N-cyclohexylbenzothiazole-2-sulfenamide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date (Animal Arrival):
05 January 2022 (Females)
11 January 2022 (Males)
Inlife Start Date: 25 January 2022
Inlife End Date: 21 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016

Deviations:

yes

Remarks:

Please see outline of deviations under 'any other information on Materials and Methods'. No deviations were considered to affected the overall interpretation of study findings nor compromise the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

The objective of the study was to screen for effects on male and female reproductive performance (i.e. gonadal function, mating behavior, conception, development of the conceptus and parturition) and offspring growth until Lactation Day 13 in the rat.

Test material

Test material information

Constituent 1

Reference substance name:

N-cyclohexylbenzothiazole-2-sulfenamide

EC Number:

202-411-2

EC Name:

N-cyclohexylbenzothiazole-2-sulfenamide

Cas Number:

95-33-0

Molecular formula:

C13H16N2S2

IUPAC Name:

N-(1,3-benzothiazol-2-ylsulfanyl)cyclohexanamine

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Sealed Container. 15 to 25°C. Protected from light.
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Formulations in corn oil at the concentrations 1.04 to 260 mg/mL were homogeneous for 8 days when stored at room temperature.
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: Not stated.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Formulations in corn oil at the concentrations 1.04 to 260 mg/mL were stable for 8 days when stored at room temperature.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Powder was made into a suspension in corn oil.
- Preliminary purification step (if any): Not applicable.
- Final concentration of a dissolved solid, stock liquid or gel: Not applicable.
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): Not applicable.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: Not applicable.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: Not applicable.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for reproduction studies due to its reproductive characteristics.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Specifications and Acclimation
Eighty-nine Crl:WI(Han) rats were obtained from Charles River Laboratories, Margate, United Kingdom, in order to provide 40 healthy animals/sex for use on study and three spare males and six spare females; one spare female was used on study as detailed in Section 3.1.4.

On arrival, males were between 8 and 10 weeks old and females were between 7 and 9 weeks old.
At the start of dosing, males weighed between 298.7 and 412.3 g, females weighed between 173.7 and 234.1 g, and animals were between 10 and 12 weeks old.

Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for 14 days prior to initiation of dosing (males) or 6 days prior to smearing (females), and an inspection was performed by the Named Animal Care and Welfare Officer (NACWO) before the start of dosing to ensure their suitability for the study.

Environmental Conditions, Diet, and Water
Housing
Animals were housed in a single, exclusive room in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied, or Used for Scientific Purposes (Home Office, 2014).

Animals were housed in groups (up to three animals/cage by sex [both sexes pre-pairing and males post-pairing] or with one female and male [pairing]), individually (mated females and any females with no confirmation of mating), or with their litter (females during lactation).

Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips or European Softwood bedding during the gestation and lactation phases (Datesand Ltd; Manchester, United Kingdom).

Each batch of bedding was analyzed for specific constituents and contaminants. No contaminants were present in the bedding at levels which might have interfered with achieving the objective of the study. Results are retained on file at Labcorp.

Water
Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants.

No contaminants were present in the water at levels which might have interfered with achieving the objective of the study. Results are retained on file at Labcorp.

Diet
Animals had ad libitum access to Rat and Mouse Breeder Diet VRF1 (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants.

No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study. Results are retained on file at Labcorp.

Environment
Rooms were air conditioned to provide a minimum of 15 air changes/hour. The temperature and relative humidity ranges were 19 to 25°C (target range: 19 to 25°C) and 22 to 70% (target range: 40 to 70%), respectively.

Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark.

Environmental Enrichment
Animals were provided with wooden Aspen chew blocks and rodent retreats as forms of environmental enrichment. During gestation and lactation, animals were provided with Aspen chew blocks and paper wool nesting materials as forms of environmental enrichment.

Animal Identification and Assignment to the Study
Upon arrival, animals were assigned to dose groups using a total randomization procedure.
Animals were individually identified by electronic implant.

Pups were uniquely identified from PND 1 by back mark with an indelible pen.

Following the first full weighing (Day 1 [males] or 8 [females] of the predose phase), group mean body weights and standard deviations were calculated and inspected to ensure no unacceptable differences occurred between groups. Following review of the data, one control female was ±20% outside of the mean body weight and was replaced with a spare female.

Cages were placed in dose group order across the batteries to avoid potential for cross contamination and to enable exposure of each cage/battery to similar environmental conditions.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

The control article (vehicle) was corn oil (batch MKCM9808, expiry date December 2023) supplied by Sigma

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared weekly.
The test article was formulated as a suspension in corn oil.
Formulations were stored at room temperature (15 to 25°C) in a sealed container, protected from the light.

Stability and Homogeneity
Formulations in corn oil at the concentrations 1.04 to 260 mg/mL were homogeneous and stable for 8 days when stored at room temperature

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on previous studies
- Lot/batch no. (if required): MKCM9808
- Expiry date: December 2023

Details on mating procedure:

During the pairing phase, one male was housed for up to 14 days with one female of the same dose group . One female administered 175 mg/kg/day (Animal R604) was initially paired with Animal R0204, but, as no positive confirmation of mating was noted after 10 days of the pairing period, it was re paired with Animal R0203. No positive confirmation of mating was recorded for the second pairing; however, Animal R0604 subsequently littered, and, based on the date of littering, the animal was considered to have mated during the initial pairing with Animal R0204.

Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal washing. Upon the confirmation of mating of the end of the pairing phase, vaginal washing was discontinued, and the male was removed. The day on which mating was confirmed was designated GD 0.

Analytical verification of doses or concentrations:

yes

Remarks:

Samples were analyzed by the Tox Analytical Services section of the Central Dispensary at Labcorp.

Details on analytical verification of doses or concentrations:

Samples for achieved concentration were taken from formulations prepared for use during Study Weeks 1 and 6. Triplicate samples (1 mL nominal) were taken from the middle of test article formulations and analyzed. A single sample (1 mL nominal) was taken from the middle of control formulations. Samples were analyzed by UPLC-UV.

