Bis(piperidinothiocarbonyl) hexasulphide

Endpoint summary

Description of key information

At 1000 mg/kg/day, the test item was clinically well-tolerated and no relevant findings were noted at clinical pathology after 28 -day and 90 -day of exposure. the No Observed Adverse Effect Level (NOAEL) was established at 1000 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

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 August 2018 - 03 January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

21 September 1998

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 Italia, Calco, Italy
- Age: 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 228 g (range: 193 g to 266 g) and the females had a mean body weight of 160 g (range: 146 g to 173 g)
- Fasting period before study: no
- Housing: the animals were housed in twos (same 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 a period of 8 days before the beginning of the study.

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: 03 October 2018 to 03 January 2019.

Route of administration:

oral: gavage

Vehicle:

other: 0.5% (w/w) methylcellulose aqueous solution

Details on oral exposure:

PREPARATION OF DOSING FORMULATIONS:
- Suspension in the vehicle
- Suitable formulation in the selected vehicle
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with UV detection (HPLC/UV).
Test item concentrations: Once in Weeks 1, 4, 5, 8 and 13. A sample was taken from control and test item dose formulations and analyzed using the validated method.
Homogeneity: homogenous formulations at 4 and 100 mg/mL
Stability: stable after 9 days at room temperature and protected from light.
The test item concentrations in the administered dose formulations analyzed in Weeks 1, 5, 8 and 13 remained within an acceptable range of -14.6% to -1.8% when compared to the nominal values (± 15% of the nominal concentrations).

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Dose / conc.:

0 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)

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, based on the results of a previous OECD 407 study.
In this study, the test item was administered daily by gavage to male and female Sprague-Dawley rats, for 4 weeks, at dose levels of 100, 300 or 1000 mg/kg/day.
At 1000 mg/kg/day, the test item was clinically well tolerated and there were no relevant findings at clinical pathology. There was a slightly higher incidence of non-adverse minimal alveolar foamy macrophages in the lungs for which the toxicological significance was considered to be doubtful. At 300 and 100 mg/kg/day, no test item effects were observed and there were no significant changes at clinical pathology or histopathology.
In absence of critical effects in the study, the same dose levels were used in the present 90-day repeated toxicity study.

- Rationale for animal assignment: computerized stratification procedure.

Positive control:

no (not required)

Observations and examinations performed and frequency:

MORTALITY/MORBIDITY:
- 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 period, including weekends and public holidays.

CLINICAL OBSERVATIONS:
- Time schedule: during the treatment period, each animal was observed at least once a day, between 1 and 3 hours post administration, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then at least once a week (between 1 and 3 hours post administration) until the end of the study.

FUNCTIONAL OBSERVATION BATTERY (FOB):
- Time schedule: each animal was evaluated once in Week 12 before the daily treatment. This evaluation included a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli, and motor activity.

BODY WEIGHT:
- 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 at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by the animals in each cage was recorded at least once a week, over a 7 day period, during the study.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule: on all animals before the beginning of the treatment period and on control and high-dose animals on one occasion at the end of the treatment period.

NEUROBEHAVIOURAL EXAMINATION:
The following parameters were assessed and graded:
- in the cage "touch escape",
- in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmia, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (two-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, convulsions, gait, arousal (hypo- and hyper- activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.

Reactivity to manipulation or to different stimuli
The following measurements, reflexes and responses were recorded:
- touch response,
- forelimb grip strength,
- pupil reflex,
- visual stimulus,
- auditory startle reflex,
- tail pinch response,
- righting reflex,
- landing foot splay,
- at the end of observation: rectal temperature.

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

HEMATOLOGY: Yes
- Time schedule for peripheral blood: the parameters were determined for all surviving animals at the end of the treatment period (Week 13)
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No. 1] were examined.

COAGULATION:
- Time schedule: the parameters were determined for all surviving animals at the end of the treatment period (Week 13). See table [No. 2].

