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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-01-17 - 2012-12-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented GLP-guideline study
Cross-reference
Reason / purpose:
reference to other study
Remarks:
range-finder 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: pilot study for GLP-guideline study
Reason / purpose:
reference to other study
Remarks:
main study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a first pilot study Rhenocure S Trocken Lohn was administered orally by gavage to 3 male and 3 female rats per group, in daily doses of 0, 500 or 1000 mg/kg b.w. per day for an intended period of 2 weeks.
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Gesundheit und Soziales Des Landes Nordrhein-Westfalen
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
other: tap water/Cremophor (98 % / 2 %, v/v)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
first study and second study (day 1 - 14)
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
first study (day 1 - 14)
Dose / conc.:
750 mg/kg bw/day (nominal)
Remarks:
first study (day 6 - 14)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
first study (day 1 - 4)
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
second study (day 1 - 14)
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
second study (day 1 - 14)
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
second study (day 1 - 14)
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Details on study design:
In a first pilot study (study No. 18082738) the test item was administered orally by gavage to 3 male and 3 female rats per group, in daily doses of 0, 500 or 1000 mg/kg b.w. per day for an intended period of 2 weeks. Due to severe toxic effects the treatment of the animals of this dose group was stopped on day 4. From day 6 of the study onwards 3 males and 3 females were included in the study and w ere treated with 750 mg/kg for a period of 10 days. Whereas females of the 750 mg/kg group slightly lost body weight after the first treatments and then gained body weight comparable to the control group, males lost body weight after 6 treatments, resulting in 19% lower body weights compared to control at the end of the study. The body weights of animals dosed with 500 mg/kg were unaffected by the treatment. Histopathology revealed in all treatment groups moderate to marked hyperplasia of the forestomach mucosa as well as hyperkeratosis/parakeratosis. Furthermore, erosions of the forestomach mucosa associated with inflammation of the forestomach and in the glandular stomach degeneration/regeneration mainly of surface epithelium and superficial part of the glands were seen.
Based on these results, doses of 0-50-125-250 mg/kg body weight were chosen for a second pilot study. The main findings in this study were changes in the forestomach: a slight hyperplasia of the epithelium was also observed in one animal at 50 mg/kg and all animals at 125 mg/kg and in a more severe grade in all animals at 250 mg/kg. This finding was accompanied by hyperkeratosis in the mid and high dose and a slight inflammation at 250 mg/kg.
Positive control:
not performed
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
first study: Females ofthe 750 mg/kg group slightly lost body weight after the first treatments and thereafter gained body weight comparable to the control group, males lost body weight after 6 treatments, resulting in 19% lower body weights compared to the control at the end ofthe study. The body weights of animals dosed with 500 mg/kg were unaffected by the treatment.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
first study: Histopathology revealed in all treatment groups moderate to marked hyperplasia of the forestomach mucosa as well as hyperkeratosis/parakeratosis. Furthermore, erosions of the forestomach mucosa associated with inflammation of the forestomach and in the glandular stomach degeneration/regeneration mainly of surface epithelium and superficial part of the glands were seen.

second study: A slight hyperplasia of the epithelium was also observed in one animal at 50 mg/kg and all animals at 125 mg/kg and in a more severe grade in all animals at 250 mg/kg. This finding was accompanied by hyperkeratosis in the mid and high dose and a slight inflammation at 250 mg/kg.
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Remarks on result:
not determinable because of methodological limitations
Critical effects observed:
not specified
Conclusions:
Based on these results 0, 10, 40, 160 mg/kg b.w. were chosen for the main study.
Executive summary:

In a first pilot study (study No. T8082738) Rhenocure S Trocken Lohn was administered orally by gavage to 3 male and 3 female rats per group, in daily doses of 0, 500 or 1000 mg/kg b.w. per day for an intended period of 2 weeks. Due to severe toxic effects at 1000 mg/kg the treatment of the animals of this dose group was stopped on day 4. From day 6 of the study onwards, 3 males and 3 females were included in the study and were treated with 750 mg/kg for a period of 10 days. Whereas females of the 750 mg/kg group slightly lost body weight after the first treatments and thereafter gained body weight comparable to the control group, males lost body weight after 6 treatments, resulting in 19% lower body weights compared to the control at the end ofthe study. The body weights of animals dosed with 500 mg/kg were unaffected by the treatment. Histopathology revealed in all treatment groups moderate to marked hyperplasia of the forestomach mucosa as well as hyperkeratosis/parakeratosis. Furthermore, erosions of the forestomach mucosa associated with inflammation of the forestomach and in the glandular stomach degeneration/regeneration mainly of surface epithelium and superficial part of the glands were seen. Based on these results, doses of 0-50-125-250 mg/kg body weight were chosen for a second pilot study. The main findings in this study were changes in the forestomach: a slight hyperplasia of the epithelium was also observed in one animal at 50 mg/kg and all animals at 125 mg/kg and in a more severe grade in all animals at 250 mg/kg. This finding was accompanied by hyperkeratosis in the mid and high dose and a slight inflammation at 250 mg/kg.

Based on these results 0, 10, 40, 160 mg/kg b.w. were chosen for the main study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Arbeit, Gesundheit und Soziales Des Landes Nordrhein-Westfalen
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: white solid
Details on test material:
- Name of test material (as cited in study report): 1,1'-dithiobis[hexahydro-2H-azepin-2-one]
- Substance type: organic
- Physical state: white solid
- Analytical purity: 99,32 %
- Stability under test conditions: until June 14, 2012
- Storage condition of test material: refrigerator, cool and dry

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS (Wistar (Hsd Cpb:WU))
- Source: Harlan Laboratories BV, Horst, The Netherlands
- Age at delivery: 5-6 weeks
- Weight at study initiation: males: 163-185 g (mean: 174,4 g); females: 135-156 g (mean: 143.3 g)
- Housing: From arrival to tattooing: individually in Makrolon cages Type Ila.
From tattooing to necropsy: in groups with 2 or 3 animals per cage in Makrolon cages Type IV.
Bedding material: Low-dust wood granules (Lignocel BK 8-15; supplier: Ssniff Spezialdiäten Inc. Soest, Westfalen, Germany;
manufacturer: J. Rettenmeier, Ellwangen-Holzmühle, Germany).
Wooden blocks (supplied by Tapvei OY. Kortteinen, Finland) for environmental enrichment will be added to each cage.
As soon as necessary, they will be replaced by new ones.
- Diet (e.g. ad libitum): ad libitum (ssniff R/M-H. 10 mm; Alleinlutter für Ratten und Mäusehaltung: ssniff Spezialdiäten GmbH, Soest, Germany.)
- Water (e.g. ad libitum): ad libitum (Tap water will be given in polycarbonate bottles)
- Acclimation period: approximately one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): Approximately 55 % (Temperature and relative humidity are recorded continuously.)
- Air changes (per hr): > 10 /hour
- Photoperiod (hrs dark / hrs light): 12h/12h (During the light period a radio program is playing.)

