at 250 and 1000 mg/kg/day) were considered to be unrelated to the test item treatment in view of the very slight severity and incidence of the other findings observed during the FOB and in the absence of correlating clinical signs during the study.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related changes in the mean organ weights (Table 5).
When compared with controls, the following changes were observed:
The mean absolute and relative liver and thyroid weights were marginally higher in females treated at 1000 mg/kg/day. In the absence of histopathological correlates, a relationship to the test item was considered to be unlikely.
The mean absolute and relative uterus weights were higher in all groups, particularly in females at 1000 mg/kg/day without reaching a statistical significance. This correlated with dilatation with translucent content in 3/5 animals at necropsy and was considered to reflect variations in the estrous cycle.
The other slight differences between the groups were considered to be part of the normal variations between groups and without relationship to the test item.
Animals sacrificed at the end of the treatment free period
The mean terminal body weight of females previously treated at 1000 mg/kg/day was higher than in controls.
When compared with controls, the mean absolute and relative liver weights were statistically significantly higher in females previously treated at 1000 mg/kg/day (p < 0.01) correlating histologically with minimal to slight glycogen content.
The absolute thyroids weights were marginally higher in females previously treated at 1000 mg/kg/day but without reaching a statistical significance. In the absence of relevant histopathological changes any relationship to the test item was excluded.
The mean relative weight of ovaries was lower in females previously treated at 1000 mg/kg/day (p < 0.05). In the absence of similar trend at the end of the treatment period, this difference was considered to be likely due to variations in the estrous cycle and unlikely related to the test item.
The mean absolute and relative adrenal weights were statistically significantly higher in males previously treated at 1000 mg/kg/day (p<0.05). In the absence of similar trend at the end of the treatment period and histopathological correlates any relationship to the test item was excluded.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Terminal sacrificed animals
No test item-related changes were seen at necropsy.
Dilation of uterus with translucent content was seen in all groups except in controls (3/5 at 50 mg/kg/day, 1/5 at 250 mg/kg/day and 3/5 at 1000 mg/kg/day). This was considered to reflect the normal variations in the estrous cycle and in the absence of dose-related trend, any relationship to the test item was considered to be unlikely (see under microscopy).
Other changes at necropsy were considered to be part of the normal background commonly seen in rats and without any relationship to the test item.
Animals sacrificed at the end of the treatment free period
No test item-related changes were seen at necropsy.
Changes observed were considered to be part of the normal background commonly seen in rats and without relationship to the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Table 6.
Terminal sacrificed animals
Test item-related changes were seen in kidneys from males.
Hyalin droplets were seen in kidneys from males at a higher incidence and severity in males treated at 250 or 1000 mg/kg/day. In the absence of associated lesions this observation was considered to be non adverse.
An immunological staining with a monoclonal anti alpha-2-u globulin antibody and using two different techniques was performed:
- a manual technique did not reveal a clear positive reaction in animals treated at 1000 mg/kg/day but a weak positive reaction in animals of the intermediate groups with hyaline droplets. The explanation for this negative reaction at 1000 mg/kg/day may be due to the masking of the epitopes by the test item and does not mean that the hyaline droplets did not contain alpha-2 u globulin.
- as a consequence of the absence of positive reaction with the commonly used manual technique, a second staining using a semi-automatic technique was performed in males treated at 1000 mg/kg/day. This additional technique showed a strong positive reaction of these hyaline droplets suggesting they effectively corresponded to alpha-2-u globulin.

There were no histopathological changes which correlated with the marginal higher liver or thyroid weights in females.
Careful examination of testes did not show any change considered to be test item-related.

Examination of the female genital tract showed a higher number of animals with dilation of uterus in animals treated at 1000 mg/kg/day than in controls. This correlated with the higher weight of uterus at necropsy and dilatation with translucent content at necropsy and corresponded to variations in the estrous cycle (these animals being in estrus or proestrus).

Dilation of uterus was also observed at necropsy and histologically in 3/5 females at 50 mg/kg/day and 1/5 females at 250 mg/kg/day correlating with higher weight of uterus at necropsy.

