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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-11-26 - 2020-11-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Organization for Economic Co-operation and Development, Testing of Chemicals Guideline No. 408 (revised 25 June 2018)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1,1'-dithiobis[hexahydro-2H-azepin-2-one]
EC Number:
245-910-0
EC Name:
1,1'-dithiobis[hexahydro-2H-azepin-2-one]
Cas Number:
23847-08-7
Molecular formula:
C12H20N2O2S2
IUPAC Name:
1,1'-dithiobis[hexahydro-2H-azepin-2-one]
Test material form:
solid: particulate/powder
Remarks:
white

Test animals

Species:
rat
Strain:
Wistar
Remarks:
RccHan:WIST
Details on species / strain selection:
The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The RccHan:WIST strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 45 to 51 days.
- Weight at study initiation: Males: 140 to 187 g, Females: 119 to 160 g
- Fasting period before study: No
- Housing:
Allocation: Non-selectively allocated on arrival.
Variations in body weight of animals did not exceed ± 20% of the mean for each sex.
Using the sequence of cages in the battery, one animal at a time was placed in each cage with the procedure being repeated until each cage held the appropriate number of animals. Each sex was allocated separately.
Cages: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals.
Cage distribution: Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
Number of animals per cage: Five of the same sex, unless reduced by mortality.
Bedding: Wood based bedding which was changed at appropriate intervals each week.
Environmental Enrichment:
Aspen gnawing material: Provided to each cage throughout the study and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study and replaced when necessary.
- Diet (e.g. ad libitum):
Diet: Teklad 2014C Diet.
Availability: Non-restricted (removed overnight before blood sampling for hematology or blood chemistry).
- Water (e.g. ad libitum):
Supply: Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability: Non-restricted.
- Acclimation period: 16 days before commencement of treatment.

DETAILS OF FOOD AND WATER QUALITY:
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the wood based bedding and Aspen gnawing material.
No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Monitored and maintained within the range of 20-24°C.
- Humidity (%): Monitored and maintained within the range of 40-70%.
Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
- Air changes (per hr): Unknown; Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark.

IN-LIFE DATES:
Animal arrival: 27 November 2019
Necropsy: 11 to 12 March 2020

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral gavage route of administration was chosen to simulate the conditions of systemic exposure.

Route: Oral, by gavage, using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg/day.
Volume dose: 10 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as the treated groups.
Frequency: Once daily at approximately the same time each day.
Formulation: Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Vehicle:
other: 98% water with 2% Kolliphor EL (cremophor) v/v
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Method of preparation :The required amount of test item was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the vehicle to form a paste. Any agglomerates were broken down. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder which had been wetted with vehicle, the mortar was rinsed a minimum of three times or until visibly clean with vehicle and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer. The suspension was stirred using a magnetic stirrer for a minimum of 20 minutes. The suspension was transferred to the final containers, via syringe whilst magnetically stirring.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2 to 8°C).
Test item accounting: Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

VEHICLE
- Justification for use and choice of vehicle (if other than water):
Comparability to previous studies / relative non-toxicity
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability and homogeneity: The suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 100 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
Results indicated that formulations were stable for one day when stored at ambient temperature (15 to 25°C) and 15 days when stored refrigerated (2 to 8°C).
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1 (first preparation), 4 and 13 (last preparation and last preparation repeat) of treatment were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once daily at approximately the same time each day.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
Dose / conc.:
10 mg/kg bw/day (nominal)
Remarks:
low dose group
Dose / conc.:
40 mg/kg bw/day (nominal)
Remarks:
mid dose group
Dose / conc.:
140 mg/kg bw/day (nominal)
Remarks:
high dose group
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels selected for this study (10, 40 and 140 mg/kg/day) have been chosen in conjunction with the Sponsor following the outcome of a previous 4-week toxicity study in Han Wistar rats performed by another laboratory. In this study oral doses of 0, 10, 40 and 160 mg/kg were administered daily. Treatment related findings indicating local irritation were observed at necropsy and histopathology in the forestomach and small intestine at 160 mg/kg. At necropsy depressions and thickening or nodules of the forestomach mucosa were found. This correlated with histopathological investigations which highlighted local irritation in the forestomach and small intestine. In the forestomach - focal hyperplasia of the squamous epithelium and erosion of the forestomach mucosa was observed. In the duodenum and jejunum, increased goblet cells were seen in the mucosa which indicated increased mucous production related to local irritancy. There was no evidence of any systemic effect related to the test item up to and including 160 mg/kg/day, and no effect on body weight, water or food consumption was observed. The NOAEL for repeated oral administration of Caprolactam disulfide to male and female Han Wistar rats was 40 mg/kg/day based on local effects in the high dose group.
Due to the erosion, focal hyperplasia and local irritancy of the small intestine observed in the 28 day repeated dose study and given the increase in study duration from four to 13 weeks, the high dose level was reduced to 140 mg/kg/day for this study. The low and intermediate doses of 10 and 40 mg/kg/day were selected to allow analysis of any dose response relationship and to aid in the determination of a No Observed Adverse Effect Level.

