Registration Dossier

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 August 2017 to 29 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

GLP compliance:

yes

Limit test:

no

Justification for study design:

The objective of the study was to screen for effects of the test article, Naugalube APAN, on male and female reproductive performance (i.e. gonadal function, mating behavior, conception, development of the conceptus and parturition) and offspring growth until LD 13.

Specific details on test material used for the study:

No further details specified in the study report.

Species:

rat

Strain:

Crj: CD(SD)

Details on species / strain selection:

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Specifications and Acclimation
Forty three male and 46 female Crl:CD(SD) rats were obtained from Charles River Laboratories, Margate, United Kingdom, in order to provide sufficient animals for study selection.
Upon arrival, males were approximately 9 to 10 weeks of age, while females were approximately 8 to 9 weeks of age. Males weighed between 368.5 and 456.2 g; females weighed between 211.9 and 274.3 g. Animals were 11 to 13 weeks old and were considered sexually mature at the start of dosing.
Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for at least 14 days prior to initiation of dosing (males) or 7 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 suitability for the study.

Environmental Conditions, Diet, and Water
Housing
Animals were housed 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).
During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation, and with their litter during lactation. Males were returned to group-housing after the pairing phase.
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 Covance.

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 Covance.

Diet
Animals had ad libitum access to VRFI diet (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 Covance.
A 50 g sample of the batch of diet used on study was collected and stored at ambient temperature until study finalization.

Environment
Animals were housed in a single, exclusive room. The room was air-conditioned to provide a minimum of 15 to 20 air changes/hour. The temperature and relative humidity ranges were maintained in the specified ranges of 19 to 25°C and 30 to 70%, respectively.
Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark, with the exception of when experimental procedures dictated.

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

Animal Identification and Assignment to Study
Upon arrival, animals were assigned to dose groups using a total randomization procedure. Animals were individually identified by electronic implant.
Cages were placed in dose group order across the batteries.

Route of administration:

oral: gavage

Vehicle:

other: Arachis oil BP,

Details on exposure:

Control Article (Vehicle)
The control article (vehicle) was Arachis oil BP, supplied by Sigma Aldrich (batch number MKBX4739V).

Test Article and Control Article (Vehicle) Formulation
Formulations were prepared weekly.
The test article was formulated as a suspension in Arachis oil BP following dispensary SOPs and the formulation method (Method 8370635_O_01D), as maintained in the study data.
Formulations were stored refrigerated (2 to 8°C) in a sealed container, protected from light.

Details on mating procedure:

During the pairing phase, one male was housed for up to 10 days with one female of the same dose group. Females without evidence of mating by Pairing Day 10 were paired for up to an additional 5 days with previous confirmed mated males of the same dose group.
Mating was confirmed by the presence of a vaginal plug in situ or of sperm in a vaginal lavage. Upon the confirmation of mating, vaginal lavage was discontinued, and the male was removed from the cage. The day on which mating was confirmed was designated GD 0.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and Homogeneity Data
Suspensions of Naugalube APAN, at 2.5 and 500 mg/mL in Arachis oil BP, were found stable and homogenous for 13 days at 15 to 25°C and 2 to 8°C (Covance Study 8370636).

Achieved Concentration
Formulations prepared for use during Weeks 1 and 6 were analyzed to determine the achieved concentration. Triplicate samples were removed from the middle of the test article formulations and were analyzed. A single sample was taken from the middle of control formulations and was analyzed.

Sample Analysis and Disposition
Samples were analyzed by the Formulations Analysis section of the Central Dispensary at Covance (refer to the Formulation Analysis Report).

Duration of treatment / exposure:

Males were dosed for 42 consecutive days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until the day before necropsy [post-pairing phase]). Females were dosed for up to 57 days (2 weeks prior to pairing [pre-pairing phase], during the pairing phase, and until LD 13, inclusive. Some females were not dosed on LD 0 if they were starting or had just completed parturition.

Frequency of treatment:

Daily

Details on study schedule:

Before females were assigned to the study, estrous cycle data were reviewed to ensure all females allocated to the study showed regular estrous cycles. Any female that did not show a regular estrous cycle was replaced by a spare female showing a regular estrous cycle.
Relevant data recorded throughout the study for the spare undosed animals is maintained within the raw data, not reported and will be archived with the study data.
Cages were appropriately identified with study information, including study number and animal number(s).

No. of animals per sex per dose:

20 animals per dose group (10 male/10 female)

Control animals:

yes, concurrent vehicle

Details on study design:

In a previous 28 day toxicity study in rats (SPL Project Number 445/297), repeated oral (gavage) administration of 15, 150 or 1000 mg/kg/day Naugalube APAN did not result in any unscheduled deaths. Females administered 1000 mg/kg/day showed a reduction in body weight gain and associated dietary intake over the first 2 weeks of dosing, with some improvement apparent thereafter. The clinical observation of hunched posture was also observed for these females from Day 9 of the dosing phase, lasting throughout the dosing phase.
Increased body weight-related liver weight was noted for males and females administered 1000 mg/kg/day and to some extent following administration of 150 mg/kg/day. Microscopic examination showed centrilobular hepatocyte enlargement (considered to be an adaptive change) in these animals, with the effect extending to some males administered 15 mg/kg/day. Elevated body weight-related spleen and adrenal weights were observed for females administered 1000 mg/kg/day, and females administered 150 mg/kg/day also showed an increase in the latter; these elevations were not associated with any microscopic changes.
Slight reductions in erythrocyte count, hemoglobin, and hematocrit were noted for males and females administered 1000 mg/kg/day, with dose-related increase in plasma bilirubin also evident for males and females of all test article-treated groups.
Based on the finding of this study, and given the longer administration period, the high dose level of 600 mg/kg/day was considered acceptable for use in this study.
The oral (gavage) route of administration was chosen because it is an acceptable and commonly used route exposure for regulatory studies of this type.

Positive control:

Not required

Parental animals: Observations and examinations:

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

Clinical Examinations
Each animal was given a detailed physical examination once daily from the start of dosing, including the day of necropsy. An individual record of the clinical condition of each animal was maintained.

Postdose Observations
Animals were observed daily for the first 3 days of dosing; upon return to the home cage; and approximately 0.5, 1, 2, and 4 hours postdose.
In the absence of any toxicologically significant postdose observations during the first 3 days of dosing, no further postdose observations were scheduled.

Body Weights
Male body weights were recorded once during acclimation (Day -7), on the first day of dosing, at weekly intervals thereafter, and on the day of necropsy.
Female body weights were recorded once during acclimation (Day -7); on the first day of dosing; weekly prior to pairing and until confirmation of mating during the pairing phase; on Gestation Day (GD) 0, 7, 14, and 20; and on LD 0 (where applicable) 1, 4, 7, 13, and 14 (prior to necropsy). Body weights for the female with no confirmation of mating (Animal R0402 [Group 1]) were recorded weekly during the post-pairing phase, including the last day of that phase.

Food Consumption
The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumption was recorded for females from GD 0 to 20 and from LD 0 to 13. Consumption was calculated as g/animal/day.