Acceptance criteria was as follows; the mean percentage nominal concentration was between 85 and 115%, with a relative standard deviation (RSD) ≤10.0%.

The standards, validation solutions and validation samples were analysed by UPLC UV.

The concentration of N-Cyclohexylbenzothiazol-2-sulfonamid in the validation solutions and samples was calculated from their UPLC responses against a calibration line.

Linearity
A linear response was observed for N-Cyclohexylbenzothiazol-2-sulfonamid with correlation coefficient (r) >0.9998.

The y-intercept was 2.79% of the middle concentration.

Precision and Accuracy
The precision and accuracy results for N-Cyclohexylbenzothiazol-2-sulfonamid were within the acceptance criteria.

Selectivity
Chromatograms of diluting solvent containing each of the control vehicles showed no interfering peak with a retention time similar to N-Cyclohexylbenzothiazol-2-sulfonamid and a response >20% LLOQ.

Sensitivity
The Lower Limit Of Quantification (LLOQ) was set at the level of the lowest standard and had a mean signal to noise ration of 8022.6

Calibration Standard and Diluted Sample Solution Stability
The calibration standard and diluted samples solutions generated mean results 5% of the mean of the initial respective validation results.

Homogeneity and Stability of N-Cyclohexylbenzothiazol-2-sulfonamid in Corn Oil
The formulations were considered to be homogenous and stable. The mean % nominal concentrations and RSD values were observed for N-Cyclohexylbenzothiazol-2-sulfonamid when stored at room temperature (15 to 25°C) as follows:


260 mg/mL
0 hour: Mean 96%, RSD 0.64%
24 hour RT: Mean 96%, RSD 0.58%
8 days RT: Mean 95%, RSD 3.29%

1.04 mg/mL
0 hour: Mean 96%, RSD 0.56%
24 hour RT: Mean 97%, RSD 0.26%
8 days RT: Mean 96%, RSD 3.01%


 

Duration of treatment / exposure:

Males were dosed once daily for 42 consecutive days (2 weeks prior to pairing, 2 weeks during the pairing phase, and 2 weeks post pairing).

Females were dosed once daily for up to 57 days (2 weeks prior to pairing, during the pairing phase, through gestation, and up to LD 13).

Dosing was omitted on one occasion for the following females as they were observed to be in or near parturition at the time of daily dosing.
• Animals R0504 and R0505 (Group 2)
• Animals R0605, R0606, R0609, and R0610 (Group 3)
• Animals R0703, R0707, R0708, and R0710 (Group 4)

Animal R0704 (Group 4) was not dosed on GD 20 due to blood observed on the animal at the time of daily dosing; however, when the animal was checked again at the mid-day parturition check, no blood was observed on the animal or in the cage.

A dose volume of 4 mL/kg was used. Individual dose volumes were calculated from the most recent individual body weight. Where an animal had completed parturition before the daily dose on LD 0, a body weight was recorded and was used to calculate the correct dose volume to be administered to avoid overdosing; then, dosing occurred.

Frequency of treatment:

Once daily

Details on study schedule:

Refer to details on mating procedure and duration of treatment/exposure sections.

Study Initiation Date: 05 January 2022
Experimental Starting Date (Animal Arrival):
05 January 2022 (Females)
11 January 2022 (Males)
Inlife Start Date: 25 January 2022
Inlife End Date: 21 March 2022
Experimental Completion Date: To be included in the Final Report
Study Completion Date: The date the Final Report is signed by the Study Director.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 - Control

Doses / concentrations 2

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

Group 2 - Low

Doses / concentrations 3

Dose / conc.:

175 mg/kg bw/day (nominal)

Remarks:

Group 3 - Intermediate

Doses / concentrations 4

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

Group 4 - High

No. of animals per sex per dose:

10 males, 10 females per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:

The high-dose level of 400 mg/kg/day was estimated to produce minor toxicity in animals. The intermediate-dose level of 175 mg/kg/day was based on geometric ratio and was estimated to produce little or no toxicity. The low-dose level of 75 mg/kg/day was estimated to produce no toxicity and was anticipated to be the NOAEL.

Background Information
In a 1997 28-day toxicity study (OECD TG 407 ; gavage) in rats at doses of 25, 80, 250 or 800 mg/kg bw/day, suppression of food consumption and body weight gain were observed following administration of ≥250 mg/kg bw/day. An increase in relative kidney weight was associated with an increase in deposition of hyaline droplets in the proximal tubular epithelium of the kidneys observed in male rats administered 250 or 800 mg/kg bw/day. Based on the result, the NOAEL was 80 mg/kg bw/day and the lowest observed adverse effect level was 250 mg/kg bw/day.

In a 1981 rat prenatal developmental (PNDT) study (similar or equivalent to OECD 414; gavage) with dose levels of 100, 300, 500, or 900 mg/kg bw/day, the NOAEL derived from this study for maternal toxicity was 100 mg/kg bw/day and was based on a decrease in mean body weight gain. The NOAEL for the offspring was 300 mg/kg bw/day and was based on a decrease in mean fetal body weight.

In a previous 14-day dose range-finding study performed in the Crl:WI(Han) rat (Labcorp Study 8476210; Labcorp Early Development Laboratories Ltd., 2022) animals were administered with dose levels of 150, 400, or 750 mg/kg/day. One animal administered 750 mg/kg/day was sacrificed in a moribund condition on the last day of dosing (Study Day 14) due to the severity of clinical observations, which included hunched posture, decreased general activity, excessive lacrimation, piloerection, and slow labored respiration.

Test article-related clinical observations of vocalization were observed in animals administered ≥150 mg/kg/day, and hunched posture (associated with piloerection) and protruding eye were each observed in a single animal administered 750 mg/kg/day.
Body weight loss, reduced body weight gain, and food consumption were observed following administration of ≥150 mg/kg/day.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

Clinical Observations
Health Monitoring
All animals were observed at the beginning and end (nominal) of the working day for signs of ill health or overt toxicity.