- Rationale for animal assignment (if not random): Non-selectively allocated on arrival.
Variations in body weight of animals did not exceed ± 20% of the mean for each sex.

- Fasting period before blood sampling for clinical biochemistry:
No overnight deprivation of food for Thyroid hormone analysis.
Blood samples were collected after overnight withdrawal of food for hematology and blood chemistry
Positive control:
not required

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: A viability check was performed near the start and end of each working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical and Behavioral Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
Signs Associated with Dosing
Daily during the treatment period, detailed observations were recorded at the following times in relation to dose administration:
• Pre-dose observation.
• On return to the home cage.
• At completion of dosing each group.
• One to two hours after completion of dosing all groups.
• As late as possible in the working day.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy.

FOOD CONSUMPTION: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study.

WATER CONSUMPTION: Yes
Fluid intake was assessed by daily visual observation. No significant effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: Yes
The eyes of the animals were examined by means of a binocular indirect ophthalmoscope as follows:
Occasion Animals
Pretreatment All animals including spares
Week 12 Groups 1 and 4
Prior to each examination, the pupils of each animal were dilated using tropicamide ophthalmic solution (Mydriacyl). The adnexae, conjunctiva, cornea, sclera, anterior chamber, iris (pupil dilated), lens, vitreous and fundus were examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food and prior to dosing at week 13
- Anaesthetic used for blood collection: Yes: Animals were held under light general anesthesia induced by isoflurane.
- Parameters as follows were examined.:
Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
• Hematocrit (Hct)*
• Hemoglobin concentration (Hb)
• Erythrocyte count (RBC)
• Absolute reticulocyte count (Retic)
• Mean cell hemoglobin (MCH)*
• Mean cell hemoglobin concentration (MCHC)*
• Mean cell volume (MCV)
• Red cell distribution width (RDW)
• Total leucocyte count (WBC)
• Differential leucocyte count:
• Neutrophils (N)
• Lymphocytes (L)
• Eosinophils (E)
• Basophils (B)
• Monocytes (M)
• Large unstained cells (LUC)
• Platelet count (Plt)
* Derived values calculated in ClinAxys.

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
• Prothrombin time (PT) - using IL PT Fibrinogen reagent.
• Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food and prior to dosing at week 13
- Animals fasted: Yes
- How many animals: All
- Parameters examined.: Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche Cobas 6000 Analyzer in respect of:
• Alkaline phosphatase (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Total bilirubin (Bili)
• Bile acid (Bi Ac)
• Urea
• Blood urea nitrogen (BUN)
• Creatinine (Creat)
• Glucose (Gluc)
• Total cholesterol (Chol)
• High density lipids (HDL)
• Low density lipids (LDL)
• Sodium (Na)
• Potassium (K)
• Total protein (Total Prot)
• Albumin (Alb)
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: All animals at termination
- Animals fasted: No
- How many animals: all
Sequence of blood sampling on each occasion: To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was randomized to allow satisfactory inter-group comparisons.
Conditions: No overnight deprivation of food.
Blood sample site: Sublingual vein.
Anesthetic: Isoflurane. The animals were not allowed to recover.
Anticoagulant: None.
Blood volume: 1.0 mL.
Blood tubes: Greiner Minicollect tubes with clotting activator.
Processing: Samples were kept at ambient temperature (15 to 25C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions: At 2000g for 10 minutes at 4°C.
All available serum were transferred to an appropriately labelled polypropylene “cryo” tubes using a plastic disposable pipette.
Serum tubes: Standard Covance tubes.
Storage conditions: Serum samples were stored at -60 to -80C, pending any future requirement for analysis.
All serum samples were discarded at the time of report finalization.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Detailed Physical Examination and Arena Observations
Before treatment commenced and during each week of treatment, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.
After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.
Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.
Sensory Reactivity Observations and Grip Strength
Sensory reactivity and grip strength assessments were performed (before dosing) on all animals during Week 12 of treatment. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.
The following measurements, reflexes and responses were recorded:
Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
1 No reaction or ignores/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.
Motor Activity
During Week 12 of treatment (before dosing), the motor activity of each animal was measured using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Covance.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