Parturition
Animals were observed three times on 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. Females were observed for signs indicating the start of parturition (for example, blood in the cage). The time and date of this observation were recorded, where possible, and marked as the end of gestation; when 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 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 (macroscopic examination for lung inflation).

Clinical Pathology
Sample Collection and Handling
Adult blood samples (2 x 0.6 mL or 1 x 1.2 mL [Serum Separator Tube], nominal) obtained for total thyroxine (T4) and thyroid stimulating hormone (TSH) analysis were withdrawn from the jugular vein on the day of necropsy (males) or from the abdominal aorta at necropsy (females); sampling was performed at a similar time on each occasion. Samples were collected after animals were fasted overnight.
Blood samples for TSH and T4 analysis from culled pups on PND 4 (1 x 0.6 mL) were withdrawn via decapitation to provide one pooled sample for each litter, where possible.
Blood samples for TSH and T4 analysis from pups sacrificed on PND 13 (2 x 0.6 mL) were withdrawn by cardiac puncture from two male and two female pups from each litter. Samples from one male and one female sample were analyzed; the remaining two samples from each litter were retained in storage until report finalization.
Each blood sample was gently inverted several times and was stored at room temperature (15 to 25°C) until centrifugation at 2300g for 10 minutes at approximately 4°C. The resultant serum was separated, transferred to uniquely labeled clear polypropylene tubes, and frozen at <-20°C. Samples were protected from light until frozen, were centrifuged and aliquoted within 2 hours of collection, and were analyzed at Covance.

Oestrous cyclicity (parental animals):

Estrous Cycle Determination
Daily vaginal lavage (washings) was conducted for all females during acclimation (predose), from 1 week after arrival until the day prior to dosing. The stage of estrous was recorded for all females, and only females with regular 4- or 5-day cycles were included on the study.
Daily vaginal lavage (washings) was conducted for females from the start of dosing until the confirmation of mating and on LD 14.

Sperm parameters (parental animals):

Not examined

Litter observations:

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 underwent a macroscopic necropsy.
On PND 4, litters were culled to 10 pups/litter, with five pups/sex, where possible.
Pups were selected randomly, with no bias to size or clinical status. Culled pups were discarded following blood sampling. Culling created a uniformly sized litter, which reduced differences in pup body weights due to litter size.

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

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

Ano-genital Distance
The ano-genital distance of all pups was recorded on PND 4.

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

Postmortem examinations (parental animals):

Males were sacrificed on Study Day 43 (Post-Pairing Day 14). Females were sacrificed on LD 14. Animals were fasted overnight prior to sacrifice.
Animals were sacrificed in a controlled randomization sequence, by isoflurane anesthesia; once a suitable deep plane of anesthesia was established; major blood vessels were severed to exsanguinate the animal. After sacrifice, macroscopic examinations were performed, and all lesions were recorded.

Necropsy and Organ Weights
Selected organs, as indicated in the following table (see "Any other information" for the table), were weighed and organ weights recorded for each adult animal. Bilateral organs were weighed together. Tissues were dissected free from fat and contiguous tissue.

Histology
Tissues denoted by E in the previous tissue list from adult animals in Groups 1 and 4 and prostate and seminal vesicles from males in Groups 2 and 3 were embedded in paraffin wax BP (block stage), sectioned at a nominal 5 μm, and stained with hematoxylin and eosin.
Sections of testes and epididymides were also stained with Periodic Acid-Schiff (PAS) for spermatogenic staging.

Microscopic Observations
The following tissues were examined microscopically by the Study Pathologist.
Groups 1 and 4: Adult males and females All tissues denoted by E in the previous tissue list
Groups 2 and 3: Adult males Prostate and seminal vesicles
Slides from PAS-stained testis and epididymis were examined by the Study
Pathologist for qualitative assessments of stages of spermatogenesis and interstitial testicular cell structure.

Postmortem examinations (offspring):

Surplus pups culled on PND 4 and remaining pups sent to necropsy on PND 13 were sacrificed using an intraperitoneal injection of sodium pentobarbitone (overdose).
Once a suitable deep plane of anesthesia was established, PND 4 pups were decapitated, and PND 13 pups were exsanguinated by severing a major blood vessel.
Surplus pups culled on PND 4 were discarded following blood sample collection and sex determination. Full macroscopic examinations were conducted for all decedents, and external examinations for gross abnormalities, with particular attention to the external reproductive genitals, were conducted for all pups sent to necropsy on PND 13.
The thyroid was removed from one pup/sex/litter on PND 13 and preserved in 10% neutral-buffered formalin.

Statistics:

Data Evaluation and Statistical Analysis
Where tables/appendices are computer-generated, rounding of individual values may have occurred 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, unless stated otherwise. Except when otherwise stated, tests were performed using a two-sided risk and were considered significant where P ≤ 0.05. By default, significant results were reported as * = P ≤ 0.05, + = P ≤ 0.01, and/or # = P ≤ 0.001.

The following data were analyzed using Tox Reporting.
-Thyroid hormones (adult males and females) - Procedure I (ANOVA)

The following data were analyzed using Pristima.
-Body weights (adult) - Procedure I (ANOVA)
-Body weight gains (adult) - Procedure I (ANOVA)
-Food consumption (gestation and lactation) - Procedure I (ANOVA)
-Absolute organ weights and organ to terminal body weight ratios - Procedure I (ANOVA)
-Male and female mating, fecundity, and fertility indices - Procedure IV (one-sided lower tail)

The following data were analyzed using SAS.
-The duration of gestation, number of implantation sites, number of pups born, number of pups alive on PND 1 and 4 (before culling), % male pups on PND 1, percent post-implantation loss, and live birth and survival indices, where appropriate - Procedure III
-Pup weights (male, female, and combined) - Procedure II (litter size as the covariate)
-Ano-genital distance (males only) - Procedure II (cube root of mean pup weight as the covariate)
-Thyroid hormones (male, female, and combined pups) - Procedure I (ANOVA)

Additional information can be found under "Any other information".

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Hunched posture was recorded on GD 0 and 1 for one female administered 600 mg/kg/day. Raised hair was noted from GD 1 to 3 for one or two females administered 600 mg/kg/day. Isolated instances of raised hair were noted during late gestation and early lactation for one female administered 600 mg/kg/day. These findings were considered related to test article-administration, however, due to the low incidences, these findings were considered to be of little toxicological importance.
No test article-related clinical observations were noted for males administered 600 mg/kg/day or either sex administered 50 or 300 mg/kg/day.
Remaining clinical observations, including purple stained bedding, staining of the skin and fur, salivation, tears in the left eye, thin fur, hair loss sores/lesions/physical injury, vocalization, and wet fur, were observed in test article-treated or control groups or lacked a dose relationship. These findings were transient or only observed in only a few animals. In the absence of a dose response, these findings were considered to have represented low incidences findings occasionally observed in laboratory maintained rats and were unrelated to test article administration.