IN-LIFE DATES: From: 17 Jan 2012 To: 24 Feb 2012

OTHER:
- identification: Tail tattoos and cards
- Wet cleaning of the floor and tables including disinfection weekly.
Detailed room cleaning and disinfection in intervals as routine.
Continuous pest control using sticky cockroach traps on pheromone basis (purchased from Killgerm GmbH, Neuss, Germany).
- Cages and drinking bottles are exchanged for clean ones routinely. Cage lids and racks are exchanged or cleaned periodically as routine.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: tap water/Cremophor (98 % / 2 %, v/v)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was administered orally by gavage in tap water / Cremophor (98% / 2%; v/v). The administration volume was 10 mL/kg body weight. The formulation were prepared as needed and taking into account the analytically determined stability. The formulations were stirred with a magnetic stirrer for several minutes (until an obviously homogenous distribution occurs) before application. They were also stirred during application.
The formulation is stored at room temperature. The formulation is at least stable for 7 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): not given
- Amount of vehicle (if gavage): 10 mL / kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before the start of treatment, the suitability of the proposed formulations was confirmed by the analyses of concentration and stability. Analyses were carried out under F0012098.
Analyses have shown that in the concentration range from 1 mg/mL to 100 mg/mL the test substance is homogenously distributed in the vehicle and is stable for at least 7 days.
- Concentration/Homogeneity: The homogeneity and the concentrations of the formulations prepared were determined twice during the study.
- Stability: The formulations prepared were analyzed 0, 3, 4, 7 and 8 days after preparation.
Duration of treatment / exposure:
30 days (males) / 31 days (females)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
10 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
40 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
160 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a first pilot study (study No. 18082738) the test item was administered orally by gavage to 3 male and 3 female rats per group, in daily doses of 0, 500 or 1000 mg/kg b.w. per day for an intended period of 2 weeks. Due to severe toxic effects the treatment of the animals of this dose group was stopped on day 4. From day 6 of the study onwards 3 males and 3 females were included in the study and w ere treated with 750 mg/kg for a period of 10 days. Whereas females of the 750 mg/kg group slightly lost body weight after the first treatments and then gained body weight comparable to the control group, males lost body weight after 6 treatments, resulting in 19% lower body weights compared to control at the end of the study. The body weights of animals dosed with 500 mg/kg were unaffected by the treatment. Histopathology revealed in all treatment groups moderate to marked hyperplasia of the forestomach mucosa as well as hyperkeratosis/parakeratosis. Furthermore, erosions of the forestomach mucosa associated with inflammation of the forestomach and in the glandular stomach degeneration/regeneration mainly of surface epithelium and superficial part of the glands were seen.
Based on these results, doses of 0-50-125-250 mg/kg body weight were chosen for a second pilot study. The main findings in this study were changes in the forestomach: a slight hyperplasia of the epithelium was also observed in one animal at 50 mg/kg and all animals at 125 mg/kg and in a more severe grade in all animals at 250 mg/kg. This finding was accompanied by hyperkeratosis in the mid and high dose and a slight inflammation at 250 mg/kg. Based on these results 0, 10, 40, 160 mg/kg were chosen for the present study.

- Rationale for animal assignment (if not random): Appointment of rats to study groups was done by randomization using the procedure within the Pristima System. Rats with extreme body weights were excluded during randomization.
Control and treated animals were handled each in the same manner.

Positive control:
not performed

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Inspections on mortality and morbidity of the animals were performed twice daily. General condition and behavior of animals were checked and recorded daily about 1 hour after administration per gavage.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A careful clinical examination was performed daily prior to treatment. Any clinical signs and abnormalities including changes in skin, fur, eyes, mucous membranes occurrence of secretions and excretions, autonomic activity, changes in gait, posture, and response to handling, clonic/tonic movements, stereotypes and bizarre behavior were recorded as well as time of onset, location and grading. Furthermore, an observation outside the home cage in a standard arena was performed once prior to treatment and weekly thereafter in all animals before treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: The individual body weights were determined just before first treatment of animals and daily thereafter. The corresponding application volumes, were filed together with the study raw data and are not reported. Furthermore, body weights were recorded immediately before scheduled necropsies, for calculation of relative organ weights.

FOOD AND WATER CONSUMPTION:
- The food and water consumption per cage were determined weekly. These data were then used to calculate the group means for each period of approximately 7 days. The weight of the food offered at the start of the measurement period minus the weight of the food at the end of the period is defined as the food consumption of the animal in g. Comparable calculation were done for the water intake.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In general, the determinations were performed using standardized procedures subjected to continuous internal and external quality control.
The blood samples for determination of glucose concentrations were taken from one of the caudal veins of non-fasted, non-anesthetized animals.
The blood samples used for determining the other parameters in peripheral blood were collected in the morning from the retro-bulbar venous plexus of non-fasted animals anesthetized with C02/room air. The blood obtained was treated as follows:
The samples for the hematological determinations were collected in tubes coated with EDTA (anticoagulant).
The samples for the determinations of the thromboplastin time (HQUICK) were collected in tubes with sodium-citrate.
The samples for the determinations of electrolyte concentrations remained untreated.
The samples for other biochemical tests were heparinized.
The blood samples for glucose determinations were mixed with perchloric acid ( 1 + 1 0) to precipitate proteins.
- Anaesthetic used for blood collection: No (for determination of glucose), Yes (all other animals)
- Animals fasted: No
- Parameters examined: Leukocytes, erythrocytes, haemoglobin, haematocrit, reticulocytes, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCI 1). mean corpuscular haemoglobin concentration (MCHC), thrombocytes, Hepato Quick, differential blood count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of sceduled necropsy
- Animals fasted: No
- How many animals: all animals of groups 1-4
- Parameters examined: Alanine aminotransferase, aspartate aminotransferase, albumin, protein (total), bile acids, cholesterol, creatinine, urea, glucose, sodium, potassium, total bilirubin.
In general, the determinations were performed using standardized procedures subjected to continuous internal and external quality control.
Occasionally, sample quantity may have been insufficient to permit determination of all intended parameters, or no determination was possible due to technical faults. The planned number of measurements per group may, therefore, not necessarily be obtained in each case.
The blood samples for determination of glucose concentrations were taken from one of the caudal veins of non-fasted, non-anaesthetized animals.
The blood samples used for determining the other parameters in peripheral blood were collected in the morning from the retro-bulbar venous plexus of non-fasted animals anesthetized with CO2/room air. The blood obtained was treated as follows:
The samples for the hematological determinations were collected in tubes coated with EDTA (anticoagulant).
The samples for the determinations of the thromboplastin time (HQUICK) were collected in tubes with sodium-citrate.
The samples for the determinations of electrolyte concentrations remained untreated.
The samples for other biochemical tests were heparinized.
The blood samples for glucose determinations were mixed with perchloric acid (1+10) to precipitate proteins.