IMMUNOLOGY: Yes
- Time schedule for examinations: At termination
- How many animals: All
- Dose groups that were examined: All
- Parameters examined.:Triiodothyronine (T3), Thyroxine (T4), Thyroid stimulating hormone (TSH)
Sequence of blood sampling on each occasion: To minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was randomized to allow satisfactory inter-group comparisons.
Conditions: No overnight deprivation of food.
Blood sample site: Sublingual vein.
Anesthetic: Isoflurane. The animals were not allowed to recover.
Anticoagulant: None.
Blood volume: 1.0 mL.
Blood tubes: Greiner Minicollect tubes with clotting activator.
Processing: Samples were kept at ambient temperature (15 to 25C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions: At 2000g for 10 minutes at 4°C.
All available serum were transferred to an appropriately labelled polypropylene “cryo” tubes using a plastic disposable pipette, then mixed by gentle 10-fold inversion. Following mixing, each serum sample was divided into two aliquots
Serum tubes: Standard Covance tubes.
Aliquot volumes: Aliquot 1: T3 and T4 - 0.2 mL of serum.
Aliquot 2: TSH - all remaining serum.
Storage conditions: Stored at -60 to -80C, pending analysis.
Fate of plasma samples: Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.

OTHER:
Vaginal Smears
Wet smears were taken as follows:
Wet smears: Using pipette lavage for four days before scheduled termination.
Smears were assessed to establish the stage of estrus (metestrus, diestrus, proestrus and estrus). These observations assisted in the histological evaluation of estrogen sensitive tissues.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All main study animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
The retained tissues were checked before disposal of the carcass.
Schedule: Animals were killed following 13 weeks of treatment.
Sequence: To allow satisfactory inter-group comparison.

HISTOPATHOLOGY: Yes
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List All animals dying prematurely.
Animals of Groups 1 and 4 killed at a scheduled interval.
Stomach and abnormalities only All animals of Groups 2 and 3 killed at a scheduled interval.
Routine staining Sections were stained with hematoxylin and eosin.
3.7.4 Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Premature deaths All animals from all groups. All specified
Scheduled kill All animals of Groups 1 and 4. All specified
All animals of Groups 2 and 3. Stomach and abnormalities only.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.

Bone Marrow
Bone marrow smears were prepared immediately following death, on completion of the scheduled treatment period during the study.
Fixation Smears were air dried and subsequently fixed in methanol.
Analysis No examinations were performed, however, the smears were retained for possible future examination.
Retention The smears were transferred to archives and will be retained for the same period as the study raw data.

Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes In modified Davidson’s fluid.
Eyes In Davidson’s fluid.
Other examinations:
Sperm Analysis
Left vas deferens : All males: sperm samples were assessed for motility using a computer assisted sperm analyzer (CASA). These data were not reported.
Samples from all animals were retained in neutral buffered formalin pending any future requirement for additional morphology analysis (see below).
Left cauda epididymis : Cauda epididymis were weighed and then retained deep frozen (-10 to 30°C) pending any future requirement for additional analysis (see below).
Left testis: Testis were weighed and then retained deep frozen (-10 to 30°C) pending any future requirement for additional analysis (see below).