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: the parameters were determined for all surviving animals at the end of the treatment period (Week 13)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No. 3] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: the parameters were determined for all surviving animals at the end of the treatment period (Week 13)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No. 4] were examined.

Sacrifice and pathology:

ORGAN WEIGHTS: see table 5
The body weight of each animal was recorded before euthanasia at the end of the treatment period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection (all animals).
The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all animals.

HISTOPATHOLOGY:
A microscopic examination was performed on all tissues listed in the Tissue Procedure Table:
- for the control and high-dose animals (groups 1 and 4) euthanized at the end of the treatment period and for group 4 male that was euthanized prematurely,
- for all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period.

In addition, a detailed examination of the testes was performed in control and high-dose males (groups 1 and 4) using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with hematoxylin-PAS in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.
According to the kidney histopathology results, immunostained kidneys from all males were also examined, and in agreement with the Sponsor, based upon the microscopic results of the high-dose group, other tissues from the low- and intermediate-dose groups (groups 2 and 3) were examined as follows:
- kidneys (males),
- mesenteric lymph nodes (males and females).

Other examinations:

SPERM PARAMETERS:
At the end of the treatment period, each male was administered with an intraperitoneal injection of sodium pentobarbital.

Epididymal sperm
Under deep anesthesia and after weighing the epididymis, sperm from the cauda of the left epididymis was collected for motility and morphology investigations. The animals were then euthanized.
The cauda of the left epididymis was separated from the corpus using a scalpel, and subsequently kept at 20°C pending further investigation.

Epididymal sperm motility
The sperm was evaluated on a slide. The numbers of motile and immotile spermatozoa from a sample of 200 spermatozoa were evaluated under a microscope using a 40-fold magnification. This was evaluated for all males and the results are expressed as the proportion of motile and non-motile spermatozoa.

Epididymal sperm morphology
Morphology was determined from a sperm smear, after eosin staining and the counting of 100 spermatozoa per slide. This was evaluated for all males.
Results are expressed as the proportion of spermatozoa in each of the following categories:
- normal,
- normally shaped head separated from flagellum,
- abnormal head separated from flagellum,
- abnormal head with normal flagellum,
- abnormal head with abnormal flagellum,
- normally shaped head with abnormal flagellum.

Epididymal sperm count
After thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
The number of spermatozoa was counted in a microscope slide counting chamber for all males, and the results are expressed as the number of spermatozoa per cauda and per gram of cauda.

Testicular sperm
The left testis was collected and frozen at -20°C for further sperm count investigation. After thawing, it was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogeneization (i.e. elongated spermatids and mature spermatozoa) were counted in a microscope slide counting chamber.
This was evaluated for all males and the results are expressed as the number of sperm heads per gram of testis. The daily sperm production rate was also calculated (using a time divisor of 6.10; Blazak et al., 1993).

MONITORING OF ESTROUS CYCLE:
The estrous cycle stage was determined daily from a fresh vaginal lavage (stained with methylene blue) for 21 consecutive days at the end of the treatment period.

THYROID HORMONES:
- Time schedule: all surviving animals euthanized at the end of the treatment period (Week 13). Thyroid hormones (T3 and T4) levels were determined by LC MS/MS for all surviving animals euthanized at the end of the treatment period.
The levels of thyroid stimulating hormone (TSH) were determined by Luminex MAP® technology for all surviving control and high dose animals euthanized at the end of the treatment period. These examinations were not carried out in low and intermediate dose animals as there was no indication of relevant changes in high dose animals.