OTHER:
Functional Observational Battery (performed once - not blind):
- These examinations were conducted on all animals in dosing week 3/4, earliest about 30 minutes after the treatment.
The following observations/examinations were performed:
- home cage observation: posture, piloerection, gait abnormalities, involuntary motor movements, vocalizations, others.
- observations during handling: ease of removing, reaction to being handled, muscle tone, palpebral closure, lacrimation. nasal discharge, salivation, stains, others.
- open field observations: piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, bizarre behavior, gait abnormalities, vocalizations, arousal, rearing, defecation, urination.
- reflex / physiological observations: approach response, touch response, auditory response, tail pinch response, pupil size, pupil response (pupil response , righting reflex, grip strength, landing foot splay, body temperature, body weight.

Motor activity:
These examinations were conducted once following the FOB examination.
Motor activity (MA) and locomotor activity (LMA) were examined as activity for the entire 60-minute session (Summary Session MA and LMA) and activity during each 10-minute interval (Summary Interval MA and LMA). Motor activity was measured as the number of beam interruptions that occurred during the test session.
Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the animal relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy
All animals living on the date of their scheduled necropsy and all animals to be killed in moribund state were sacrificed by exsanguination under deep isoflurane anaesthesia, necropsied and their organs and tissues subjected to thorough gross pathological examination. Changes were described in terms of localization, size, color and consistency whenever appropriate.
If animals died spontaneously during the study they were necropsied at the earliest opportunity. From these animals the organs and tissues were handled as described above.

Organ Weights
From all animals necropsied as scheduled, the organs listed in the following Table 2 marked with "X" in the column "Organ weights" was trimmed and weighed wet, as soon as possible after dissection. The ratio of organ weight to body weight was calculated.