Clinical Examinations
Each animal was given a detailed physical examination once during the predose phase, then once daily from the start of dosing (performed at the time of dosing), up to and including the day of necropsy . Unscheduled clinical examinations were recorded when necessary. An individual record of the clinical condition of each animal was maintained.

Postdose Observations
Animals were observed daily for the first 7 days of dosing; animals were observed upon return to the home cage and 2 hours postdose. Some animals were observed on Pairing Day 2.

Body Weights
Male body weights were recorded once during acclimation, on the first day of dosing, at weekly intervals thereafter, and on the day before necropsy. Three control males were weighed on the day of necropsy in addition to requirement (see Protocol Deviations). Female body weights were recorded once during acclimation; on the first day of dosing; weekly prior to pairing, on GD 0, 7, 14, and 20; and on LD 1, 4, 7, 13, and 14 (prior to necropsy).

Where an animal had completed parturition before the daily dose on LD 0, a body weight was recorded and was used to calculate the correct dose volume to be administered to avoid overdosing; then, dosing occurred.

Food Consumption
The amount of food consumed was determined twice weekly prior to pairing from Day 1 of the study (both sexes) and during the post-pairing phase for males. Food consumptions were recorded for females from GD 0 to7, 7 to 14, and 14 to 20 and on LD 1 to 4, 4 to 7, and 7 to 13. Consumption was calculated as g/animal/day.


Parturition
Animals were observed three times each day (at the beginning, middle, and end of each working day), starting when the first females reached GD 21 and until the last female had littered. The female (R0704 administered 400 mg/kg/day) that had a confirmation of mating but did not litter, was sent to necropsy on GD 26.

Females were observed for signs of the start of parturition (for example, blood in the cage). The time and date of this observation was recorded, where possible, and marked the end of gestation; when this was not observed, the end of gestation was the day when the completion of parturition was recorded or on the day prior to when LD 1 observations were made.

Abnormal observations of nesting, parturition, or nursing were recorded. It was considered important that the dam was disturbed as little as possible on LD 0. Any dead pups were removed and sent to necropsy to establish if they were born alive or dead (by looking for lung inflation).

Oestrous cyclicity (parental animals):

Estrous Cycle Determination
Daily vaginal washings were taken from all females during acclimation (predose) for 2 weeks prior to the start of dosing; the stage of estrous was recorded, and only females with regular 4 or 5 day cycles were included on study.

Daily vaginal washings were taken from females from the start of dosing until confirmation of mating or until the end of the pairing period for the female with no sign of mating, and on LD 14.

Daily vaginal washings were recorded for Animal R0604 up until Day 14 of the pairing phase.
However, data from Day 6 through 14 was unable to be back-entered into the Pristima system as the date of littering meant the animal was in the gestation phase at this time. The raw data are retained in the study file, but not reported.

Sperm parameters (parental animals):

Additional sections of testes and epididymides from Group 1 and 4 were also stained with Periodic Acid Schiff (PAS) and assessed for qualitative stages of spermatogenesis and interstitial testicular cell structure.

Litter observations:

Offspring Procedures
Litter Size and Sex Determination
On PND 1, 4, 7, and 13, litter size and pup sex were recorded.

Pups were uniquely identified from PND 1.
Daily records of mortality and changes in litter sizes were maintained. Where possible, pups found dead or in a moribund condition were given a macroscopic necropsy.

On PND 4, litters were culled to ten pups/litter, with five pups/sex, when possible. Pups were selected randomly, with no bias as to size or clinical status. Culled pups were sexed but not examined. Culling created a uniformly sized litter, which reduced differences in pup body weights due to litter size.

Clinical Observations
Each pup was given a detailed clinical examination daily from PND 1.

Body Weights
On PND 1, 4, 7, and 13, individual pup body weights were recorded.

Ano-genital Distance
Ano-genital distance of all pups was recorded on PND 4 post-cull.

Nipple/Areolae Count
The number of nipples/ areolae for male pups was counted on PND 13.

Postmortem examinations (parental animals):

Clinical Laboratory Procedures
Clinical Pathology
Sample Collection and Handling
Blood samples for thyroid hormone (1 x 1.2 mL [serum separator tubes], nominal) were withdrawn from the jugular vein of adult males prior to necropsy on Day 43 of dosing (Post-Pairing Day 15). Blood samples for thyroid hormone (1 x 1.2 mL [serum separator tubes], nominal) were withdrawn from the abdominal aorta of adult females at necropsy on LD 14. Samples were collected after animals were fasted overnight. Sampling was performed at a similar time on each occasion (between 09:00 and 13:00). Serum samples were split into two aliquots (Set 1 [minimum of 300µL] used for analysis; Set 2 [remaining serum] was retained as a backup) ; samples from adult females were retained until finalization but not analyzed. Blood samples were collected by controlled randomization (i.e., Groups 1, 4, 2 then 3 and repeated in the same sequence until all animals were sampled). The Set 2 backup sample was used for analysis for Animal R0004 due to insufficient sample for analysis for total thyroxine.


Thyroid Hormone Tests
Total thyroxine (T4)
Thyroid stimulating hormone (TSH)
Total triiodothyronine (T3)

Terminal Procedures
With the exception of fasting, these procedures were also followed for unscheduled sacrifices.

All animals that died prior to their scheduled sacrifice were examined macroscopically for abnormalities of the thoracic and abdominal viscera. The uterus was examined for implantations.
In addition, the brain, heart, lungs, liver, and kidneys were retained in the appropriate fixative.

Females
Females were sacrificed on LD 14 (those that littered) or on Day 26 post coitum (Animal R0704 administered 400 mg/kg/day, which did not litter), or immediately following total litter loss
. Animals were administered isoflurane anesthesia. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Littered females were sacrificed in a controlled randomization order when possible. Scheduled necropsies were performed after an overnight period without food.

Blood sampling information is detailed in clinical pathology section. Upon sacrifice, macroscopic examinations were performed under the general supervision of a Pathologist, and all lesions were recorded. For tissue collection see necropsy and organ weights.