Statistics:

Citox software was used to perform the statistical analyses of body weight, food consumption, motor activity, estrus cycle, sperm parameters, hematology, blood biochemistry, urinalysis and thyroid hormone data.
PathData software was used to perform the statistical analysis of organ weight data.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related clinical signs were observed in surviving animals.
Clinical signs recorded during the study (abnormal whitish color of teeth, abnormal growth of teeth and loss of teeth, together with hunched posture, thinning of hair, alopecia, scabs, soiled head and/or cheeks, reflux at administration, bent tail, chromodacryorrhea and/or eye opacity), were of isolated occurrence, observed both in control and test item-treated animals, with no dose-relationship, commonly seen when a test item is administered by gavage, and/or are common findings in this strain of rats when housed under laboratory conditions. They were therefore considered to be unrelated to the test item.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item-related deaths occurred during the study.
One male given 1000 mg/kg/day was euthanized prematurely for humane reasons on Day 57 (Week 9). Prior to euthanasia, this male showed signs of poor clinical condition (i.e. thin appearance together with hunched posture from Day 55, and piloerection and hypoactivity on Day 57) with a body weight loss of 106 g between Days 50 and 57 (Week 8) associated with reduced food consumption in the cage (18.8 g/animal/day). At necropsy, the major findings were enlargement of the liver and spleen, correlating to a malignant lymphoma, enlargement of the adrenals, correlating to cortical hypertrophy, and reduced size of the thymus, correlating to lymphoid atrophy. The latter two observations on thymus were considered stress related (Everds et al, 2013).
Malignant lymphomas are commonly observed in young control rats. It is therefore considered spontaneous and not related to the test item.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

See table 2.
Mean body weight and mean body weight change were unaffected by the test item in males and females.
When compared with controls, slight but statistically significant, lower or higher mean body weight gain was recorded in Weeks 2, 7 and/or 13 in females given 100 mg/kg/day and males given 300 mg/kg/day.
These transient variations were considered to be fortuitous as they were not dose-related and of opposite trends.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean food consumption was unaffected by the test item in males and females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related findings at the end of the treatment period.
Lens opacity was observed in one eye of a control female at the end of the study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg/day in males, when compared with controls, statistically significant, higher mean cell volume (+3%) together with statistically significant and higher mean reticulocyte count (+44%) were noted.
In view of the low magnitude, in absence of correlation with microscopic lesions and/or as mean values remained within the range of the Historical Control Data, the findings detailed above were considered to be of no toxicological importance.
The other differences between control and test item-treated animals, namely in white blood cell count (females given 300 mg/kg/day), basophil and lymphocyte counts (females given 300 mg/kg/day) and prothrombin time (males given 100 mg/kg/day), were considered to be of no toxicological importance as they were noted with no dose-relationship.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See table 4.
At 300 and 1000 mg/kg/day, when compared with controls, increased mean biliary acid level was noted in females (2.6-fold and 3.3-fold, respectively), reaching statistical significance at the high-dose level. In the absence of effects in the other sex and relationships to other changes (e.g. total bilirubin and cholesterol) and microscopic findings (e.g. on liver), these findings were considered to be of minor importance.
At 1000 mg/kg/day, when compared with controls, a statistically significant increase in creatinine level was noted in males (+13% vs. controls). These changes could be suggestive of a possible glomerular filtration problem as they were associated with a minimally higher urea levels in these animals (+12% vs. controls).
In view of the slight magnitude, and/or absence of relationships to renal microscopic findings, and/or as mean values were within the range of the HCD, these findings were not considered as adverse.

At the intermediate and high dose levels, decreased alkaline phosphatase activity was noted in males (-11% and -20% vs. controls, respectively) and females (-25% and -34% vs. controls, respectively). In view of the direction of these changes and in absence of any correlating microscopic findings (e.g. on liver), these differences were considered to be of no biological importance.
The other statistically significant differences between control and test item-treated animals consisted of high sodium (males at 1000 mg/kg/day), high potassium (males at 300 mg/kg/day) and high chloride (females from 300 mg/kg/day) levels and high glucose level (females at 300 mg/kg/day). As these variations were of slight magnitude, without a dose relationship and without any correlating microscopic findings, they were considered to be incidental and of no toxicological importance.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See table 5.
There were no differences from controls in the specific gravity of test item treated animals.
At 1000 mg/kg/day, when compared with controls, there were statistically significant, lower mean pH values in males and females (-15% and -9%, respectively). These differences were of low magnitude, within the range of the HCD and/or did not correlate with any other urinary changes. Therefore they were considered to be of minor toxicological importance.
Although the presence of water was suspected in the urine of one female at 1000 mg/kg/day, mean values of volume, specific gravity and pH were within the range of the HCD but the individual values of this female were excluded from the calculation.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