Considering the low number of animals (n = 5), the absence of dose-related trend between animals treated at 50 or 250 mg/kg/day), and the absence of changes during monitoring of the estrous cycle at the end of the treatment period, a relationship of these differences with the test item was considered to be doubtful.

Other changes were considered to be part of the normal background commonly seen in rats and without relationship to the test item.

Animals sacrificed at the end of the treatment-free period
Following a 2-week treatment-free period, minimal increased hyaline droplets were seen in kidneys of males previously treated at 1000 mg/kg/day suggesting an ongoing recovery.

Minimal tubular basophilia was seen in 2/5 control males and 4/5 males previously treated at 1000 mg/kg/day.
This was mainly focal, except one previously treated male where it was multifocal. As this observation is commonly seen in rat kidneys, any relationship to the test item was considered to be unlikely.

In the liver of females previously treated at 1000 mg/kg/day, minimal to slight glycogen content was observed in 3/5 animals and correlated with the higher liver weight at necropsy. Although of unknown origin, and not observed at the end of the treatment period, a relationship to the test item seems improbable.

No test item-related changes were seen in the thyroids of females previously treated at 1000 mg/kg/day. As a consequence, the marginal higher weight seen at necropsy was not considered to be related to the test item.

Similarly, in the absence of relevant histopathological changes the higher adrenal weight in males previously treated at 1000 mg/kg/day was not considered to be related to the test item.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle: no effects observed
No test item-related effects were noted on mean estrous cycle length or on the mean number of cycles.

Thyroid hormones: effects observed, non-treatment-related
TSH, T3 and T4 levels were unaffected by the test item at the end of the treatment and treatment-free periods.

Higher mean TSH values were noted in females given 250 or 1000 mg/kg/day at the end of the treatment period. As these differences were not dose-related, not statistically significant and/or were due to the contribution of one female, these variations were considered to be incidental and of no toxicological importance.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Mean body weight (BW) gain/ Mean body weight (expressed in g)

 

Sex	Male				Female
Dose-level (mg/kg/day)	0	50	250	1000	0	50	250	1000
Treatment period
Mean BW gain Days 1/28	+212	+195	+199	+205	+60	+63	+58	+63
% compared to controls	-	-8	-6	-3	-	+5	-3	+5
Mean body weight
. Day 1	258	251	252	256	194	195	192	200
. Day 28	469	445*	451	461	254	258	250	263
% compared to controls	-	-5	-4	-2	-	+2	-2	+4
Treatment-free period
Mean BW gain Days 28/42	+56	-	-	+62	+22	-	-	+23
Mean BW on Day 42	533	-	-	524	272	-	-	281
% compared to controls	-	-	-	-2	-	-	-	+3

Statistically significant from controls: *: p < 0.05; -: not applicable.

 

Table 2: Motor activity

 

Sex		Male					Female
Dose-level (mg/kg/day)		0		50		250		1000		0		50		250		1000
Horizontal movements		413		563		386		635		692		919		517		588
% from controls		-		+36		-7		+54		-		+33		-25		-15
Rearing		109		165		129		245		221		228		252		194
% from controls		-		+51		+18		+125		-		+3		+14		-12

-: not applicable.

 

Table 3:Hematology parameter changes

 

Sex	Female
Dose-level (mg/kg/day)	0	50	250	1000	HCD
End of treatment period
White blood cell count (G/L)	16.28 -	15.22 (-7)	13.08 (-20)	11.88* (-27)	8.43 3.36-15.30
Platelet count (G/L)	574 -	697 (+21)	737 (+28)	957** (+67)	1068 567-1852
Lymphocyte count (G/L)	13.36 -	12.06 (-10)	10.30 (-23)	9.63* (-28)	7.22 2.45-13.57
End of treatment-free period
White blood cell count (G/L)	12.11 -	- -	- -	13.52 (+12)	-
Platelet count (G/L)	951 -	- -	- -	730 (-23)	-
Lymphocyte count (G/L)	9.76 -	- -	- -	11.18 (+15)	-

% from controls are expressed in brackets. Statistically significant from controls: *: p<0.05; **: p<0.01.; -: not applicable.