The uterus of the apparently non pregnant female (Animal R0704 administered 400 mg/kg/day, which did not litter), was immersed in a 10% ammonium sulfide solution to reveal any evidence of implantation.

Males
Males were sacrificed on Day 43 of the study (Post-Pairing Day 15) by isoflurane anesthesia after an overnight period without food. Necropsies were carried out in controlled randomization order. Once a suitable deep plane of anesthesia was established, the major blood vessels were severed to exsanguinate the animal. Blood sampling information is detailed in clinical pathology. Upon sacrifice, macroscopic examinations were performed under the general supervision of a Pathologist, and all lesions were recorded.

Necropsy and Organ Weights
Organ weights, as indicated in the following table, were recorded at each scheduled sacrifice, excluding the non-littered female (Animal R0704 [Group 4]). Paired organs were weighed together.

The following tissues from each animal were retained in 10% neutral buffered formalin, unless otherwise indicated.

Organ/Tissue
animal identification
Cowper’s glands (bulbourethral gland)
epididymisa W E
glans penis
gross lesions (variable) E
levator ani plus bulbocavernosus muscle complex
mammary gland E
ovary Wb E
oviducts E
pituitary W
prostate W E
seminal vesicle with coagulating gland W E
testis (including tunica albuginea)a W E
thyroid with parathyroid Wc
uterus with cervix W E
vagina E

E = Processed and examined microscopically; W = Weighed.
a Modified Davidson’s fixative and processed to at least block stage.
b Tissue weighed with oviducts.
c Tissues weighed post-fixation.

Histopathology
All tissues denoted by E in the previous tissue list from each adult animal in Groups 1 and 4 and all decedents were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 µm, stained with hematoxylin and eosin, and were examined microscopically by the Study Pathologist.

Additional sections of testes and epididymides from Group 1 and 4 were also stained with Periodic Acid Schiff (PAS) and assessed for qualitative stages

Postmortem examinations (offspring):

Pups culled on PND 4 had blood samples (1 x 0.8 mL [serum separator tube], nominal) for thyroid hormone analysis withdrawn via decapitation to provide one pooled sample for each litter or one sample pooled by dose group. All serum was placed in a single aliquot and was retained until finalization, but not analyzed

Pups sacrificed on PND 13 had blood samples (2 x 0.8 mL [serum separator tube], nominal) for thyroid hormone analysis withdrawn by cardiac puncture following injection of sodium pentobarbitone to provide two samples for each litter for analysis and two samples for backup (two samples from two males and two sample from two females, when possible). Where blood volume was limited, some samples were pooled to reach the required volume - (see Protocol Deviations).
Pup blood samples were collected in group order.
Each blood sample was gently inverted several times (approximately 10) ensuring that the blood travels all the way to the top and bottom of the tube each time to mix with the clot activator, were allowed to clot for at least 30 minutes at room temperature (15 to 25°C), and were protected from light. Samples were centrifuged and aliquoted within 2 hours of blood sampling at 2300g for 10 minutes at 4°C. The resultant serum was aliquoted into uniquely labeled amber polypropylene tubes and protected from light until frozen at <10°C (-20°C nominal).


Pups
Surplus pups culled on PND 4 (to standardize litter size) and pups sent to necropsy on PND 13 were sacrificed by an intraperitoneal injection of sodium pentobarbitone (overdose). Once a suitable deep plane of anesthesia was established, major blood vessels were severed to exsanguinate the animal (decapitation for PND 4 pups).

Pups culled on PND 4 were examined to determine sex only; no macroscopic examination was performed, and tissues were discarded following blood sample collection.

After sacrifice on PND 13, full macroscopic examinations were conducted for one pup/sex/litter. Remaining PND 13 pups were examined externally, with particular attention to the external reproductive genitals.

The thyroid from one pup/sex/litter sacrificed on PND 13 (animals subjected to a full macroscopic examination) was preserved in 10% neutral-buffered formalin and weighed (post-fixation); the tail was retained as animal identification. No microscopic examination was required.

All decedent pups were examined macroscopically.

Statistics:

Data Evaluation and Statistical Analysis
Where tables/appendices are computer-generated, rounding of individual values may occur during the calculation of derived values. Therefore, recalculation of derived values from the individual data, as presented in this report, may have, in some instances, yielded minor variations.

Data from test article-treated animals were compared with control data. Statistical analyses were performed, where appropriate.

Data for each sex were analyzed separately; only data collected on or after the first day of dosing were analyzed statistically.
Please also refer to "Any other information on materials and methods"

The pairwise comparisons of interest were: Group 1 versus Groups 2, 3, and 4.

All statistical tests were evaluated at the 5.0, 1.0, and 0.1% probability levels.

Due to system limitations, additional statistical analyses may be run but were not reported or used to interpret study data.

Please also refer to "Any other information on materials and methods"

Reproductive indices:

Please refer to "Any other information on materials and methods"

Offspring viability indices:

Please refer to "Any other information on materials and methods"

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 400 mg/kg/day, test article-related clinical observations of increased activity, piloerection, and red/pink staining around the muzzle and/or chin were occasionally noted for the males during the pairing and post-pairing phases. For females at 400 mg/kg/day, clinical observations of piloerection, and red/pink staining around the muzzle and/or chin were occasionally noted during the pairing and gestation phases, with mildly increased activity also observed during the pairing and gestation phase. Individual females from this group also had piloerection during the pre-pairing (Animal R0702) and lactation (Animal R0701) phases, with wet eyelids and pelage on Pairing Day 2 and GD 0 also noted for the latter female. These observations were considered to represent an adverse effect of the test article.
Post dose observations of salivation, mouth rubbing, and/or jaw chomping were observed on the study in test article-treated males and females during the pre-paring and pairing phases. These post dose observations are commonly noted on orally dosed studies and were considered likely due to the unpalatability of the formulations and therefore considered not to be of toxicological significance.
Any other observations noted were considered incidental, as they were noted transiently, lacked a dose relationship, and/or were within the usual spectrum of observations noted for rats of this age and strain at this laboratory.