See table 1.
There were no relevant test item-related effects on functional observation battery tests or motor activity data in any group.
At 1000 mg/kg/day in males, differences from controls were noted in the mean number of horizontal (+22%) and rearing (-19%) movements. These values were considered to be of no toxicological importance as they were of opposite trends, were not statistically significant, were poorly dose-related and/or the values remained close to or within the standard deviation of control values.
Grooming was noted in 1/10 males given 100 mg/kg/day and in 2/9 males given 1000 mg/kg/day (vs. in 0/10 control males), defecation was recorded in 2 males from each test item-treated group (vs. in 0/10 control males) and urination was observed in 5 males and 2 females given 300 or 1000 mg/kg/day (vs. in 3/10 control males and 0/10 control females). In view of the very low magnitude of these findings, in the absence of correlation between findings and/or with other changes seen during the study and as the findings were poorly dose-related, they were considered to be unrelated to the test item.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no relevant differences in organ weights between test item-treated and control groups.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Unscheduled death
One high dose male was killed moribund on Day 57 (Week 09). At necropsy, the major findings were enlargement of the liver and spleen and enlargement of the adrenals.

Terminal sacrifice
All macroscopic observations belonged to the spectrum of spontaneous changes in rats of this age and strain, and bore no relationship to treatment. This includes red discoloration of the mesenteric or mandibular lymph nodes, observed almost exclusively in high dose animals, which correlated microscopically with sinus erythrocytes, a common background histological finding in rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See table 8.

Unscheduled death
One high dose male (1000 mg/kg/day) was killed moribund on Day 57. At necropsy, the major findings were enlargement of the liver and spleen, correlating to a malignant lymphoma involving the liver and spleen but also bone marrow and mandibular lymph node; enlargement of the adrenals, correlating to cortical hypertrophy; and reduced size of the thymus, correlating to lymphoid atrophy. The observations on the adrenals and thymus were considered stress-related (Everds et al, 2013).
Malignant lymphomas are commonly observed in young control rats. It is therefore considered spontaneous and not related to the test item.

Terminal sacrifice
A dose related minimal to moderate increase in hyaline droplets in the kidneys was diagnosed in males at all doses. Hyaline droplets are composed of alpha 2u-globulin, and this was confirmed by immuno-histo chemistry (IHC). The average score for IHC positivity was 1.5, 1.7, 2.1 and 2.6 in control, low-, mid- and high-dose groups, respectively. These findings, which were directly related to the test item, are part of the so-called alpha 2u-globulin nephropathy, which is specific to male rats and irrelevant to human risk assessment (Swenberg 1993). It was considered non adverse, given the absence of associated clinical or clinical pathology changes.
Pigmented macrophages were observed in the mesenteric lymph node in 3/10 high-dose males, with a minimal or slight severity. A relationship to treatment cannot be excluded, but such a minor focal change has no toxicological significance and is considered non adverse.
All other observations belonged to the spectrum of spontaneous changes in rats of this age and strain and bore no relationship to test item administration. The testicular morphology was similar in control and high dose animals, with no evidence of disruption of the spermatogenic cycle.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Details on results:

Estrus cycle
See table 3.
There were no test item-related effects on the mean length of the estrus cycle or the mean number of cycles.

Thyroid hormones
See table 6.
There were no relevant differences from controls in T3 or TSH concentrations of test item-treated animals.
At 1000 mg/kg/day, when compared with controls, there were slightly decreased mean T4 concentrations in males (-22%; p<0.01) and females (-23%; not statistically significant). These differences were observed in both sexes but were less pronounced in females. They were considered to be test item-related but non-adverse as they did not correlate with variations in mean TSH concentrations (males), as they resulted from higher mean control values (T4 concentration was outside the range of the HCD in two control males: 64.19 and 81.21 ng/mL), as they were within the range of the HCD and not associated with any thyroid or pituitary microscopic findings.