HCD: Historical Control Data; mean and minimum-maximum values from Historical Control Data.

 

Table 4:Blood biochemistry parameter changes

 

Sex	Female
Dose-level (mg/kg/day)	0	50	250	1000	HCD
End of treatment period
Chloride (mmol/L) % compared to controls	106.1 -	108.2** +2	107.9** +2	107.4* +1	109.6-111.9
Inorganic phosphorus (mmol/L) % compared to controls	2.22 -	2.17 -2	2.08 -6	2.00* -10	1.72-2.08
ALAT (U/L) % compared to controls	33 -	31 -6	28* -15	28* -15	21-35

Statistically significant from controls: *: p<0.05; **: p<0.01.; -: not applicable.

ALAT: alanine aminotransferase activity.

HCD: Historical Control Data; minimum-maximum values from Historical Control Data.

 

 

Table 5: Organ weights

 

Animals sacrificed at the end of the treatment period

Sex	Male			Female
Group	2	3	4	2	3	4
Dose-level (mg/kg/day)	50	250	1000	50	250	1000
Number of animals	5	5	5	5	5	5
- Final body weight	-4	-2	-1	-1	-4	+3
- Liver
.absolute	-6	-4	-2	+4	+6	+10
.relative	-2	-2	0	+5	+11	+8
- Thyroid glands
.absolute	-5	-5	+5	-5	+6	+14
.relative	-2	-4	+6	-3	+12	+12
- Uterus
.absolute				+55	+36	+91
.relative				+58	+42	+84

 

Animals sacrificed at the end of the treatment free period

Sex	Male	Female
Group	4	4
Dose-level (mg/kg/day)	1000	1000
Number of animals	5	5
- Final body weight	-3	+9*
- Adrenal glands
. absolute	+19*	+5
. relative	+23*	-3
- Liver
. absolute	-6	+26**
. relative	-4	+16**
- Thyroid glands
. absolute	+6	+15
. relative	+10	+6

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

The significance concerned the organ weights values and not the percentages.

 

Table 6: Microscopic examination

Terminal sacrificed animals

Group	1	2	3	4
Dose-level (mg/kg/day)	0	50	250	1000
Number of animals	5	5	5	5
Hyaline droplets (in Hematoxilin/eosin sections)
Minimal (grade 1)	1	1	2	1
Slight (grade 2)	-	-	1	3
Moderate (grade 3)	-	-	-	1
Total	1	1	3	5
Hyaline droplets (immunostaining with antibody for alpha-2globulin protein using the manual technique)
Minimal (grade 1)	-	1	1	-
Slight (grade 2)	-	-	1	-
Moderate (grade 3)	-	-	-	-
Total	0	1	2	0
Hyaline droplets (immunostaining with antibody for alpha-2globulin protein using the semi-automatic technique, group 4 only)
Moderate (grade 3)	NA	NA	NA	4
Marked (grade 4)	NA	NA	NA	1
Total	NA	NA	NA	5

-: not seen in this group; NA: Not Applicable

 

Animals sacrificed at the end of the treatment-free period

Group	1	4
Dose-level (mg/kg/day)	0	1000
Hyaline droplets (in Hematoxilin/eosin sections)
Number of animals	5	5
Minimal (grade 1)	1	1
Slight (grade 2)	-	-
Moderate (grade 3)	-	-
Total	1	1

-: not seen in this group.

 

Applicant's summary and conclusion

Conclusions:

The test item was administered daily by oral gavage to male and female Sprague-Dawley rats at the dose-levels of 50, 250 or 1000 mg/kg/day for 4 weeks. On completion of the treatment period, designated animals were held for a 2-week treatment-free period in order to evaluate the reversibility of any findings.
The test item was clinically well tolerated at all dose-levels and no effects were observed on FOB and estrous cycle parameters.
At 1000 mg/kg/day, the test item administration did not induce any adverse changes in clinical pathology. Non-adverse histopathological findings were noted (i.e. hyaline droplets in kidneys) in males which were partially reversible at the end of the treatment-free period.
At 250 mg/kg/day, the test item administration did not induce any relevant clinical pathology changes. Hyaline droplets were observed in males.
At 50 mg/kg/day, the test item did not induce any changes in clinical pathology or in either macroscopic or microscopic evolutions.
Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) after the 4-week treatment period was established at 1000 mg/kg/day.