Assessment of sperm count and sperm morphology was not required, all retained fixed or frozen samples were discarded when the report was finalized.
Statistics:
yes, see below, due to limitations of this free-text field

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs or signs observed following dose administration that were considered test item-related. As the high incidence of chin rubbing was only seen on Day 28 of the study for both sexes receiving 40 or 140 mg/kg/day and it is a commonly observed sign in studies where the test item is administered by gavage, it is considered not test item-related.
Due to limitations within this free-text field, please see „Overall remarks“ for the tabular presentation of observed effects, see table “Signs associated with dosing - group distribution of observations”.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There was one premature death during the study; Male No. 12 that received 140 mg/kg/day died following the administration of dose on Day 68 of treatment. Macroscopic examination revealed a perforated oesophagus with abnormal contents (clear fluid) in the thoracic cavity. These findings are consistent with a dose administration-related trauma and this death was considered not related to treatment with Caprolactam disulfide.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and overall body weight gain (Weeks 1-13) were not affected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Overall food consumption (Weeks 1-13) was unaffected by treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment related ophthalmoscopic findings in Week 12.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment related hematology findings.
All inter-group differences from controls, including those that attained statistical significance, were minor, seen in one sex only or were without dose-relationship and were, therefore considered to represent normal biological variation. This includes the high mean corpuscular hemaglobin and mean corpuscular hemaglobin concentration, low activated partial thromboplastic time for males given 140 mg/kg/day and low hematocrit, hemaglobin and red blood cell counts for females that received 10, 40 or 140 mg/kg/day. It also include the low neutrophil count for males that received 40 mg/kg/day, low eosinophil counts for all treated females and high prothrombin time for females given 10 mg/kg/day.
Due to limitations within this free-text field, please see „Overall remarks“ for the tabular presentation of observed effects, see tables “Hematology - group mean values during Week 13 of treatment”
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The biochemical examination of the blood plasma during Week 13 revealed, when compared to the controls, slightly low alkaline phosphatase (ALP) for males that received 40 or 140 mg/kg/day (0.73 or 0.80X control, respectively), although there was no dose relationship.
The plasma concentration of urea (which is equivalent to BUN) were lower, when compared to the controls, for females that received 10, 40 or 140 mg/kg/day (0.87, 0.81 or 0.89X control, respectively), though the extent of these findings were generally slight and there was no dose relationship. Males were not similarly affected.
All other inter-group differences from controls, including those that attained statistical significance, were minor, seen in one sex only or were without dose-relationship and were, therefore considered to represent normal biological variation. This includes the high group mean bile acid values for females that received 40 mg/kg/day and the low plasma concentration of total protein and albumin for males that received 140 mg/kg/day when compared to the controls.
Due to limitations within this free-text field, please see „Overall remarks“ for the tabular presentation of observed effects, see tables “Blood chemistry - group mean values during Week 13 of treatment”
Endocrine findings:
no effects observed
Description (incidence and severity):
There were no treatment related effects on serum T3, T4 or TSH concentrations.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no treatment related findings at the sensory activity and grip strength assessments in Week 12. Statistically significant increases in forelimb grip strength for females that received 40 or 140 mg/kg/day was evident however, there was no dose-relationship with the same pattern seen in the hindlimb values also and the individual values are within the concurrent control range. It is likely that these differences from Controls were due to natural biological variation.
Motor activity in Week 12 was unaffected by treatment.
Due to limitations within this free-text field, please see „Overall remarks“ for the tabular presentation of observed effects, see table “Sensory reactivity observations and grip strength - summary of findings during Week 12 of treatment“
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The organ weight analysis after 13 weeks of treatment revealed, when compared to the controls, slightly high absolute and body weight adjusted kidney and liver weights for males that received 140 mg/kg/day, however the majority of individual weights were within the concurrent control (effects not consistant with pathology and histopathology).
All other inter-group differences from controls were minor, occurred in one sex (where appropriate) or were without dose-relationship and were therefore considered to represent normal biological variation.
Due to limitations within this free-text field, please see „Overall remarks“ for the tabular presentation of observed effects in tables “Organ weights - group mean absolute and adjusted (g) values for animals killed after 13 weeks of treatment“
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The macroscopic examination performed after 13 weeks of treatment revealed a few changes in the stomach.
Stomach
Depression of the nonglandular mucosa was seen in males and females receiving 140 mg/kg/day. Furthermore, incidences of a thickened stomach were evident only for males receiving 140 mg/kg/day.
Summary of findings in the stomach for animals killed after 13 weeks of treatment
Group/sex 1M 4M 1F 4F
Dose (mg/kg/day) 0 140 0 140
Depression, Nonglandular Mucosa 0 3 0 4
Thickening, Nonglandular Mucosa 0 3 0 0
Number of tissues examined 10 10 10 10
The incidence and distribution of all other findings were considered to be unrelated to treatment.
Due to limitations within this free-text field, please see „Overall remarks“ for the tabular presentation of observed effects, see table “Macropathology - group distribution of findings for animals killed after 13 weeks of treatment“
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with Caprolactam disulphide given by oral gavage to Han Wistar rats were seen in the stomach.
Stomach
Minimal to slight epithelial hyperplasia of the nonglandular region was seen in seven males and three females treated with 140 mg/kg/day Caprolactam disulphide.
Summary of treatment related findings in the stomach for animals killed after 13 weeks of treatment
Group/sex 1M 2M 3M 4M 1F 2F 3F 4F
Dose (mg/kg/day) 0 10 40 140 0 10 40 140
Hyperplasia, Epithelial, Nonglandular Region
Minimal 0 0 0 3 0 0 0 0
Slight 0 0 0 4 0 0 0 3
Total 0 0 0 7 0 0 0 3
Number of tissues examined 10 10 10 10 10 10 10 10
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.
Incidental Findings
The incidence and distribution of all other findings were considered to be unrelated to treatment.