Please refer to the attached table: "Summary of clinical observations"

Mortality:

mortality observed, treatment-related

Description (incidence):

One female administered 400 mg/kg/day (Animal R0704) was sent to necropsy on GD 26 as it had shown no signs of littering. At necropsy, the animal was confirmed to have been pregnant, but only had three implantations (all early resorptions) and no viable fetuses.

Five females administered 400 mg/kg/day were sent to necropsy during the lactation phase due to having a total litter loss, as follows.

• Animal R0701; LD 7. Clinical observations for this female noted on the same day as necropsy included piloerection and abnormal color of the muzzle.
• Animal R0703; LD 0. No clinical observations were noted.
• Animal R0705; LD 2. No clinical observations were noted.
• Animal R0707; LD 1. Clinical observations for this female noted on the same day as necropsy included abnormal color of both front legs and muzzle.
• Animal R0709; LD 4. No clinical observations were noted.

The range of macroscopic and microscopic findings in these females was generally similar to that in animals that survived to the terminal sacrifice on LD 14.

All other parental animals survived to their scheduled sacrifice on Day 42 (males) or LD 14 (females).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A test article-related, dose-dependent reduction in mean body weight gain was noted for males between Pre-Pairing Days 1 and 8, compared with controls, by 28%, 32%, and 98% for males administered 75, 175, or 400 mg/kg/day, respectively; this was statistically significant for males administered 400 mg/kg/day (P≤0.001). Subsequent improvement was noted for males administered 75 or 175 mg/kg/day, although mean body weight gains generally remained lower than controls for males administered 400 mg/kg/day, which resulted in a statistically significant reduction of 46% in overall mean body weight gain between Pre Pairing Day 1 and Post-Pairing Day 14 for these animals when compared with the controls (P≤0.001).

Statistically significant body weight loss was noted between Pre-Pairing Days 1 and 8 for females administered 400 mg/kg/day (P≤0.001). As a result, overall body weight gain during the pre-pairing phase was significantly lower by 74% when compared with the controls for females administered 400 mg/kg/day (P≤0.001), and significantly lower absolute mean body weights were noted on Pre-Pairing Days 8 and 15 for these females (P≤0.05). Throughout the gestation phase, dose dependent and occasionally significant reduction in mean body weight gain was noted at all dose levels, which resulted in dose dependent reduction in overall mean body weight gain by 12%, 18% and 38% for females administered 75, 175 or 400 mg/kg/day, respectively; differences from controls at 175 or 400 mg/kg/day were statistically significant (P<0.01 or P<0.001). Body weight loss was noted on LD 0 through 1 and 4 through 7, and reduced body weight gain on LD 1 through 4 (70% lower than controls) for females administered 400 mg/kg/day (significant on LD 4 through 7; P≤0.05), which resulted in an overall reduction in body weight gain of 47% compared with controls from LD 0 to 13. Reduced body weight gain was also noted throughout the lactation phase for females administered 175 mg/kg/day which resulted in overall significantly lower body weight gain of 50% compared with controls (P≤0.05) from LD 0 to 13. Significantly lower mean body weights were noted throughout the lactation phase for females administered 75 (P≤0.01-0.05) or 400 (P≤0.001-0.01) mg/kg/day, and on LD 4 through 13 for females administered 175 mg/kg/day (P≤0.01-0.05).

There were no effects on body weight gain at 175 or 75 mg/kg/day in the pairing and post-pairing phases in the males or at 75 mg/kg/day in the females in the lactation phase.

Please refer to the attached table "Summary of bodyweight changes - Parental generation"

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A slight, dose-dependent reduction in mean food consumption was noted during the pre-pairing phase for males administered 175 or 400 mg/kg/day (8 and 19% lower than controls, respectively) and overall during the post-pairing phase for males administered 400 mg/kg/day (7% lower than controls).

Statistically significantly lower mean food consumption was noted during all phases for females administered 400 mg/kg/day (26, 24, and 36% lower than controls during the pre-pairing, gestation, and lactation phases, respectively; P≤0.001-0.01). During the gestation phase, significantly lower mean food consumption was noted from GD 7 through 20 for females administered 75 mg/kg/day (P≤0.05) and from GD 0 through 20 for females administered 175 mg/kg/day (P≤0.01-0.05); this resulted in overall mean food consumption that was significantly lower than controls for females administered 175 mg/kg/day (12%; P≤0.01). Lower food consumption was also noted for females administered 75 or 175 mg/kg/day (significant at 175 mg/kg/day; P≤0.01-0.05) during the lactation phase; overall food consumption between LD 1 and 13 was 14 (P≤0.05) and 21% (P≤0.01) lower than controls for females administered 75 or 175 mg/kg/day, respectively.

There were no effects on food consumption in the males at 75 mg/kg/day in the pre-pairing phase and at 175 or 75 mg/kg/day in post-pairing phase. There were no effects on food consumption in the females at 175 or 75 mg/kg/day in the pre-pairing phase.

Please refer to the attached table "Summary of food consumption - Parental generation"

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

no effects observed

Description (incidence and severity):

No effect on thyroid hormones was noted for adult males at 75, 175 or 400 mg/kg/day.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Upon microscopic examination, no test article-related findings were recorded at 75, 175 or 400 mg/kg/day.

Microscopic findings in tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age.

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No effect was noted on mean length or number of estrous cycles at 75, 175 or 400 mg/kg/day.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant increase in mean duration of gestation was recorded for females administered 400 mg/kg/day, compared with controls (P<0.001). No such effect was observed at 75 or 175 mg/kg/day.

All females delivered liveborn offspring with the exception of one female administered 400 mg/kg/day (Animal R0704). The animal was confirmed to have been pregnant, but had only three implantations (all early resorptions) in the uterus and no viable fetuses; this incidence was within the historical control range (0 to 2; data compiled January 2021 from 31 studies) and, was therefore, considered not related to the test article.