Seminology
See table 7.
No test item-related effects were noted on epididymal sperm count, motility or morphology.
Some differences were observed in epididymal sperm morphology. Higher mean percentages of spermatozoids with normal head separated from flagellum were recorded in males given 300 or 1000 mg/kg/day (2.7-fold and +76% vs. controls). As these differences were not dose related, were not statistically significant and had only minimal impact on the percentage of normal spermatozoids, they were considered to be incidental.
Statistically significant lower mean values were recorded in the testicular sperm count of males at 1000 mg/kg/day (-20% vs. controls; p<0.05). As these test item-related differences were of minor magnitude, were inside the range of the HCD and had no epidydimal and microscopic correlates, they were considered to be non-adverse.

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

Critical effects observed:

no

Table 1. Functional Observation Battery

 

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Horizontal movements	418	456	436	509	746	717	609	761
% from controls	-	+9	+4	+22	-	-4	-18	+2
Rearing	153	134	119	124	166	165	149	177
% from controls	-	-12	-22	-19	-	-1	-10	+7

-: not applicable.

 

Table 2. Body weight and body weight gain

 

	Sex		Male					Female
Dose level (mg/kg/day)		0		100	300	1000	0		100	300	1000
Mean BW gain Weeks 1/13		+319		+336	+322	+326	+132		+143	+143	+125
% from controls		-		+5	+1	+2	-		+8	+8	-5
Mean body weight Week 1		228		226	228	231	158		161	162	158
Mean body weight Week 13		548		562	549	559	290		303	305	283
% from controls		-		+3	0	+2	-		+4	+5	-2

Statistical analysis: no significance.

 

Table 3. Estrus cycle

 

Dose level (mg/kg/day)	0	100	300	1000
Number of cycles	3.2	3.1	3.2	3.6
Cycle length (days)	5.7	7.1	4.9	5.9
Number of females having a mean average cycle of 4-5 days	4	3	7	7

Table 4. Blood biochemistry

 

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Biliary acids (µmol/L)	11.14	2.31	2.46	12.98	6.76	9.68	17.48	22.41*
HCD	Not available due to insufficient number of values
Creatinine (µmol/L)	26.22	28.09	27.49	29.58**	31.61	32.60	33.54	32.53
HCD	Mean: 30.46 [23.19-45.05]				Mean: 34.23 [25.30-47.27]
Urea (mmol/L)	4.1	4.3	4.0	4.6	5.3	5.1	5.3	5.3
HCD	Mean: 4.8 [3.6-7.6]				Mean: 5.4 [3.3-7.7]
Alkaline Phosphatase Activity (U/L)	202	188	179	162*	128	135	96*	84**
HCD	Mean: 234 [133-426]				Mean: 129 [56-258]

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

HCD: Historical Control Data [minimum and maximum values]

 

Table 5: Urinalysis

 

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
Volume (mL)	12 [4-28]	15 +25%	12 0%	14 +17%	8 [1-33]	11 +38%	6 -25%	8 (6) 0%
Specific gravity	1031 [1015-1050]	1026 0%	1032 0%	1031 0%	1034 [1010-1050]	1026 -1%	1034 0%	1035 (1037) 0%
pH	7.4 [6.5-8.5]	7.5 +1%	7.6 +3%	6.3** -15%	6.8 [5.0-8.0]	7.0 +3%	6.7 -1%	6.2* (6.1) -9%

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

[ ]: minimum-maximum values vs. Historical Control Data (HCD).

( ): mean values without values of one female.

In italic: % vs. controls.