Due to limitations within this free-text field, please see „Overall remarks“ for the tabular presentation of observed effects, see tables “Histopathology - group distribution of findings for animals killed after 13 weeks of treatment“
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of treatment on estrous cycles at the end of the treatment period.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
140 mg/kg bw/day (nominal)
Based on:
test mat.
Remarks:
nominal
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
immunology
mortality
ophthalmological examination
serum/plasma biochemistry
serum/plasma hormone analyses

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

Formulation Analysis

The mean concentrations of Caprolactam Disulfide in test formulations analyzed during the study were within 35% of the nominal concentration. The difference from mean values remained within 30%.

The overall mean concentrations were within 26% of nominal concentration.

For all occasions that were analyzed within the confirmed stability period, a 1 × 10 mL sample was taken from the formulation and submitted for analysis. This was then mixed by magnetic stirring prior to taking 2 aliquots for analysis. The usual procedure for suspensions is for 4 × 1 mL samples to be taken, of which two samples are analyzed in their entirety. It is considered that the poor results seen were due to the 10 mL sample that was received not being adequately mixed prior to the two aliquots being taken for analysis. 

The following achieved concentrations were calculated based on the mean concentrations of analysed samples from Week 1, Week 4 and Week 13:

Group/Concentration:

Week of formulation/Mean analysed concentration:

Group 2 (1 mg/mL)

Group 3 (4 mg/mL)

Group 4 (14 mg/mL)

 

 

 

 

Week 1

0.969

3.13

14.4

Week 4

0.96

3.45

12.8

Week 13

0.98

2.61

9.32

Achieved concentration

0.97

3.06

12.17

Applicant's summary and conclusion

Conclusions:
The study was performed according to OECD 408 under GLP and is well-documented. So, the results are considered to be highly reliable.
It is concluded that the oral administration of Caprolactam disulfide to Sprague Dawley rats for 13 weeks at concentrations of up to 140 mg/kg/day did not cause any adverse findings. Epithelial hyperplasia of the nongladular region was seen in males and females treated at 140 mg/kg/day and was consistent with local irritation of the forestomach due to the test item. These findings correlated with macroscopic observations comprising depression and thickening of nonglandular mucosa. Due to low severity and lack of associated inflammatory changes or necrosis this finding was considered to be non-adverse. The no-observed-adverse-effect level (NOAEL) in this study was therefore considered to be 140 mg/kg/day (equivalent to 121.7 mg/kg/day).
Further, no necessity was obvious to classify Caprolactam disulfide as STOT RE according to Regulation 1272/2008 and amendments.
Executive summary:

The purpose of this study according to OECD TG 408 under GLP was an assessment of systemic toxic potential of a vulcanizing agent Caprolactam disulfide in a 13 week oral gavage study in the Han Wistar rat.