HISTOPATHOLOGY: Yes
Preservation of Tissues
The fixed material was retained (pathology no. 8107). Histological procedures, organs and tissues fixed and evaluated are given in details in the Pathology Report in the Annex (Section 7.8). The scope of organs histopathologically evaluated is shown in Table 2.
Histopathological Technique
Histopathological technique will be performed on tissues marked with "X" in the following Table 2 in the column "Histotechnique up to slides"".
Sections of all tissues will be stained with hematoxylin and eosin (1 l&L ). Cryocuts from liver sections will additionally be stained with Oil Red O.
Osseous tissues will be decalcified before further processing.
Histopathological Evaluation
The tissues in the following Table marked with "X" in the column "Histopathological evaluation" from animals (from decedent animals as far as possible) will be histopathologieally evaluated. If possible treatment-related effects occur this organ will be examined in the lower dose groups. This includes also organs being morphologically connected or fixed with them. The organs of animals of groups 2, 3, which died or were killed prematurely, will be histopathologieally evaluated as described for animals of group 4. If additional organs have to be examined according to these rules, this will be noted in the raw data without amending the study plan..
Statistics:
The statistical evaluation of data related to clinical chemistry, haematology, body and organ weights were performed using routines of the validated Pristima System. Statistical evaluations on FOB, body weight and absolute organ weight data were done using the Dunnett-test. For relative organ weights the Dunnett exact homogenous test after logarithmic transformation were used. Data for food/water intake were statistically evaluated using the adjusted Mann-Whitney U-Test. Clinical pathology data were evaluated using a Dunnett exact homogenous or heterogeneous test, a Dunnett exact homogenous test after logarithmic transformation or the Bonferroni/Mann-Whitney U-test.
All variables that are not dichotomous are described by sex, dose group and date using appropriate measures of central tendency (mean, median) and general variability (standard deviation, minimum, maximum). Statistical tests were not performed for groups, which are smaller than 3.
Statistics of MA/LMA were generated with an evaluation step of the SPADER application. Generation of results was done for duration and interval analysis separately. A preliminary step determined whether there exist significant interactions at all for duration and interval analysis. If so, a Dunnett test based on the GLM Procedure were executed to determine which specific groups differ in a significant way on basis of the computed means with regard to the comparison group.
For statistical evaluations of histopathological data, if any, the PATHDATA program was used and described in detail in the Pathology Report.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No animal died unscheduled. Therefore, survival was not affected by the treatment with the test substance. At clinical observations no findings were observed.
Mortality:
no mortality observed
Description (incidence):
No animal died unscheduled. Therefore, survival was not affected by the treatment with the test substance. At clinical observations no findings were observed.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight development as well as food and water intake in treated groups was comparable to the respective control group.
Haematological findings:
no effects observed
Description (incidence and severity):
Neither hematology nor clinical chemistry gave evidence for treatment-related effects up to 160 mg/kg.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Neither hematology nor clinical chemistry gave evidence for treatment-related effects up to 160 mg/kg.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Determination revealed slightly increased absolute & relative weights of livers at 160 mg/kg in females & decreased weights of thymus in males of this dose group. The increased mean weight of uterus at 10 mg/kg is mainly caused by a high value of animal27
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related findings indicating reduced local tolerance were observed at necropsy and at histopathology in the forestomach % small intestine at 160 mg/kg. At necropsy depression/s, thickening or nodule/s of the forestomach mucosa were described.
Details on results:
CLINICAL SIGNS AND MORTALITY
Survival was not affected by the treatment with the test substance. At clinical observations no findings were observed.

BODY WEIGHT AND WEIGHT GAIN
Body weight development as well as food and water intake in treated groups was comparable to the respective control group. Body weight development of males and females was not retarded by the treatment up to 160 mg/kg.

FOOD CONSUMPTION
Mean food intake over the dosing period was comparable to the respective control. Food and water intake in treated groups was comparable to the respective control group.

WATER CONSUMPTION:
The water intake was not relevantly affected by the treatment with the test substance. Food and water intake in treated groups was comparable to the respective control group. The water intake was not relevantly affected by the treatment with the test substance.

FUNCTIONAL OBSERVATIONAL BATTERY:
In week 3/4 a FOB was conducted including home cage observation, observation during handling and in an open field, reflex/physiological observations and measurement of grip strength. The investigation gave no evidence for treatment-related findings.

MOTOR ACTIVITY ASSESSMENT:
Motor activity measurements of male and female rats were made in week 3/4, respectively. The activity determination over the entire 60 minute observation period failed to reveal a significant effect on motor (MA) and locomotor activity (LMA). The results of the 10-minute intervals indicate that there were no relevant differences between treated and control groups In particular, there were no significant differences in the first 10-minute intervals which are regarded as best indicator of increases or decreases in activity before the animals habituate to a minimal level of activity.

HAEMATOLOGY
Hematology gave no evidenced for treatment-related effect on red or white blood or blood coagulation. Neither hematology nor clinical chemistry gave evidence for treatment-related effects up to 160 mg/kg.

CLINICAL CHEMISTRY
Clinical chemistry gave no evidence for treatment-related changes. The only conspicuous findings were decreased activities of ALAT at 10 mg/kg and of ASAT at 160 mg/kg in females. However, due to missing dose dependence, relatively slight differences to control and/or due to the fact that generally increases of these activities indicate adverse effects these changes are not regarded as toxicologically relevant.