 

Table 6: Thyroid hormones

 

Sex	Male				Female
Dose level (mg/kg/day)	0	100	300	1000	0	100	300	1000
T3 (ng/mL)	0.39 -	0.39 0%	0.43 +10%	0.38 -3%	0.48 -	0.53 +10%	0.53 +10%	0.55 +15%
HCD	0.36 [0.27-0.46]				0.48 [0.35-0.65]
T4 (ng/mL)	52.39 -	49.87 -5%	47.36 -10%	40.79** -22%	30.40 -	37.87 +25%	31.56 +4%	23.39 -23%
HCD	45.21 [29.02-61.07]				28.87 [17.22-38.89]
TSH (pg/mL)	2532 -	- -	- -	2230 -12%	1006 -	- -	- -	756 -25%
HCD	2345 [686-5442]				817 [271-1395]

Statistically significant versus controls:**: p<0.01

HCD: Historical Control Data [5 - 95 percentiles].

In italic: % vs. controls.

-: not applicable.

 

Table 7: Seminology

 

Dose level (mg/kg/day)	0	100	300	1000
% of motile epididymal sperm % from controls	98.4 /	98.2 0	98.5 0	98.3 0
% of morphologically normal epididymal sperm % from controls	95.3 /	94.1 -1	87.9 -8	92.0 -3
Mean number of epididymal sperm (10^6/cauda) % from controls	118.3 /	131.9 +11	131.9 +11	125.3 +6
Mean number of epididymal sperm (10^6/g cauda) % from controls	400.0 /	417.8 +4	420.5 +5	410.3 +3
Mean number of testicular sperm heads (10^6/g testis) % from controls	113.3 /	108.3 -4	103.2 -9	90.7* -20
Daily sperm production rate (10^6/g testis/day) % from controls	18.6 /	17.8 -4	16.9 -9	14.9* -20

/  : not applicable. Statistically significant: *: p<0.05.

Table 8: Microscopic examination

 

Incidence and severity of selected microscopic findings
Gender	Males				Females
Group	1	2	3	4	1	2	3	4
Dose (mg/kg)	0	100	300	1000	0	100	300	1000
Concentration (mg/mL)	0	10	30	100	0	10	30	100
Number of animals	10	10	10	9	10	10	10	10
Kidney
Increased hyaline droplets
Minimal	0	2	3	1	0	-	-	0
Slight	0	0	1	3	0	-	-	0
Moderate	0	0	0	2	0	-	-	0
Positive for alpha 2u IHC
Minimal	5	4	1	1	0	-	-	0
Slight	5	5	5	2	0	-	-	0
Moderate	0	1	4	6	0	-	-	0
Lymph node, Mesenteric
Pigmented macrophages
Minimal	0	0	0	1	0	0	0	0
Slight	0	0	0	2	0	0	0	0

Conclusions:

The test item was administered daily for 13 weeks by gavage to male and female Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/day in 0.5% (w/w) methylcellulose aqueous solution.

There were no relevant test item-related or adverse effects on mortality, clinical signs, ophthalmology, body weight, food consumption, clinical pathology, estrous cycle, sperm parameters, hormone levels or histopathology parameters.

Under the experimental conditions and according to the results of this study, the No Observed Adverse Effect Level (NOAEL) was set at 1000 mg/kg/day.

Executive summary:

The objective of this GLP study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 13 weeks.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item, daily, by the oral route, (gavage) for 13 weeks. The test item was administered at dose levels of 100, 300 or 1000 mg/kg/day as a suspension in the vehicle [0.5% (w/w) methylcellulose aqueous solution] under a constant dosage volume of 10 mL/kg/day. A control group of ten males and ten females received the vehicle, alone, under the same experimental conditions.

The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 4, 5, 8 and 13 were determined using a High Performance Liquid Chromatography with UV detectionmethod (HPLC/UV).

The animals were checked daily for mortality, morbidity and clinical signs. Detailed clinical observations were performed once a week. Body weight was recorded pre-test, on the first day of treatment and then once a week. Food consumption was recorded weekly.

Ophthalmological examinations were performed on all animals before the beginning of the treatment period and on control and high-dose animals at the end of the treatment period (Week 13). A functional observation battery was performed in Week 12.