Three groups, each comprising 10 male and 10 female RccHanTM;WIST rats, received Caprolactam disulfide at doses of 10, 40 or 140 mg/kg/day. A similarly constituted control group received the vehicle, 98% water with 2% Kolliphor EL (cremophor) v/v, at the same dose volume as treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, water consumption (visual assessment), ophthalmic examination, T3, T4 and TSH, hematology (peripheral blood), blood chemistry, organ weight, macropathology and histopathology investigations were undertaken.

Results

The following achieved concentrations were calculated based on the mean concentrations of analysed samples from Week 1, Week 4 and Week 13:

Group/Concentration:

Week of formulation/Mean analysed concentration:

Group 2 (1 mg/mL)

Group 3 (4 mg/mL)

Group 4 (14 mg/mL)

 

 

 

 

Week 1

0.969

3.13

14.4

Week 4

0.96

3.45

12.8

Week 13

0.98

2.61

9.32

Achieved concentration

0.97

3.06

12.17

 

The findings in this study demonstrated that concentrations up to 140 mg/kg/day (achieved concentration 121.7 mg/kg/day) were tolerated and did not result in any adverse responses. Dose level selection was based on the results of a previous repeated dose 28-day toxicity study conducted according to TG OECD 407 protocol and GLP principles. In the 28-day study,treatment related adverse effects of local irritation were observed at necropsy and histopathological examination in the forestomach and small intestine at the top dose of 160 mg/kg/day. More specifically, in the forestomach focal hyperplasia of the squamous epithelium and erosion of the forestomach mucosa was observed. In the duodenum and jejunum, increased goblet cells were seen in the mucosa which indicated increased mucous production related to local irritancy. Consequently, the top dose of 160 mg/kg/day the 28-day study was considered as too high for the prolonged exposure duration of the present study (90 days instead of 28 days). Such a high dose level is expected to lead to more severe local effects, severe suffering or induce premature death of the high dose animals for exposure durations exceeding 28-days.

In the present study,epithelial hyperplasia of the nonglandular region of the stomach was noted at low severity in males and coupled with low incidence in females at 140 mg/kg/day. This finding is consistent with the local irritation potential of the forestomach by the test item and correlated with the macroscopic observation of depression and thickening of nonglandular mucosa. Due to the low severity and lack of associated inflammatory changes or necrosis, this finding is considered to be non-adverse. Nevertheless these findings are precursors to the adverse irritation and hyperplasia noted in the 28-day study, demonstrating the appropriate dose level selection for the present study.

The general appearance and behaviour of the animals and sensory activity, grip strength and motor activity were unaffected by treatment. There was one premature death throughout the duration of study however, this was due to a perforated oesophagus with abnormal contents in the thoracic cavity, a finding which is commonly attributed to the dosing procedure and was considered not related to treatment with Caprolactam disulphide.

There was no effect of treatment on group mean body weight gain or food intake.

Serum triiodothyronine (T3), thyroxine (T4) and thyroid stimulating hormone (TSH) concentrations were unaffected by treatment.

There were no treatment-related ophthalmoscopic findings.

The biochemical examination of the blood plasma during Week 13 revealed, treatment related reductions in low alkaline phosphatase for males that received 40 or 140 mg/kg/day and treatment-related reductions in the plasma concentration of urea for females that received 10, 40 or 140 mg/kg/day. Given that these findings were minor and only seen in one sex they were not considered adverse. All other inter-group differences from controls, including those that attained statistical significance were minor, seen in one sex only or were without dose‑relationship.

Estrous cycles at the end of the treatment period were unaffected.

The organ weight analysis after 13 weeks of treatment revealed high kidney and liver weights in males given 140 mg/kg/day. These findings did not correlate with any macroscopic or microscopic abnormalities.

Conclusion

It is concluded that the oral administration of Caprolactam disulfide to Sprague Dawley rats for 13 weeks at concentrations of up to 140 mg/kg/day did not cause any toxicologically significant finding. Epithelial hyperplasia of the nongladular region was seen in males and females treated at 140 mg/kg/day and was consistent with local irritation of the forestomach due to administration of the test item. These findings correlated with macroscopic observations comprising depression and thickening of nonglandular mucosa. Due to low severity and lack of associated inflammatory changes or necrosis this finding was considered to be non-adverse. The no-observed-adverse-effect level (NOAEL) in this study was therefore considered to be 140 mg/kg/day (equivalent to 121.7 mg/kg/day).

No necessity for classification is given.