The mean number of implantation sites was significantly lower for females administered 400 mg/kg/day (P=0.05) at 10.60 compared with 13.40 in the control animals, and mean post-implantation loss (%) was significantly increased for females administered 175 (P=0.01) or 400 (P=0.001) mg/kg/day at 18.46% and 31.38%, respectively. This resulted in significantly smaller number of pups delivered per litter (P=0.01) and mean number of liveborn pups in each litter (P=0.001) for dams administered 400 mg/kg/day, compared with controls; therefore, these effects were considered an adverse effect of the test article. In the absence of an effect on the mean number implantation sites and litter size, the finding of increased post-implantation loss at 175 mg/kg/day was considered not adverse. There was no effect of test article at 75 mg/kg/day on the mean number of implantation sites and litter size when compared with the controls.

Reproductive indices
No effect on fertility was noted at 75, 175 or 400 mg/kg/day.

Males mated with one female within 4 days of pairing, and all females became pregnant. One female administered 175 mg/kg/day (Animal R0604) was initially paired with Animal R0204, but, as no positive confirmation of mating was noted, it was re paired with Animal R0203. No positive confirmation of mating was recorded for the second pairing; however, Animal R0604 subsequently littered and, based on the date of littering, the animal was considered to have mated during the initial pairing with Animal R0204.

Please refer to the attached table "Summary of fertility and reproductive performance"

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

175 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related clinical observations were noted for offspring of dams administered 400 mg/kg/day and included no milk in the stomach, thin, weak, cold to touch, and/or small appearance which often preceded the early sacrifice/death of these pups. One pup (Animal R0707 7) was also noted as having blue coloration to the neck.

Small appearance was also noted for one pup in one litter of a dam administered 75 mg/kg/day and two pups in two litters of dams administered 175 mg/kg/day; however, at these dose levels this observation was generally transient and not accompanied by other observations or effects on pup body weight, and as such, it was considered not to be adverse at these dose levels.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Two pups from one litter of a dam administered 75 mg/kg/day, two pups from two litters of dams administered 175 mg/kg/day, and 11 pups from five litters of dams administered 400 mg/kg/day were stillborn. In total, between PND 0 and 4, five pups from three litters of dam administered 75 mg/kg/day, 10 pups from three litters of dams administered 175 mg/kg/day, and 39 pups from six litters of dams administered 400 mg/kg/day died, were sacrificed in a moribund condition, or were missing and/or cannibalized. This resulted in significantly lower livebirth (P=0.05) and Day 4 viability (P=0.01) indices for dams administered 400 mg/kg/day. An increase in the number of still births, pups found dead, or missing and/or cannibalized was also noted at 75 or 175 mg/kg/day when compared with the controls. However, values were within the historical control ranges, and were, therefore, considered not to be adverse.

One further pup from a litter of a dam administered 400 mg/kg/day (Animal R0701 2 [female]) was sent to necropsy on PND 7 due to the severity of clinical observations, including small, thin, cold to touch and weak.
As a result of the mortality observed, only four of nine litters of dams administered 400 mg/kg/day survived to PND 13. All liveborn pups in these litters survived until PND 13.


Please refer to the following table "Summary of unscheduled pup deaths PND 0-4" in "Any other information on results"
and "Summary of Partuition and Litter data" attached

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Lower mean pup body weights were noted from Day 1 through 13 for pups of dams administered 400 mg/kg/day, reaching statistical significance on Days 1 and 4 (both pre and post cull) when adjusted for litter size (P=0.001-0.01). No significant effects were noted for litters of dams administered 75 or 175 mg/kg/day.

Please refer to the attached table

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No effect of maternal dosing on ano-genital distance was noted at 75, 175 or 400 mg/kg/day.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No effect of maternal dosing on nipple count of male offspring was noted at 75, 175 or 400 mg/kg/day.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No N-Cyclohexylbenzothiazole-2-sulfenamide-related changes in thyroid weights or thyroid weight ratios were recorded for test article-treated animals at 75, 175 or 400 mg/kg/day, compared with concurrent controls.
All thyroid weight and thyroid weight ratio differences were attributed to normal biological variation and were considered not test article related as they were small in magnitude, not dose dependent, inconsistent between sexes, due to normal inter-animal variability, and/or lacked a microscopic correlate.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Upon macroscopic examination, no test article-related findings were recorded at 75, 175 or 400 mg/kg/day.

Most tissues were macroscopically unremarkable or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.

Histopathological findings:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Pup Thyroid Hormone Analysis
Increased mean total T4 was noted for female pups sampled on PND 13, compared with controls, which was statistically significant for those animals from litters of dams administered 175 (P=0.01) or 400 (P=0.05) mg/kg/day; however, the response was not dose-dependent. Group means for T4 fell within the historical control ranges (63 to 110 nmol/L), and, in the absence of an effect on thyroid weights or macroscopic abnormalities, this was considered incidental. No effects on thyroid hormones noted at 75, 175, or 400 mg/kg/day were considered to be test article related.
Please refer to the following tables "Thyroid Hormone Data" in "Any other information on results".

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Target system / organ toxicity (F1)

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects occurring together with other toxic effects, but not as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Any other information on results incl. tables

Achieved Concentration

The achieved concentration and relative standard deviation results were within specification.

 

Formulation Analysis Results: Achieved Concentration

Occasion / Group	Concentration mg/mL	Results as % Nominal Concentration			Mean (%)	RSD (%)
Week 1
1	0		NS
2	18.75	91	88	93	91	2.63
3	43.75	94	91	92	92	1.41
4	100	97	95	95	96	1.02
Week 6
1	0		ND
2	18.75	101	96	95	97	3.26
3	43.75	94	94	95	94	0.75
4	100	94	94	95	94	0.72
Mean and RSD are calculated from the database values, not the rounded values presented above. NS = No significant test article peak detected. ND = No test article peak detected.