Executive summary:

The potential toxicity of di-tert-dodecyl disulfide was evaluated following daily oral administration (gavage) to rats for 4 weeks. On completion of the treatment period, designated animals were held for a 2-week treatment-free period in order to evaluate the reversibility of any findings.

This GLP study was carried out according to OECD test guideline No. 407 (03 October 2008). Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item for four weeks as follows: two groups of five males and five females at dose-levels of 50 (group 2) or 250 (group 3) mg/kg/day and another group of ten males and ten females at the dose-level of 1000 mg/kg/day (group 4). One group of ten males and ten females received the vehicle only (corn oil) under the same experimental conditions, and acted as a control group (group 1). A constant dosage volume of 5 mL/kg/day was used. At the end of the treatment period, the animals were sacrificed, except for the first five group 1 and 4 animals per sex, which were kept for a 2-week treatment-free period. The actual test item concentrations in the dose formulations prepared for use in Weeks 2, 3 and 4 were determined using a gas chromatography with analytical method using mass spectrometry detection. The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed weekly and a Functional Observation Battery (FOB) was conducted in Week 4. Body weight was recorded once pre-test, on the first day of treatment and then once a week. Food consumption was recorded weekly. Hematology, blood biochemistry, urinalysis and thyroid hormones investigations were performed at the end of the treatment period. Hematological (females) and thyroid hormones parameters were also determined at the end of treatment-free period. The estrous cycle stage was determined for all females, over 4 consecutive days, before sacrifice at the end of the treatment period. On completion of the treatment or treatment-free period, the animals were sacrificed and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on designated tissues from control- and high-dose animals sacrificed at the end of the treatment period, on kidney slides immunostained with an antibody for alpha-2-u globulin protein from all males sacrificed at the end of the treatment period, on kidneys from all main and recovery males, on adrenals from recovery males and on liver and thyroids from recovery females.

Actual concentrations of the test item in the dose formulations administered to the animals during the study remained within an acceptable range (-1.2% to +8.9%) compared to the nominal concentrations. No unscheduled deaths occurred during the study. No test item treatment-related clinical signs were noted during the study. The FOB results were unaffected by the test item treatment. Body weight change, body weight and food consumption were unaffected by the test item treatment in both sexes. The estrous cycle was not altered by the test item treatment. No treatment-related and/or adverse changes were observed in hematology, blood biochemistry and urinary parameters at the end of the treatment period. Thyroid hormone levels were unaffected by the test item at the end of the treatment and treatment-free periods. At pathology investigations, non-adverse increased incidence and severity of hyaline droplets were seen in the kidneys of males at 250 and 1000 mg/kg/day. When using a manual technique of staining, these droplets were weakly positive for alpha-2-u globulin (immunostaining) at 250 mg/kg/day but not at 1000 mg/kg/day. A semi-automatic technique of staining performed in kidneys from males treated at 1000 mg/kg/day showed a strong positive staining of these droplets suggesting they corresponded to alpha-2-u globulin. Following a two week treatment-free period, minimal increased hyaline droplets were still present in kidneys of males previously treated at 1000 mg/kg/day when compared to controls.  

Di-tert-dodecyl disulfide was clinically well tolerated at all dose-levels and no effects were observed on FOB and estrous cycle parameters.

At 1000 mg/kg/day, the test item administration did not induce any adverse changes in clinical pathology. Non-adverse histopathological findings were noted (i.e. hyaline droplets in kidneys) in males which were partially reversible at the end of the treatment-free period.

At 250 mg/kg/day, the test item administration did not induce any relevant clinical pathology changes. Hyaline droplets were observed in males.

At 50 mg/kg/day, the test item did not induce any changes in clinical pathology or in either macroscopic or microscopic evolutions. 

Consequently, under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) after the 4-week treatment period was established at 1000 mg/kg/day.