GROSS PATHOLOGY / HISTOPATHOLOGY:
At necropsy treatment-related findings were observed in the stomach of males and females treated with 160 mg/kg. One male and 3 females showed depressions of the stomach, in one male it was thickened and in one females a nodule was observed.
Determination of organ weights revealed slightly increased absolute and relative weights of livers at 160 mg/kg in females and decreased weights of thymus in males of this dose group.
The increased mean weight of uterus at 10 mg/kg is mainly caused by the high value of animal No. 27.

In the forestomach, focal or diffuse hyperplasia of the squamous epithelium were observed in most animals dosed at 160 mg/kg. In females, in addition inflammatory infiltrates and edema were seen at a slightly increased incidence at that dose level. One female showed an erosion of the forestomach mucosa. In the duodenum or jejunum, increased goblet cells were seen in the small intestine at 160 mg/kg. No signs of inflammation were present in the small intestine. In one female at 40 mg/kg, an inflammatory infiltrate and minimal edema were observed at the limiting ridge between forestomach and glandular stomach. This lesion is likely of spontaneous origin since it was not associated with any changes of the forestomach epithelium.
There was no evidence of any systemic effect related to dosing with the test compound up to and including 160 mg/kg.
In conclusion, treatment-related findings indicating reduced local tolerance were observed at necropsy and at histopathologically in the forestomach and small intestine at 160 mg/kg. At necropsy depression/s, thickening or nodule/s of the forestomach mucosa were described. These findings correlated with diffuse hyperkeratosis, focal hyperplasia and erosion of the forestomach mucosa. Inflammatory infiltrates and edema were seen at slightly increased incidences at 160 mg/kg. In the duodenum and jejunum, increased goblet cells were seen in the mucosa at 160 mg/kg. Furthermore, in the duodenum or jejunum, increased goblet cells were seen in the small intestine. This indicates increased mucous production related to decreased local tolerance.
There was no evidence of any systemic effect related to dosing with the test compound up to and including 160 mg/kg.

Daily oral treatment of rats with the test item at doses of 0, 10, 40, 160 mg/kg b.w. for a period of 4 weeks resulted in local effects in the forestomach and small intestine.
Under the conditions described the no observed adverse effect level (NOAEL) for administration of the test substance to male and female Wistar rats was 40 mg/kg bw.
This NOAEL is set due to local irritating effects. There was no evidence of any systemic effect related to dosing with the test compound up to and including 160 mg/kg.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: local irritation in forestomach
Dose descriptor:
NOAEL
Effect level:
> 160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There was no evidence of any systemic effect related to dosing with the test compound up to and including 160 mg/kg.
Remarks on result:
other: highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table‎ 3: Body Weights (g)

Sex

Males

Females

Dose

 (mg/kg)

0

10

40

160

0

10

40

160

Day

 

 

 

 

 

 

 

 