 

 

Summary of Unscheduled Pup Deaths PND 0-4

Dose Level (mg/kg/day)	Dam ID	Pup ID(Sex)
		Stillborn	Found Dead	Found Cannibalized	Moribund	Missing/Presumably Cannibalized
75	R0503	-	-	11(U) 12(U)	-	-
	R0504	10(F) 11(F)	-	-	-	-
	R0505	-	-	-	-	9(M)
	R0508	-	7(M) 12(F)	-	-	-
Total	4	2	2	2	0	1
Affected Litters		1	1	1	0	1
175	R0601	13(F)	-	-	-	-
	R0602	-	6(M) 7(M)	-	-	5(M) 13(F) 14(F) 15(F)
	R0604	-	16(F)	-	-	-
	R0608	13(F)	8(M) 9(M) 14(F)	-	-	-
Total	4	2	6	0	0	4
Affected Litters		2	3	0	0	1
400	R0701	1(M) 7(F)	8(F)	-	-	3(F) 4(F) 5(F) 6(F)
	R0703	1(M)	2(M) 3(F) 4(F) 5(F) 6(F) 7(F) 8(F) 9(F) 10(F)	-	-	-
	R0705	12(F)	1(M) 2(M) 3(M) 4(M) 5(M) 8(F) 9(F) 10(F) 11(F)	-	-	6(F) 7(F)
	R0707	3(M) 4(M) 8(F) 9(F) 10(F)	1(M) 2(M)	5(M) 11(F)	-	6(F) 7(F)
	R0708	6(F) 7(F)	-	-	-	-
	R0709	-	-	3(F)	2(F)	1(F) 4(U) 5(U)
	R0710	-	-	-	-	5(M) 10(F) 11(F)
Total	7	11	21	3	1	14
Affected Litters		5	4	2	1	5
Historical Control Range (Number of Pups)		0-6	0-6	0-7	0-2	0-9
- = none; F = female; M = male; U = Unknown (sex unknown due to pup having been cannibalized).

 

Thyroid Hormone Data

 

Summary of Thyroid Hormone Data: Male Pups

		PND 13
		IMT4 nmol/L	TSHI μIU/mL	TT3S nmol/L
Group 1	Mean	92	0.22	1.34
	SD	5.7	0.055	0.063
	N	5	5	5
Group 2	Mean	113	0.22	1.50
	SD	18.0	0.053	0.061
	N	3	3	3
Group 3	Mean	98	0.39	1.42
	SD	6.0	0.278	0.231
	N	5	5	5
Statistics		P3	P1	P3
PND = Postnatal Day P1 = ANOVA and Dunnett's P3 = Kruskal-Wallis and Wilcoxon

 

 

 

 

Summary of Thyroid Hormone Data: Female Pups

 

		PND 13
		IMT4 nmol/L	TSHI μIU/mL	TT3S nmol/L
Group 1	Mean	92	0.24	1.21
	SD	3.3	0.097	0.106
	N	5	5	5
Group 2	Mean	100	0.18	1.37
	SD	2.1	0.035	0.057
	N	2	2	2
Group 3	Mean	108+H	0.29	1.34
	SD	4.7	0.087	0.335
	N	3	3	3
Group 4	Mean	102*H	0.26	1.41
	SD	4.2	0.106	0.445
	N	2	2	2
Statistics		P1	P1	P3
PND = Postnatal Day *H = Dunnett Exact Homogeneous Test Significant at 0.05 level +H = Dunnett Exact Homogeneous Test Significant at 0.01 level #H = Dunnett Exact Homogeneous Test Significant at 0.001 level P1 = ANOVA and Dunnett's P3 = Kruskal-Wallis and Wilcoxon

Applicant's summary and conclusion

Conclusions:

Following once daily oral (gavage) administration of 75, 175, or 400 mg/kg/day N Cyclohexylbenzothiazole-2-sulfenamide to male rats for 42 consecutive days and to female rats for up to 57 days, a dose-dependent reduction in mean body weight gain was observed, which was considered adverse for animals administered 400 mg/kg/day. No effect on estrous cyclicity or fertility was noted; however, a lower mean number of implantation sites and increased post-implantation loss (%) were noted for females administered 400 mg/kg/day, which resulted in a significantly smaller mean litter number of pups delivered and mean number of liveborn pups per litter. Additionally, a test article-related, dose-dependent increase in pup mortality was noted, which resulted in significantly lower livebirth and Day 4 viability indices for animals administered 400 mg/kg/day, compared with controls. Lower pup body weights and adverse clinical observations were also noted for litters of dams administered 400 mg/kg/day. Based on the observations noted, the no observed adverse effect level (NOAEL) for adult reproduction and offspring development is 175 mg/kg/day.

Executive summary:

 

Introduction

The purpose of the study was to investigate the effect of the test item, N‑Cyclohexylbenzothiazole-2-sulfenamide (an industrial chemical), on male and female reproductive performance (i.e. gonadal function, mating behavior, conception, development of the conceptus and parturition) and offspring growth until Lactation Day 13 in the rat. This study is both a screening study for reproductive and developmental toxicity, and as a bridging study to support a read-across strategy.

 

Study Design

Four groups of 10 rats/sex/group were administered 0 (control article [vehicle]), 75, 175, or 400 mg/kg/day by oral gavage at a volume of 4 mL/kg. Males were dosed once daily for 42 consecutive days (2 weeks prior to pairing, during the pairing period, and until the day before necropsy). Females were dosed once daily for up to 57 days (2 weeks prior to pairing, during the pairing period, through gestation, and until Lactation Day [LD] 13). The control article (vehicle) was Corn oil.

 

Assessment of toxicity for adults was based on mortality, clinical and postdose observations, body weights, food consumption, estrous cycles, fertility and pregnancy indices, parturition, and offspring development. For pups, clinical observations, litter size, sex, and body weights were recorded. Ano-genital distance was recorded on Postnatal Day (PND) 4, and nipple retention was recorded for male pups on PND 13. One pup/sex/litter/dose group was selected for recording of thyroid weights.

 

Macroscopic examinations were performed for all animals (except pups culled on PND 4), and any macroscopic abnormalities were recorded. Blood samples for thyroid hormone assessments were collected from all surviving adults at the end of the study. Blood samples for thyroid hormone assessment were also collected at necropsy from all selected pups on PND 4 and 13. Selected organs were weighed, and microscopic examinations were performed.