Hematology, blood biochemistry and urinalysis investigations were performed in all animals at the end of the treatment period (Week 13). Additional blood samples were collected in Week 13 for the analysis of TSH (control and high-dose groups) and T4 and T3 thyroid hormone (all groups) levels in animals euthanized at the end of the treatment period.

The estrus cycle was determined over 21 consecutive days for all females at the end of the treatment period. At the end of the treatment period, seminology investigations (morphology, count and motility) were performed in all males.

On completion of the treatment period, all animals were euthanized and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissues were preserved.A microscopic examination (including a detailed examination of the testes) was performed on designated tissues from control and high-dose animals euthanized at the end of the treatment period and from animals that were euthanized prematurely, and on all macroscopic lesions from low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period. A microscopic examination was also performed on kidney slides (immunostained with an antibody for a2u-globulin protein) from all control and test item-treated males euthanized at the end of the treatment period. The kidneys (males) and mesenteric lymph nodes (males and females) from low- and intermediate-dose animals (groups 2 and 3) euthanized at the end of the treatment period were also microscopically examined, as changes were noted in these organs at the high-dose level.

 

Results

The test item concentrations in the administered dose formulations analyzed in Weeks 1, 5, 8 and 13 remained within an acceptable range of -14.6% to -1.8% when compared to the nominal values (±15% of the nominal concentrations).

No test item-related unscheduled deaths occurred during the study.

No test item-related clinical signs were noted during the study.

 

The results of the functional observation battery were unaffected by the test item treatment.

 

There were no significant effects on body weight, body weight gain or food consumption in either sex.

 

No test item-related ophthalmological findings were observed at the end of the treatment period.

 

No relevant test item-differences were observed in estrous cycle, epididymal sperm motility, morphology, and epididymal sperm count. Low testicular sperm count was noted in males given 1000 mg/kg/day. This variation was considered to be of minor toxicological importance as no histological correlates were noted.

 

There were no relevant differences from controls in the hematological parameters.

 

Dose-related increases in biliary acid levels were noted from 300 mg/kg/day in females, and increased creatinine and urea levels were recorded in males given 1000 mg/kg/day. All these changes were considered to be of minor toxicological importance as there were no corresponding histological findings.

The lower pH value of the urine noted in males and females given 1000 mg/kg/day was considered to be of minor toxicological importance due to its low magnitude and isolated nature.

When compared with control values, T4 thyroid hormone levels were decreased in males (-22%; p<0.01) and females (-23%; not statically significant) at 1000 mg/kg/day.These results were due to higher control values in males and remained within the range of the Historical Control Data and did not correlate with any other findings. They were therefore considered to be of minor toxicological importance. No relevant differences from controls were observed in T3 or TSH concentrations of test item-treated animals.

A dose-related alpha 2u-globulin nephropathy was observed in the kidney of male rats at all dose levels, and accumulation of pigmented macrophages was noted in the mesenteric lymph node in a few male rats at the high-dose only. None of these changes were considered adverse in absence of associated clinical or clinical pathology changes.

 

Conclusion

The test item was administered daily for 13 weeks by gavage to male and female Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/day in 0.5% (w/w) methylcellulose aqueous solution.

 

There were no relevant test item-related or adverse effects on mortality, clinical signs, ophthalmology, body weight, food consumption, clinical pathology, estrous cycle, sperm parameters, hormone levels or histopathology parameters.

 

Under the experimental conditions and according to the results of this study, the No Observed Adverse Effect Level (NOAEL) was set at 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The 9-day study is considered to be reliable because it was performed according to the OECD guideline (klimisch score = 1).

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

90 -day repeated toxicity study (2019):

Three groups of ten male and ten female Sprague-Dawley rats received the test item, daily, by the oral route, (gavage) for 13 weeks. The test item was administered at dose levels of 100, 300 or 1000 mg/kg/day as a suspension in the vehicle [0.5% (w/w) methylcellulose aqueous solution] under a constant dosage volume of 10 mL/kg/day. A control group of ten males and ten females received the vehicle, alone, under the same experimental conditions.