1

174.0

176.0

175.2

172.2

142.2

143.0

143.6

144.2

2

180.0

179.4

182.4

178.2

144.8

148.6

146.4

146.2

3

189.0

188.6

190.4

184.6

146.8

149.2

150.2

149.8

4

196.4

195.2

197.2

191.4

151.2

152.6

153.6

154.0

5

203.2

201.0

205.4

200.2

154.8

155.8

157.6

159.2

6

211.6

210.0

213.0

207.2

156.6

161.8

160.8

164.0

7

218.0

215.0

219.8

214.8

158.0

163.0

163.2

164.8

8

224.8

221.8

226.8

222.0

162.2

167.0

169.2

169.0

9

232.2

229.4

234.6

229.2

168.4

170.6

172.4

175.6

10

238.2

235.6

241.2

236.8

169.4

173.6

177.0

178.4

11

247.0

242.6

248.6

242.6

172.0

176.0

175.8

177.8

12

250.2

248.8

253.0

248.8

176.2

180.8

182.6

183.6

13

257.6

252.2

259.2

254.4

180.0

184.2

185.2

187.6

14

266.6

260.0

268.0

263.8

181.6

187.8

189.6

192.2

15

269.8

264.8

273.2

267.4

184.0

187.4

188.6

190.8

16

278.2

270.8

280.6

274.4

189.4

190.6

195.2

195.2

17

279.4

274.2

284.4

277.2

192.4

194.6

197.0

199.8

18

287.4

279.0

290.8

282.4

193.0

198.4

199.4

203.0

19

289.4

285.0

293.8

283.8

194.6

195.0

199.8

200.6

20

295.6

288.2

299.6

289.8

198.4

199.2

204.8

205.2

21

299.2

294.6

304.8

295.6

203.2

203.2

205.4

211.4

22

303.6

295.4

307.6

297.2

202.0

208.0

209.2

212.8

23

306.2

300.2

311.0

302.0

202.4

205.4

207.4

210.6

24

311.2

304.0

316.8

308.2

206.0

205.6

212.6

214.6

25

314.8

306.6

320.0

310.4

209.6

210.2

214.8

217.6

26

317.8

312.8

324.2

313.2

210.2

213.8

216.0

220.4

27

323.6

314.8

329.4

316.6

211.8

214.8

217.4

219.2

28

326.6

318.0

332.2

321.8

213.0

214.8

221.4

221.8

29

331.2

324.2

335.4

326.4

218.4

218.8

222.2

226.4

30

336.6

328.8

344.0

331.8

217.4

223.8

224.8

228.0

31

 

 

 

 

219.0

221.6

223.8

224.0

Table 4: -Mean Daily Food Intake

Group means

Dose

Days

g/animal

g/kg body weight

ppm

 

per day

per day

Male

0

1-29

25.0

89.9

10

1-29

23.9

87.8

40

1-29

25.0

88.8

160

1-29

24.8

90.5

Female

0

1-29

17.4

91.4

10

1-29

18.0

93.2

40

1-29

17.5

89.5

160

1-29

18.8

95.2

Table5: -Mean Daily Water Intake

Group means

Dose

Days

g/animal

g/kg body weight

ppm

 

per day

per day

Male

0

1-29

28.8

103.8

10

1-29

30.9

113.5

40

1-29

31.4

112.7

160

1-29

30.9

111.4

Female

0

1-29

21.1

116.7.

10

1-29

22.1

114.3

40

1-29

21.4

109.4

160

1-29

22.6

114.2

Table 6: Haematology

Dose

ERY

HB

HCT

MCH

MCHC

MCV

RETI

THRO

Hep-Quick

(mg/kg)

T/l

g/l

l/l

pg

g/l Ery

fl

%

G/l

sec.

Males - Day 31

0

8.398

156.0

0.5218

18.58

299.0

62.14

2.10

1075.0

34.18

10

8.484

158.0

0.5328

18.60

296.2

62.84

1.90

 981.2

36.34

40

8.396

157.6

0.5300

18.80

297.2

63.22

2.08

 978.2

34.16

160

8.426

156.0

0.5260

18.54

297.0

62.44

2.42

1058.8

33.34

Female - Day 32

0

8.276

150.4

0.5126

18.20

293.8

61.94

1.84

1150.8

32.10

10

8.470

155.0

0.5350

18.30

289.8

63.18

2.12

 975.4*

33.50

40

8.368

151.4

0.5112

18.10

296.2

61.12

1.74

1119.4

32.00

160

8.534

154.6

0.5234

18.12

295.4

61.34

1.82

1100.2

32.20

 

Table 7: Differential Blood Count

Dose

LEUCO

LYM

NEUTRO

Basophils

EOS

MONO

Atypical

(mg/kg)

G/l

G/l

G/l

G/l

G/l

G/l

G/l

Males - Day 31

0

12.126

10.742

0.828

0.100

0.100

0.272

0.082

10

10.948

 9.740

0.738

0.072

0.112

0.198

0.088

40

11.066

 9.852

0.706

0.070

0.106

0.234

0.100

160

10.950

 9.622

0.844

0.072

0.102

0.218

0.090

Females - Day 32

0

 9.112

 8.074

0.608

0.070

0.086

0.194

0.076

10

 8.362

 7.426

0.580

0.056

0.076

0.154

0.076

40

 8.966

 7.854

0.700

0.068

0.106

0.160

0.072

160

 8.636

 7.596

0.650

0.074

0.074

0.174

0.078

Table 8: Clinical Chemistry:

Dose

ALAT

ASAT

(mg/kg)

U/l

U/l

Males Day 31

0

55.72

60.58

10

61.16

63.44

40

59.58

58.64

160

61.66

60.54

Females Day 32

0

55.88

64.22

10

45.82**

57.68

40

51.12

54.32

160

48.68

56.92**

**   = significantly different at p <= 0.01

Table 9: Clinical Chemistry: Substrates

Dose

GLUCOSE

CHOL

CREA

UREA

Bili-t

S-Bile ac.