Postdose Observations
No toxicologically significant postdose observations were noted.
The only postdose observation was the presence of mouth rubbing immediately after dosing on Pre-Pairing Day 3 for three females administered 600 mg/kg/day. This finding is occasionally observed following the oral administration of an unpleasant test article formulation and was considered unrelated to systemic toxicity; as such, postdose observations were discontinued after the first 3 days of dosing.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No adult mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males
No adverse effect on body weight was noted for males.
Body weight gain during the first week of dosing was significantly reduced for males administered 600 mg/kg/day, compared with controls (P ≤ 0.01). Values were comparable with controls thereafter. Although a dose-response was not clear, overall body weight was reduced during the dosing period for males administered 300 mg/kg/day (-10%) or 600 mg/kg/day (-7%), compared with controls; this finding was considered to have arisen incidentally and was of no toxicological significance.

Females
Lower body weight gain was evident during the first 2 weeks of dosing for females administered 300 or 600 mg/kg/day, compared with controls; a dose-response was not evident.
Females administered 600 mg/kg/day showed significantly lower body weights from GD 7, compared with controls (P ≤ 0.01). Body weights during lactation were also adversely affected, compared with those of controls, following administration of 600 mg/kg/day (P ≤ 0.01 to P ≤ 0.001). Overall body weight gain from the start of the study to LD 13 was approximately 30% lower than that of controls.
A similar body weight effect to that observed following administration of 600 mg/kg/day was evident following administration of 300 mg/kg/day, albeit to a lesser extent. Lower body weights were observed during gestation, compared with controls, although statistical significance was only achieved on GD 7 (P ≤ 0.05). Body weights were significantly lower than controls up to LD 7, compared with controls (P ≤ 0.05 to P ≤ 0.01). Overall body weight gain from the start of the study to LD 13 was approximately 20% lower than that of controls.
From the start of dosing to LD 13, overall body weight gain for females administered 50 mg/kg/day was 12% lower than that of controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males
No adverse effect on food consumption was observed for males.
Food consumption was marginally lower during the first week of dosing for males administered 600 mg/kg/day, compared with controls (-12%). Improvement was observed, with values comparable with controls thereafter.

Females
Food consumption of females administered 50, 300, or 600 mg/kg/day was lower than that of controls.
For females administered 600 mg/kg/day, food consumption was reduced by 33%, compared with controls, during the first 2 weeks of dosing. Food consumption was 21% lower than controls during the first week of gestation, 15% lower than controls during the second week of gestation, and 11% lower than controls from GD 14 through 20. Food consumption was approximately 15% lower than controls during lactation. Over the duration of the study, mean food consumption was approximately 18% lower than that of controls.
For females administered 300 mg/kg/day, food consumption was reduced by 24% during the first 2 weeks of dosing and was 18% lower during the first week of gestation, compared with controls. Values were still approximately 11% lower than controls during the remainder of gestation, and were 13% lower that controls during the first week of lactation. Over the duration of the study, mean food consumption was 12% lower than that for controls.
A similar food consumption effect was noted for females administered 50 mg/kg/day, albeit to a lesser extent than that observed following administration of 300 or 600 mg/kg/day. Food consumption during the first 2 weeks of dosing was approximately 25% lower than that for controls. Improvement was observed thereafter; however, over the duration of the study, mean food consumption was 7% lower than that for controls.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Thyroid hormone assessments showed lower thyroxine (T4) for males administered 300 or 600 mg/kg/day, compared with controls (P ≤ 0.001).
Elevated TSH was observed for males from all test article treated groups, compared with controls. However, a convincing dose-response was not evident, and statistical significance was not achieved.
No such changes were noted for females.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Against a variable background, decreased secretion was recorded in prostate in a few control males and a few males administered 50, 300 or 600 mg/kg/day. While there was a degree of correlation with reduced prostate weights within groups, the variability seen precluded a definite relationship to Naugalube APAN. Decreased secretion was characterized by a reduced prostate size with little secretion in the lumen of acini mainly located in the ventral portion of the prostate.
Qualitative testis examination did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle.
No microscopic findings that suggested Naugalube APAN-related effects were noted in the testis, epididymis, seminal vesicle, or ovary.
Most tissues were microscopically unremarkable, and the findings observed were generally consistent with the usual pattern of findings in rats of this strain and age.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Parturition and Litter Data
No statistically significant difference was noted in gestation length; however, three females administered 300 mg/kg/day and three females administered 600 mg/kg/day had gestation lengths of 24 days, while remaining females of these groups had gestation lengths of 23 days. The longer gestation lengths were considered to be attributable to test article administration. The gestation length of females administered 50 mg/kg/day was identical to that of controls (23 days).
Smaller litter sizes (number of pups alive on PND 1) of -16% for groups administered 300 or 600 mg/kg/day, which attained statistical significance, compared with controls (P ≤ 0.05). The smaller litter sizes were attributable to lower number of mean implantation sites noted at these dose levels, although statistical significance was not achieved.
One stillborn pup was evident from each of three litters maternally administered 600 mg/kg/day. Two stillborn pups were observed from each of two litters maternally administered 300 mg/kg/day. No stillborn pups were evident in the control group. The higher incidence of stillborn pups in these dose groups was considered to be test article-related. Statistical analysis was not performed on these data.
No test article-related effects on parturition or the subsequent litters were noted following maternal administration of 50 mg/kg/day.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No overt difference was noted in the number or duration of estrous cycles of test article-treated females, compared with controls.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No effect was noted on mating or fertility.
With the exception of one pair administered 50 mg/kg/day and one pair administered 600 mg/kg/day, all pairs mated within 5 days of pairing.
All animals from all dose groups mated and achieved pregnancy, and all females from all dose groups gave birth to a live litter.

Clinical signs:

no effects observed

Description (incidence and severity):

All clinical observations recorded are occasionally observed in juvenile animals of this species and were unrelated to test article toxicity.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

A higher incidence of pup mortality was evident for litters from groups administered 600 or 300 mg/kg/day, compared with controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No overt difference in pup body weights was noted for those maternally administered the test article, compared with controls.
Following the cull on PND 4, mean body weights, adjusted for litter size were marginally lower for pups maternally administered 300 or 600 mg/kg/day, compared with control; however, statistically significance was not achieved.
On PND 7, mean adjusted body weights were lower for male pups maternally administered 300 or 600 mg/kg/day, with statistically significant differences noted for mean male and combined sex body weights (adjusted for litter size), compared with controls (P ≤ 0.05).
By PND 13, mean adjusted pup body weights for males, females, and combined sexes maternally administered 600 mg/kg/day were significantly lower than those of control pups (P ≤ 0.001). The mean adjusted weight for male pups maternally administered 300 mg/kg/day was also significantly lower than that of controls (P ≤ 0.05), albeit to a lesser extent than that for male pups from the high-dose group.
Body weights of pups maternally administered 50 mg/kg/day were essentially similar to those of controls.