 

Results

Test article-related clinical observations of increased activity, piloerection, red/pink staining around the muzzle and/or chin, and/or wet eyelids were noted for animal administered 400 mg/kg/day. These observations were mostly noted during the pairing phase and thereafter; piloerection, staining and/or wet eyelids were considered to be a manifestation of overall clinical condition of the animals and, as such, considered adverse. Incidences of salivation, mouth rubbing, and jaw chomping noted at all dose levels were considered due to the unpalatability of the formulations and of no toxicological significance.

 

A test article-related, dose-dependent reduction in mean body weight gain was noted for males between Pre-Pairing Days 1 and 8, compared with controls. Improvement was noted thereafter for males administered 75 or 175 mg/kg/day, although mean body weight gain remained lower than controls for males administered 400 mg/kg/day, which resulted in a statistically significant reduction in overall mean body weight gain between Pre‑Pairing Day 1 and Post-Pairing Day 14 for these animals. Body weight loss was noted between Pre-Pairing Days 1 and 8 for females administered 400 mg/kg/day which resulted in lower overall body weight gain during the pre-pairing phase and lower absolute mean body weights on Pre-Pairing Days 8 and 15. Lower body weights gains that were occasionally statistically significant, compared with controls, were noted for test article-treated females during the gestation phase, which resulted in a dose-dependent decreased overall body weight gains for these females over the gestation phase. During the lactation phase, body weight loss was noted on LD 0 through 1 and 4 through 7, and reduced body weight gain on LD 1 through 4 for females administered 400 mg/kg/day, which resulted in lower mean body weights throughout the lactation phase and a reduction in overall body weight gain from LD 0 through 13 for these animals. Reduced body weight gain was also noted throughout the lactation phase for females administered 175 mg/kg/day, which resulted in lower mean body weight on LD 4 through 13 and an overall reduction in overall body weight gain from LD 0 to 13. Mean body weight gain at 75 mg/kg/day remained unaffected during the lactation phase. These effects at 400 mg/kg/day were considered to be adverse.

 

A slight, dose-dependent reduction in mean food consumption was noted during the pre-pairing phase for males administered 175 or 400 mg/kg/day, and overall during the post-pairing phase for males administered 400 mg/kg/day. Statistically significantly lower mean food consumption was noted during all phases for females administered 400 mg/kg/day. No effect on food consumption was noted during the pre-pairing phase for females administered 75 or 175 mg/kg/day; however, lower mean food consumption was noted from GD 7 through 20 and throughout the lactation phase for females administered 75 mg/kg/day, and from GD 0 through 20 and throughout the lactation phase for females administered 175 mg/kg/day. The effect on food consumption was only considered to be adverse at 400 mg/kg/day due to the effects on body weight.

 

No effects on estrous cyclicity or fertility were noted. Males generally mated with one female within 4 days of pairing, and all females became pregnant.

 

No effect on thyroid hormones was noted for the adult males. No test article-related organ weight changes and no macroscopic or microscopic observations were noted for the adults.

 

The mean number of implantation sites was significantly lower and mean post-implantation loss (%) was significantly increased for females administered 400 mg/kg/day; this finding was considered to be adverse. As a result of these effects, the mean number of pups delivered and mean number of liveborn pups per litter was statistically significantly smaller for dams administered 400 mg/kg/day, compared with controls. There was also an increase in mean post-implantation loss (%) for dams administered 175 mg/kg/day; however, in the absence of an effect on litter size, this finding was considered not to be adverse.

 

An increased incidence of stillbirths was noted for litters of test article-treated dams, which was higher than the historical control range for dams administered 400 mg/kg/day. Additionally, between PND 0 and 4, 39 pups from litters of dams administered 400 mg/kg/day died, were sacrificed in a moribund condition, or were missing and/or cannibalized. As a result of this mortality, only four of nine litters of dams administered 400 mg/kg/day survived to PND 13, with the remaining five dams in this group sent to necropsy due to total litter loss. In turn, this resulted in lower livebirth and Day 4 viability indices for animals administered 400 mg/kg/day, compared with controls. One additional pup from a litter of a dam administered 400 mg/kg/day was sent to necropsy on PND 7 due to the severity of clinical observations. These findings were higher than the historical control range and considered to be adverse.

 

Test item‑related clinical observations which were associated with the increase in pup mortality at 400 mg/kg/day included no milk in the stomach, thin, weak, cold to touch and/or blue coloration to the neck, as well as an increased incidence of small appearance which was associated with lower mean body weights for pups in litters of dams administered 400 mg/kg/day. These findings were considered to be adverse. Small appearance was also noted for one pup in one litter of a dam administered 75 mg/kg/day and two pups in two litters of dams administered 175 mg/kg/day. However, at these dose levels this observation was generally transient and not accompanied by other observations or effects on pup body weight, and as such, it was considered not adverse at these dose levels.

 

No effect on thyroid hormones was noted for female pups sampled on PND 13. Additionally, no effect of maternal on nipple count of male offspring or pup ano-genital distance was observed. No test article-related effects on organ weights or macroscopic observations were noted.

 

Conclusions

In conclusion, following once daily oral (gavage) administration of the test item at 75, 175, or 400 mg/kg/day to male rats for 42 consecutive days and to female rats for up to 57 days, a dose-dependent reduction in mean body weight gain was observed, which was considered adverse for animals administered 400 mg/kg/day. No effect on estrous cyclicity or fertility was noted; however, a lower mean number of implantation sites and increased post-implantation loss (%) were noted for females administered 400 mg/kg/day, which resulted in a significantly smaller mean litter number of pups delivered and mean number of liveborn pups per litter. Additionally, a test article-related, dose-dependent increase in pup mortality was noted, which resulted in significantly lower livebirth and Day 4 viability indices for animals administered 400 mg/kg/day, compared with controls. Lower pup body weights and adverse clinical observations were also noted for litters of dams administered 400 mg/kg/day.

 

Based on the observations noted, the no observed adverse effect level (NOAEL) for adult reproduction and offspring development is 175 mg/kg/day.