Protein

Albumin

(mg/kg)

mmol/l

mmol/l

µmol/l

mmol/l

µmol/l

µmol/l

g/l

g/l

Males - Day 31 

0

5.620

2.412

51.2

7.436

0.10

23.52

67.24

36.44

10

5.294

2.056

54.4

6.878

0.02

27.64

67.68

36.86

40

5.454

2.080

51.0

6.640

0.00

22.92

66.32

36.44

160

5.334

2.364

51.0

7.478

0.06

31.02

65.78

36.14

Females - Day 32

0

5.148

1.728

54.8

7.184

0.06

21.68

65.84

37.54

10

4.978

1.974

53.8

7.032

0.04

27.76

64.84

36.50

40

5.124

2.140

53.2

6.790

0.02

13.66

65.80

37.14

160

5.192

2.100

52.6

8.074

0.00

10.16

66.90

37.36

  

Table 10: Clinical Chemistry: Electrolytes

 

Na

K

Dose

mmol/l

mmol/l

Males

Day 31

 

0 mg/kg

147.6

6.28

10 mg/kg

147.2

6.12

40 mg/kg

147.6

6.10

160 mg/kg

147.4

6.04

Females

Day 32

 

0 mg/kg

145.6

5.44

10 mg/kg

145.6

5.34

40 mg/kg

146.0

5.42

160 mg/kg

146.4

5.14

Applicant's summary and conclusion

Conclusions:
The GLP-study was performed according to the OECD Guideline 407 in rats without deviations and is considered to be reliable without restrictions (reliability Klimisch 1). The test material did induce a response and the results show that the test item has a NOAEL of 40 mg/kg bw.
Executive summary:

In a repeated dose study (Schadt, 2012, according to OECD 407), the substance was administered orally by gavage to 5 male and 5 female Wistar (Hsd Cpb:WU) rats per dose group using tap water / Cremophor (98% / 2%; v/v)as vehicle, in daily doses of 0, 10, 40, 160 mg/kg bw for a period of 4 weeks. The animals were regularly observed and weighed and feed and water intakes were determined. In addition, clinical laboratory investigation of blood samples was performed. Organs and tissues were subjected to gross and histopathological investigation.

Survival was not affected by the treatment with the test substance. At clinical observations no findings were observed. Body weight development as well as food and water intake in treated groups was comparable to the respective control group. Neither haematology nor clinical chemistry gave evidence for treatment-related effects up to 160 mg/kg. Treatment-related findings indicating reduced local tolerance were observed at necropsy and at histopathologically in the forestomach and small intestine at 160 mg/kg. At necropsy depression/s, thickening or nodule/s of the forestomach mucosa were described. These findings correlated with diffuse hyperkeratosis, focal hyperplasia and erosion of the forestomach mucosa. Inflammatory infiltrates and oedema were seen at slightly increased incidences at 160 mg/kg. One female showed an erosion of the forestomach mucosa. The forestomach is particularly sensitive to local irrtancy since the test compound remains there for prolonged time period after oral uptake. These findings are an indication of reduced local tolerance. Furthermore, in the duodenum and jejunum, increased goblet cells were seen in the mucosa at 160 mg/kg. Furthermore, in the duodenum or jejunum, increased goblet cells were seen in the small intestine. This indicates increased mucous production related to local irritation.

In conclusion, daily oral treatment of rats with the test item resulted in local effects in the forestomach and small intestine. Under the conditions described the no observed adverse effect level (NOAEL) for administration of 1,1'dithiobis[hexahydro-2H-azepin-2 -one to male and female Wistar rats was 40 mg/kg bw. This NOAEL is set due to local irritating effects. There was no evidence of any systemic effect related to dosing with the test compound up to and including 160 mg/kg. Therefore, the NOAEL based on systemic effects is >160 mg/kg bw/day.