No test item-related unscheduled deaths occurred during the study. No test item-related clinical signs were noted during the study. The results of the functional observation battery were unaffected by the test item treatment. There were no significant effects on body weight, body weight gain or food consumption in either sex. No test item-related ophthalmological findings were observed at the end of the treatment period. No relevant test item-differences were observed in estrous cycle, epididymal sperm motility, morphology, and epididymal sperm count. Low testicular sperm count was noted in males given 1000 mg/kg/day. This variation was considered to be of minor toxicological importance as no histological correlates were noted. There were no relevant differences from controls in the hematological parameters. Dose-related increases in biliary acid levels were noted from 300 mg/kg/day in females, and increased creatinine and urea levels were recorded in males given 1000 mg/kg/day. All these changes were considered to be of minor toxicological importance as there were no corresponding histological findings. The lower pH value of the urine noted in males and females given 1000 mg/kg/day was considered to be of minor toxicological importance due to its low magnitude and isolated nature.

When compared with control values, T4 thyroid hormone levels were decreased in males (-22%; p<0.01) and females (-23%; not statically significant) at 1000 mg/kg/day.These results were due to higher control values in males and remained within the range of the Historical Control Data and did not correlate with any other findings. They were therefore considered to be of minor toxicological importance. No relevant differences from controls were observed in T3 or TSH concentrations of test item-treated animals.

A dose-related alpha 2u-globulin nephropathy was observed in the kidney of male rats at all dose levels, and accumulation of pigmented macrophages was noted in the mesenteric lymph node in a few male rats at the high-dose only. None of these changes were considered adverse in absence of associated clinical or clinical pathology changes.

In conclusion, there were no relevant test item-related or adverse effects on mortality, clinical signs, ophthalmology, body weight, food consumption, clinical pathology, estrous cycle, sperm parameters, hormone levels or histopathology parameters. Under the experimental conditions and according to the results of this study, the No Observed Adverse Effect Level (NOAEL) was set at 1000 mg/kg/day.

28 -day repeated toxicity study (2012):

Three groups of five male and five female Sprague-Dawley rats received the test item by daily oral administration for 28 days at dose-levels of 100, 300 or 1000 mg/kg/day. The test item was administered as a suspension in the vehicle (0.5% methylcellulose aqueous solution) at a constant dosage-volume of 10 mL/kg/day. A control group of five males and five females received the vehicle alone under the same experimental conditions.

No mortalities or test item treatment-related clinical signs were observed.The mean body weight, body weight gains andfood consumption in males and females were not affected by the test item treatment.There were no test item-related effects on FOB assays, including motor activity.

At hematology, no test item-related effects were observed. At blood biochemistry, at 1000 mg/kg/day, a statistically significant lower alkaline phosphatase activity was observed in females and at 300 mg/kg/day slightly higher chloride levels were noted in males and minimally higher sodium level observed in females.These changes in blood biochemistry parameters were considered as unrelated to the test item (very slight amplitude of variation and without any dose-relationship). At urinalysis, no relevant changes were recorded. At the histopathological examination, there was a slightly higher incidence of minimal alveolar foamy macrophages in lungs. This non adverse increased incidence was considered to be of doubtful toxicological significance.

To conclude, at 1000 mg/kg/day, the test item was clinically well-tolerated and no relevant findings were noted at clinical pathology. At the histopathological examination, there was a slightly higher incidence of minimal alveolar foamy macrophages in lungs. This non adverse increased incidence was considered to be of doubtful toxicological significance. At 300 and 100 mg/kg/day, no test item effects were observed. No significant changes were observed at clinical pathology or histopathology. Under the experimental conditions of the study, the No Observed Adverse Effect Level (NOAEL) was established at 1000 mg/kg/day.

Justification for classification or non-classification

Due to the absence of adverse effect in the 28 -day and 90 -day repeated toxicity studies, no classification for repeated toxicity is required for Dipentamethylenethiuram hexasulfide according to the Regulation EC n°1272/2008.