Food consumption and compound intake (if feeding study):

not examined

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:

effects observed, non-treatment-related

Description (incidence and severity):

Pup thyroid hormone levels were unaffected by maternal exposure to the test article.
Thyroid hormone assessment on samples from PND 4 showed T4 values that were below the lower limit of quantification (LLOQ; <26 nmol/L) for all dose groups, including controls. The T4 was significantly higher for PND 13 male pups maternally administered 300 or 600 mg/kg/day, compared with controls (P ≤ 0.01 or P ≤ 0.05, respectively). Combined male and female pups maternally administered 300 mg/kg/day also showed significantly higher T4 on PND 13, compared with controls (P < 0.01); however, no such effect was noted in the 600 mg/kg/day dose group. In the absence of a dose response, these increases were considered attributable to normal biological variation and were unrelated to test article toxicity.
On PND 4, mean TSH levels for pups maternally administered the test article were higher than those of controls; however, all values were within the historical background control data for this parameter (0.16 to 0.63 μIU/mL). In the absence of a similar effect observed in pups on PND 13, this was finding considered a result of normal biological variation and was unrelated to test item toxicity.

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No Naugalube APAN-related macroscopic abnormalities were noted for pups.

Histopathological findings:

not examined

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Summary of Organ Weight %-Difference, Compared with Concurrent Controls – Terminal Sacrifice

		Males
		2M	3M	4M
	Level (mg/kg/day)	50	300	600
		% difference
Seminal vesicle	Group mean unadjusted weights Body weight ratio	+7.704 +10.739	-6.274 -4.405	-17.923 -16.118
Prostate	Group mean unadjusted weights Body weight ratio	-3.653 -1.562	-10.033 -8.124	-23.556 -21.550

M = Male

Comments on the Data

The summary table for observations indicates the number of animals within a period for which a condition was observed, without regard to the number of incidences/animal or the length of time the condition persisted.

The individual table for post-dose observations only presents animals with observations. All other animals were recorded as No Abnormalities Detected.

The individual table for pup clinical observations only presents pups with observations. All other pups were recorded as No Abnormalities Detected.

Pairing Day 15 and Post-pairing Day 1 were the same calendar day. Data for this calendar day were recorded only as Pairing Day 15.

Summary of Clinical Observations

Males

Pre-pairing

Group #

Category, Observations

Day of Phase:

1

2

3

4

5

6

7

Control

Excretion, salivation, minimal

Skin and fur, sores/lesions, minimal, ear left

Skin and Fur, staining, abdomen, yellow

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, pink

Skin and Fur, thinning fur, back

Number Examined:

Number Normal:

10

9

0

1

0

10

9

0

1

0

10

9

0

1

0

10

6

1

0

1

0

10

7

0

1

0

1

10

7

0

1

0

1

10

7

0

1

0

1

2

Number Examined:

Number Normal:

10

3

Eyes, tears, eye left

Skin and Fur, staining, eyelid left, red

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, shoulder right, yellow

Skin and Fur, thinning fur, head

Number Examined:

Number Normal:

10

0

10

0

10

0

10

7

0

1

10

8

1

0

1

10

8

1

0

1

10

8

1

0

1

4

Skin and Fur, staining, abdomen, yellow

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, pink

Skin and Fur, wet fur, abdomen

Skin and Fur, wet fur, uro-genital area

Number Examined:

Number Normal:

10

0

10

0

10

0

10

6

1

2

1

10

9

0

1

0

10

0

10

0

Males

Pre-pairing

Group #

Category, Observations

Day of Phase:

8

9

10

11

12

13

14

15

Control

Behaviour, vocalisation

Skin and Fur, sores/lesions, minimal, face

Skin and Fur, staining, abdomen, yellow

Skin and Fur, staining, muzzle, brown

Skin and Fur, thinning fur, back

Skin and Fur, thinning fur, face

Number Examined:

Number Normal:

10

7

0

1

0

10

0

10

0

10

9

0

1

10

9

0

1

10

9

0

1

0

1

10

9

0

1

0

1

10

8

1

0

1

2

Number Examined:

Number Normal:

10

3

Eyes, tears, eye left

Skin and Fur, staining, eyelid left, red

Skin and Fur, thinning fur, head

Number Examined:

Number Normal:

10

8

1

10

9

0

1

10

9

0

1

10

9

0

1

10

9

0

1

10

9

0

1

10

9

0

1

10

9

0

1

4

Behaviour, vocalisation

Skin and Fur, staining, neck, red

Number Examined:

Number Normal:

10

0

10

0

10

0

10

0

10

0

10

0

10

0

10

8

1

Males

Pairing

Group #

Category, Observations

Day of Phase:

1

2

3

4

5

6

7

Control

Behaviour, vocalisation

Skin and fur, sores/lesions, minimal, face

Skin and Fur, staining, mouth, pink

Skin and Fur, staining, neck, red

Skin and Fur, staining, nose, brown

Skin and Fur, staining, nose, red

Skin and Fur, thinning fur, face

Skin and Fur, thinning fur, lower part of back

Number Examined:

Number Normal:

10

6

1

0

1

0

1

10

6

1

0

1

0

1

10

4

1

0

1

10

7

1

0

1

0

1

0

10

7

1

0

1

0

1

0

10

7

1

0

1

0

1

0

10

6

1

0

1

0

2

Skin and Fur, sores/lesions, minimal, ear right, small

Skin and Fur, staining, ear right, red

Number Examined:

Number Normal:

10

0

10

0

10

9

1

0

10

9

1

10

9

1

10

9

1

10

9

1

3

Skin and Fur, staining, mouth pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, nose, red

Skin and Fur, thinning fur, head

Number Examined:

Number Normal:

10

8

0

1

0

1

10

0

10

8

1

0

1

0

10

0

10

9

0

1

0

10

9

0

1

0

10

0

4

Appearance, damaged, minimal, ear left

Skin and Fur, staining, neck, red

Skin and Fur, staining, nose, brown

Skin and Fur, staining, nose, red

Skin and Fur, thinning fur, face

Number Examined:

Number Normal:

10

9

0

1

0

10

0

10

8

1

0

1

10

8

1

0

1

0

10

7

1

0

1

0

10

7

1

0

1

0

10

8

1

0

1

0

Males

Pairing

Group #

Category, Observations

Day of Phase:

8

9

10

11

12

13

14

15

Control

Behaviour, vocalisation

Skin and Fur, sores/lesions, minimal, face

Skin and Fur, sores/lesions, minimal, shoulder right

Skin and Fur, sores/lesions, minimal, side right

Skin and Fur, staining, cheek, orange

Skin and Fur, staining, muzzle, orange

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, nose, brown

Skin and Fur, staining, nose, red

Skin and Fur, thinning fur, face

Number Examined:

Number Normal:

10

6

1

0

1

10

8

0

1

0

1

10

0

10

0

10

6

0

1

0

1

0

1

10

6

0

1

0

1

10

7

0

1

0

1

10

6

0

1

0

1

2

Skin and Fur, sores/lesions, minimal, ear right, small

Skin and Fur, sores/lesions, minimal, neck

Skin and Fur, staining, neck, orange

Skin and Fur, thinning fur, face

Number Examined:

Number Normal:

10

9

1

0

10

9

1

0

10

9

1

0

20

9

1

0

10

9

1

0

10

4

1

0

2

3

10

6

1

2

1

10

6

1

2

1

3

Skin and Fur, staining, eyelid right, brown

Skin and Fur, staining, face, orange

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, orange

Skin and Fur, staining, nose, brown

Number Examined:

Number Normal:

10

9

0

1

10

0

10

0

10

0

10

0

10

8

1

0

10

9

0

1

0

10

9

0

1

0

4

Appearance, damaged, minimal, ear left

Appearance, damaged, moderate, ear left

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, nose, red

Number Examined:

Number Normal:

10

8

1

0

1

10

8

1

0

1

0

10

8

1

0

1

0

10

8

1

0

1

0

10

8

1

0

1

0

10

8

0

1

0

10

8

0

1

0

10

8

0

1

0

Males

Post-pairing

Group #

Category, Observations

Day of Phase:

2

3

4

5

6

7

8

Control

Skin and Fur, sores/lesions, minimal, muzzle, small

Skin and Fur, sores/lesions, minimal, nose, small

Skin and Fur, sores/lesions, minimal, paw fore left

Skin and Fur, sores/lesions, minimal, shoulder, right

Skin and Fur, staining, cheek, orange

Skin and Fur, staining, eyelids both, brown

Skin and Fur, staining, muzzle, orange

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, pink

Skin and Fur, staining, nose, brown

Skin and Fur, staining, nose, red

Skin and Fur, thinning fur, face

Number Examined:

Number Normal:

10

6

2

0

1

0

10

7

2

0

1

0

1

0

10

7

2

0

1

0

1

0

10

1

2

1

0

1

0

1

0

2

0

2

10

1

2

1

0

1

0

1

0

2

0

2

10

4

2

1

0

1

0

1

0

1

0

10

5

2

1

0

1

0

1

0

1

0

1

0

2

Skin and Fur, sores/lesions, minimal, neck

Skin and Fur, staining, neck, orange

Skin and Fur, staining, nose, red

Skin and Fur, thinning fur, neck

Number Examined:

Number Normal:

10

7

1

2

0

10

8

0

2

0

10

8

0

2

0

10

8

0

2

0

10

9

0

1

0

10

8

0

1

0

10

9

0

1

3

Skin and Fur, staining, legs fore both, orange

Skin and Fur, staining, nose, brown

Skin and Fur, thinning fur, face

Skin and Fur, thinning fur, leg hind left

Skin and Fur, thinning fur, legs fore both

Number Examined:

Number Normal:

10

0

10

0

10

9

1

0

10

8

1

0

10

9

0

1

0

10

8

0

1

0

1

10

7

0

1

4

Appearance, damaged, moderate, ear left

Skin and Fur, hair loss, paws fore both

Skin and Fur, sores/lesions, minimal, leg hind right, small

Skin and Fur, staining, face, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, nose, red

Skin and Fur, staining, uro-genital area, [ink

Number Examined:

Number Normal:

10

9

1

0

10

9

1

0

10

7

1

0

1

0

10

7

1

0

1

0

10

7

1

0

1

0

10

6

1

0

1

0

10

7

1

0

1

0

1

Males

Post-pairing

Group #

Category, Observations

Day of Phase:

9

10

11

12

13

14

Control

Behaviour, vocalisation

Skin and Fur, sores/lesions, minimal, cheek

Skin and Fur, sores/lesions, minimal, leg hind right

Skin and Fur, sores/lesions, minimal, muzzle, small

Skin and Fur, sores/lesions, minimal, nose, small

Skin and Fur, staining, cheek, orange

Skin and Fur, staining, eyelids both, brown

Skin and Fur, staining, muzzle, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, nose, brown

Number Examined:

Number Normal:

10

3

1

0

2

1

0

1

10

4

0

2

1

0

1

10

5

0

2

1

0

1

10

4

0

1

2

0

1

10

7

0

1

0

1

0

1

10

7

0

1

0

1

0

1

2

Skin and Fur, thinning fur, neck

Number Examined:

Number Normal:

10

9

1

10

9

1

10

9

1

10

9

1

10

9

1

10

9

1

3

Skin and Fur, thinning fur, face

Skin and Fur, thinning fur, leg hind left

Skin and Fur, thinning fur, legs fore both

Number Examined:

Number Normal:

10

7

1

10

7

1

10

7

1

10

7

1

10

7

1

10

7

1

4

Appearance, damaged, minimal, ear left

Appearance, damaged, moderate, ear left

Skin and Fur, sores/lesions, minimal, leg hind right, small

Skin and Fur, staining, face, pink

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, nose, red

Skin and Fur, staining, uro-genital area, pink

Number Examined:

Number Normal:

10

7

0

1

0

1

10

7

0

1

0

1

10

7

0

1

0

1

10

7

0

1

0

1

10

7

1

0

1

0

1

10

6

1

0

1

Females

Pre-pairing

Group #

Category, Observations

Day of Phase:

1

2

3

4

5

6

7

Control

Skin and fur, sores/lesions, minimal, shoulder right

Skin and Fur, staining, abdomen, yellow

Skin Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, nose, pink

Skin and Fur, staining, shoulders both, brown

Skin and Fur, thinning fur, abdomen

Skin and Fur, thinning fur, side both

Number Examined:

Number Normal:

10

8

0

1

0

1

10

7

0

1

0

1

10

7

0

1

0

1

10

3

1

2

1

10

3

1

2

1

10

4

1

0

1

2

1

10

4

1

0

1

2

1

0

1

2

Skin and Fur, sores/lesions, minimal, ear left, small

Skin and Fur, sores/lesions, minimal, ears both, small

Skin and Fur, staining, ears both, brown

Skin and Fur, staining, head, pink

Skin and Fur, staining, nose, pink

Skin and Fur, thinning fur, back

Number Examined:

Number Normal:

10

7

0

1

0

2

10

7

0

1

0

2

10

7

1

0

2

10

7

1

0

1

0

10

6

1

0

1

10

6

1

0

1

10

6

1

0

1

3

Skin and Fur, staining, head, red

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, pink

Skin and Fur, staining, neck, red

Skin and Fur, thinning fur, neck

Number Examined:

Number Normal:

10

0

10

8

1

0

1

0

10

8

1

0

1

0

10

7

0

2

1

0

10

7

0

2

1

0

10

7

0

2

1

0

1

10

7

0

2

1

0

1

4

Skin and Fur, staining, head, red

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, nose, brown

Skin and Fur, thinning fur, back

Number Examined:

Number Normal:

10

9

0

1

10

8

1

0

1

10

8

1

0

1

10

5

1

2

1

0

10

5

1

2

1

0

10

5

1

2

1

0

10

5

1

2

1

0

Females

Pre-pairing

Group #

Category, Observations

Day of Phase:

8

9

10

11

12

13

14

15

Control

Skin and Fur, hair loss, head

Skin and Fur, hair loss, shoulders both

Skin and Fur, hair loss, side both

Skin and Fur, sores/lesions, minimal, shoulder left

Skin and Fur, sores/lesions, minimal, shoulder right

Skin and Fur, staining, head, pink

Skin and Fur, staining, head, red

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, red

Skin and Fur, staining, nose, pink

Skin and Fur, thinning fur, abdomen

Skin and Fur, thinning fur, head

Skin and Fur, thinning fur, sides both

Skin and Fur, thinning fur, legs hind both

Number Examined:

Number Normal:

10

4

0

1

0

2

0

1

0

1

0

10

5

0

1

2

0

1

0

1

0

10

6

0

1

0

1

0

10

5

0

1

0

1

0

10

9

0

1

0

1

0

1

0

10

9

0

1

0

1

0

1

0

10

9

0

1

0

1

0

1

0

10

9

1

0

1

0

1

0

1

2

Skin and Fur, sores/lesions, minimal, ear left, small

Skin and Fur, staining, ears, both, brown

Skin and Fur, staining, head, pink

Skin and Fur, staining, neck, red

Skin and Fur, staining, nose, pink

Skin and Fur, thinning fur, back

Number Examined:

Number Normal:

10

6

1

0

1

10

5

1

2

0

1

10

5

1

2

0

1

10

5

1

3

0

1

10

7

1

0

1

10

8

1

0

1

0

1

10

8

1

0

1

0

1

10

8

0

1

0

1

3

Skin and Fur, staining, head, red

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, pink

Skin and Fur, staining, neck, red

Skin and Fur, thinning fur, neck

Number Examined:

Number Normal:

10

7

0

2

0

1

0

1

10

9

0

1

0

1

10

9

0

1

0

1

10

7

0

1

0

2

1

10

9

0

1

0

1

10

8

0

1

0

1

10

5

1

3

0

1

10

5

1

2

0

1

4

Skin and Fur, staining, eyelids both, red

Skin and Fur, staining, eyelid left, red

Skin and Fur, staining, head, orange

Skin and Fur, staining, head, pink

Skin and Fur, staining, head, red

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, Staining, muzzle, red

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, red

Skin and Fur, staining, nose, brown

Skin and Fur, thinning fur, leg fore right

Number Examined:

Number Normal:

10

5

0

1

2

0

1

0

10

7

0

1

0

1

0

10

7

0

1

0

1

0

10

7

0

1

0

1

0

10

8

0

1

0

1

0

10

5

1

0

1

0

2

0

1

0

10

3

1

0

1

0

4

0

2

0

10

2

0

1

0

2

1

0

1

Females

Pairing

Group #

Category, Observations

Day of Phase:

1

2

3

4

5

6

7

Control

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, pink

Skin and Fur, staining, head, red

Skin and Fur, staining, muzzle, brown

Skin and Fur, thinning fur, body

Number Examined:

Number Normal:

6

2

1

2

0

1

5

1

2

1

2

1

0

1

0

1

0

1

0

1

0

1

0

1

0

1

0

1

0

1

0

2

Skin and Fur, staining, ears both, brown

Ski and Fur, staining, head, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, pink

Skin and Fur, thinning fur, back

Number Examined:

Number Normal:

10

8

1

0

1

7

4

1

0

1

5

3

0

1

0

1

0

1

0

1

0

1

0

3

Skin and Fur, staining, head, red

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, red

Skin and Fur, thinning fur, head

Skin and Fur, thinning fur, neck

Number Examined:

Number Normal:

8

2

1

3

1

2

7

3

1

2

1

5

3

1

0

1

0

4

Skin and Fur, staining, eyelid left, red

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, red

Skin and Fur, thinning fur, leg fore right

Number Examined:

Number Normal:

7

1

2

1

5

0

3

0

1

0

1

0

1

0

1

0

1

0

1

0

Females
Pairing
Group #	Category, Observations	Day of Phase:	8	9	10	11	12	13	14	15
Control	Skin and Fur, staining, head, pink Skin and Fur, staining, head, red Skin and Fur, staining, muzzle, brown	Number Examined: Number Normal:	1 0 0 1 1	1 0 0 1 1	1 0 0 1 1	1 0 0 1 1	1 0 1 0 1	1 0 1 0 1	1 0 1 0 1	1 0 1 0 1

Females

Gestation

Group #

Category, Observations

Day of Phase:

0

1

2

3

4

5

6

Control

Skin and Fur, hair loss, head

Skin and Fur, hair loss, shoulders both

Skin and Fur, hair loss, sides both

Skin and Fur, sores/lesions, minimal, shoulder left

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, pink

Skin and Fur, staining, head, red

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, nose, brown

Skin and Fur, staining, nose, pink

Skin and Fur, staining, nose, red

Skin and Fur, thinning fur, abdomen

Skin and Fur, thinning fur, back

Skin and Fur, thinning fur, body

Skin and Fur, thinning fur, legs hind both

Number Examined:

Number Normal:

9

3

1

2

1

0

1

0

1

0

1

9

2

1

0

1

4

0

2

0

1

0

1

0

1

9

2

1

0

1

5

0

2

0

1

0

1

0

1

9

2

1

0

1

5

0

2

1

0

1

0

1

0

1

9

1

0

1

5

0

2

1

0

2

1

0

1

0

1

9

1

0

1

4

0

2

1

0

2

0

1

0

1

0

1

9

1

0

1

4

0

2

1

0

2

0

1

0

1

2

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, pick

Skin and Fur, staining, nose, pink

Skin and Fur, thinning fur, back

Number Examined:

Number Normal:

10

7

2

0

1

0

1

10

6

3

0

1

0

1

10

3

2

0

3

0

1

10

5

2

0

2

0

1

10

5

3

0

2

0

1

10

5

3

0

1

2

0

1

10

2

4

1

3

1

3

Skin and Fur, staining, head, orange

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, neck, red

Skin and Fur, thinning fur, head

Number Examined:

Number Normal:

10

5

0

1

2

0

1

10

4

0

2

1

2

0

1

0

10

4

0

3

1

0

1

0

10

4

0

3

1

0

1

0

10

1

4

2

1

0

1

0

10

1

5

2

1

0

10

1

5

3

0

2

1

0

4

Appearance, hunched

Skin and Fur, raised hair

Skin and Fur, staining, eyelid left, red

Skin and Fur, staining, head, orange

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, nose, red

Skin and Fur, thinning fur, leg fore right

Skin and Fur, thinning fur, legs fore both

Number Examined:

Number Normal:

10

2

1

0

1

0

4

1

0

1

0

10

3

1

2

0

3

1

0

1

0

10

2

0

2

1

3

1

0

1

0

10

5

0

1

0

1

2

1

0

1

0

10

4

0

1

0

1

2

1

0

10

4

0

1

0

1

2

1

0

10

3

0

1

0

1

2

1

2

0

1

Females

Gestation

Group #

Category, Observations

Day of Phase:

7

8

9

10

11

12

13

Control

Skin and Fur, hair loss, head

Skin and Fur, hair loss, shoulders both

Skin and Fur, hair loss, sides both

Skin and Fur, staining, ears both, red

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, orange

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, red

Skin and Fur, staining, nose, pink

Skin and Fur, thinning fur, back

Skin and Fur, thinning fur, head

Skin and Fur, thinning fur, legs hind both

Number Examined:

Number Normal:

9

2

1

0

1

0

4

2

1

0

1

0

2

1

0

1

9

1

0

1

4

2

0

1

0

2

1

0

1

9

2

1

2

3

2

0

2

0

1

0

1

9

2

1

2

3

2

0

2

0

1

0

1

9

1

2

3

2

0

2

1

0

1

9

0

1

2

3

2

1

0

2

1

0

1

9

0

1

2

4

1

2

1

0

1

2

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, pink

Skin and Fur, staining, nose, pink

Skin and Fur, thinning fur, back

Skin and Fur, thinning fur, muzzle

Number Examined:

Number Normal:

10

2

1

4

1

3

1

0

10

2

1

4

1

2

3

1

0

10

3

1

4

1

2

4

0

1

0

10

3

0

4

1

2

4

0

10

1

0

5

1

2

5

0

1

10

1

0

5

1

2

5

0

1

10

2

0

5

0

2

4

0

1

3

Skin and Fur, staining, ears both, brown

Skin and Fur, staining, head, orange

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, neck, red

Skin and Fur, staining, paws fore both, red

Number Examined:

Number Normal:

10

1

0

1

5

3

2

1

0

10

1

0

1

5

3

2

1

0

10

0

1

5

4

2

3

1

0

10

1

0

3

2

3

1

0

10

1

0

3

2

3

1

0

10

3

0

4

2

3

2

1

10

4

1

0

3

1

3

2

0

4

Skin and Fur, staining, chin, pink

Skin and Fur, staining, eyelid left, red

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, nose, red

Skin and Fur, thinning fur, legs fore both

Number Examined:

Number Normal:

10

3

0

1

2

0

1

2

1

10

3

0

1

2

0

1

2

1

10

2

0

1

3

1

10

2

1

3

0

2

1

10

2

0

1

6

0

2

1

0

1

10

1

0

1

5

0

2

0

1

10

3

0

1

3

0

2

1

0

1

Females

Gestation

Group #

Category, Observations

Day of Phase:

14

15

16

17

18

19

20

Control

Skin and Fur, hair loss, shoulders both

Skin and Fur, hair loss, sides both

Skin and Fur, sores/lesions, minimal neck

Skin and Fur, sores/lesions, minimal, paw hind right, small

Skin and Fur, staining, back, pink

Skin and Fur, staining, ears both, red

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, orange

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, neck, red

Skin and Fur, staining, nose, brown

Skin and Fur, staining, nose, pink

Skin and Fur, staining, nose, red

Skin and Fur, thinning fur, head

Skin and Fur, thinning fur, legs hind both

Skin and Fur, thinning fur, shoulders both

Skin and Fur, thinning fur, sides both

Number Examined:

Number Normal:

9

1

0

1

2

4

1

0

1

0

1

0

9

2

0

1

0

1

2

3

1

0

1

0

2

0

1

9

2

0

1

2

3

1

0

1

0

2

0

1

9

1

0

1

2

3

1

0

1

2

1

0

1

9

1

0

1

0

1

3

1

0

1

2

1

0

1

0

1

9

1

0

1

0

1

3

2

0

1

0

1

9

1

0

1

0

1

0

1

2

3

0

1

0

1

2

Skin and Fur, sores/lesions, minimal, paws hind both, small

Skin and Fur, staining, ear left, brown

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, neck, red

Skin and Fur, thinning fur, lower part of back

Skin and Fur, thinning fur, muzzle

Number Examined:

Number Normal:

10

2

0

5

0

2

0

4

0

1

10

3

0

5

0

1

0

3

0

10

0

1

0

6

0

2

0

3

1

0

10

1

0

6

0

2

0

3

1

0

10

5

0

3

0

2

0

2

0

10

6

0

2

1

0

10

5

0

1

2

0

1

0

1

0

3

Skin and Fur, staining, ears both, brown

Skin and Fur, staining, face, orange

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, tail, brown

Number Examined:

Number Normal:

10

4

1

0

3

1

2

0

10

5

1

0

1

0

2

1

0

10

3

1

0

2

1

2

3

1

10

4

1

0

2

0

10

4

1

0

2

0

10

4

1

0

2

1

10

4

1

0

2

1

4

Eyes, tears, eye left, clear

Skin and Fur, raised hair

Skin and Fur, sores/lesions, minimal, paw hind left, small

Skin and Fur, staining, ears both, red

Skin and Fur, staining, eyelid left, pink

Skin and Fur, staining, eyelid left, red

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, tail, brown

Skin and Fur, thinning fur, legs fore both

Number Examined:

Number Normal:

10

3

1

0

1

0

1

3

2

1

0

1

10

3

1

0

1

0

1

0

1

3

2

0

1

10

4

0

1

0

1

0

1

2

0

1

10

5

0

1

0

1

3

0

1

10

5

0

1

0

1

0

1

2

0

2

1

10

5

0

1

0

1

0

1

2

0

4

1

10

5

0

1

0

1

2

0

2

0

Females

Gestation

Group #

Category, Observations

Day of Phase:

21

22

Control

Skin and Fur, sores/lesions, minimal, paw hind right, small

Skin and Fur, staining, ears both, red

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, neck, pink

Skin and Fur, staining, neck, red

Skin and Fur, staining, nose, brown

Skin and Fur, staining, nose, pink

Skin and Fur, staining, nose, red

Skin and Fur, thinning fur, legs hind both

Skin and Fur, thinning fur, shoulders both

Skin and Fur, thinning fur, sides both

Number Examined:

Number Normal:

9

3

1

2

1

0

2

Skin and Fur, staining, ear left, brown

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, pink

Number Examined:

Number Normal:

10

6

1

2

1

0

1

0

1

3

Skin and Fur, staining, ears both, brown

Skin and Fur, staining, face, orange

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, pink

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, tail, brown

Number Examined:

Number Normal:

10

4

1

2

1

2

0

1

0

4

Skin and Fur, raised hair

Skin and Fur, staining, ears both, red

Skin and Fur, staining, eyelid left, red

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, orange

Skin and Fur, staining, tail, brown

Skin and Fur, thinning fur, legs fore right

Number Examined:

Number Normal:

10

5

1

3

2

1

3

0

1

0

1

3

1

0

Females

Lactation

Group #

Category, Observations

Day of Phase:

0

1

2

3

4

5

6

Control

Skin and Fur, sores/lesions, minimal, paw fore left, small

Skin and Fur, sores/lesions, minimal, paw hind right, small

Skin and Fur, staining, ears both, brown

Skin and Fur, staining, ears both, red

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, pink

Skin and Fur, staining, neck, red

Skin and Fur, staining, nose, brown

Skin and Fur, staining, nose, pink

Skin and Fur, staining, nose, red

Skin and Fur, staining paws fore both, brown

Skin and Fur, thinning fur, legs hind both

Skin and Fur, thinning fur, paws fore both

Skin and Fur, thinning fur, shoulders both

Skin and Fur, thinning fur, sides both

Number Examined:

Number Normal:

10

2

1

0

1

2

3

0

1

2

1

0

1

0

1

10

2

1

0

1

3

0

1

2

1

0

1

10

3

1

0

1

3

0

1

2

0

1

0

1

10

2

1

0

1

0

3

0

2

1

2

0

1

10

2

1

0

1

0

3

0

2

1

2

0

1

0

2

0

1

0

3

0

2

1

2

0

1

10

1

0

1

0

3

1

2

1

2

0

1

2

Skin and Fur, staining, ear left, brown

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, pink

Skin and Fur, staining, tail, brown

Skin and Fur, thinning fur, shoulder right

Number Examined:

Number Normal:

9

6

1

2

0

1

0

1

0

10

4

1

2

1

10

5

0

2

1

0

1

0

1

10

5

0

2

1

0

1

0

1

10

5

0

2

1

0

2

1

0

1

10

6

0

2

1

0

1

0

10

6

0

2

1

0

1

0

3

Skin and Fur, staining, ears both, brown

Skin and Fur, staining, face, orange

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, orange

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, tail, brown

Number Examined:

Number Normal:

10

4

1

0

1

2

1

10

5

1

0

1

0

1

2

1

10

6

1

0

1

0

1

0

2

1

10

6

1

0

1

0

1

0

2

1

10

6

1

0

1

0

1

2

0

1

10

6

1

0

1

0

1

2

0

1

10

6

0

1

0

1

0

1

2

0

1

4

Skin and Fur, raised hair

Skin and Fur, staining, ears both, red

Skin and Fur, staining, eyelid left, red

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, orange

Skin and Fur, staining, tail, brown

Skin and Fur, thinning fur, legs all

Number Examined:

Number Normal:

10

4

1

0

1

3

1

10

3

1

3

4

1

10

3

1

0

1

3

4

1

10

3

1

0

1

0

3

4

1

10

5

0

1

0

1

0

3

2

4

1

10

5

0

1

0

1

0

1

4

1

10

5

0

1

0

1

0

1

4

1

Females

Lactation

Group #

Category, Observations

Day of Phase:

7

8

9

10

11

12

13

Control

Skin and Fur, staining, ears both, brown

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, pink

Skin and Fur, staining, neck, red

Skin and Fur, staining, nose, brown

Skin and Fur, staining, nose, pink

Skin and Fur, staining, nose, red

Skin and Fur, staining paws fore right, brown

Skin and Fur, thinning fur, paws fore both

Skin and Fur, thinning fur, shoulders both

Skin and Fur, thinning fur, sides both

Number Examined:

Number Normal:

10

1

3

1

2

1

2

0

1

10

1

3

1

2

1

2

0

1

10

0

1

3

1

2

1

2

1

10

0

1

3

1

2

1

2

1

10

0

1

3

1

2

1

2

1

10

0

1

3

2

1

2

1

10

0

1

3

1

3

1

0

1

2

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, pink

Skin and Fur, staining, nose, brown

Skin and Fur, staining, nose, pink

Skin and Fur, staining, nose, red

Skin and Fur, staining, paws fore both, brown

Teeth, broken, lower

Teeth, cut, lower

Number Examined:

Number Normal:

10

5

2

1

0

1

0

1

0

10

5

2

1

0

1

0

1

0

10

3

2

1

0

1

0

2

1

0

10

1

2

1

2

1

0

1

0

3

1

0

10

1

2

0

2

1

0

2

0

1

10

2

1

2

1

2

1

0

1

10

2

1

2

1

2

3

1

0

3

Skin and Fur, staining, face, orange

Skin and Fur, staining, head, orange

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, red

Skin and Fur, staining, nose, red

Skin and Fur, staining, tail, brown

Skin and Fur, thinning fur, paws fore both

Number Examined:

Number Normal:

10

6

1

2

0

1

0

10

6

1

2

0

1

0

10

6

1

2

0

1

0

10

6

1

2

0

1

0

10

5

1

2

0

1

10

2

1

0

6

3

1

10

0

9

3

0

1

4

Skin and Fur, hair loss, uro-genital area

Skin and Fur, staining, eyelid left, red

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, orange

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, orange

Skin and Fur, staining, nose, red

Skin and Fur, staining, tail, brown

Skin and Fur, thinning fur, leg hind right

Skin and Fur, thinning fur, legs all

Skin and Fur, thinning fur, legs fore both

Skin and Fur, thinning fur, lower part of back

Number Examined:

Number Normal:

10

5

0

1

0

1

0

1

0

4

0

1

0

10

5

0

1

0

1

0

1

0

4

0

1

0

10

5

0

1

0

2

0

1

0

4

0

1

0

10

3

0

1

0

2

0

1

4

0

1

0

1

10

3

0

1

0

1

0

1

3

0

1

0

1

10

2

0

1

0

2

1

2

1

3

0

1

0

1

10

2

1

0

1

0

5

1

0

3

1

0

1

Females

Lactation

Group #

Category, Observations

Day of Phase:

14

Control

Skin and Fur, staining, ears both, brown

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, pink

Skin and Fur, staining, neck, red

Skin and Fur, staining, nose, brown

Skin and Fur, staining, nose, pink

Skin and Fur, staining, nose, red

Skin and Fur, thinning fur, paws fore both

Skin and Fur, thinning fur, shoulders both

Skin and Fur, thinning fur, sides both

Number Examined:

Number Normal:

10

1

3

1

2

3

1

2

Skin and Fur, staining, ear left, red

Skin and Fur, staining, face, pink

Skin and Fur, staining, head, pink

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, pink

Skin and Fur, staining, nose, brown

Number Examined:

Number Normal:

10

2

1

2

3

1

2

1

3

Skin and Fur, staining, muzzle, brown

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, neck, orange

Skin and Fur, staining, neck, pink

Skin and Fur, staining, tail, brown

Skin and Fur, thinning fur, paws fore both

Number Examined:

Number Normal:

10

5

1

3

2

1

4

Skin and Fur, hair loss, uro-genital area

Skin and Fur, staining, muzzle, pink

Skin and Fur, staining, muzzle, red

Skin and Fur, staining, neck, orange

Skin and Fur, staining, tail, brown

Skin and Fur, thinning fur, leg hind right

Skin and Fur, thinning fur, legs fore both

Skin and Fur, thinning fur, lower part of back

Number Examined:

Number Normal:

10

3

1

4

1

3

1

Conclusions:

Once daily oral gavage administration of 50, 300, or 600 mg/kg/day Naugalube APAN to male rats for 42 consecutive days and to female rats for up to 57 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation) resulted in test article-related findings at all dose levels. Adverse findings were confined to 300 and 600 mg/kg/day Naugalube APAN in females.
Effects for males included lower thyroxine levels and lower prostate and seminal vesicle weights following administration of 300 or 600 mg/kg/day. Fertility was not affected in any of the dose levels used, therefore a ‘no observed adverse effect level’ (NOAEL) for male reproductive toxicity was established as 600 mg/kg/day.
For females, lower body weight gain and reduced food consumption may have affected pregnancy due to the smaller liter sizes along with pup mortality and lower pup weights following administration of 300 or 600 mg/kg/day; as such, the NOAEL for female reproductive toxicity and offspring development is considered 50 mg/kg/day.

Executive summary:

The objective of the study was to screen for effects of the test article, Naugalube APAN, on male and female reproductive performance (i.e. gonadal function, mating behaviour, conception, development of the conceptus and parturition) and offspring growth until Lactation Day (LD) 13.

Naugalube APAN was administered to rats by oral gavage for a minimum of 28 days, following the OECD 421 test guideline.

Four groups of 10 male and 10 female sexually mature Crl:CD (SD) strain rats were administered 0 (control article [vehicle]), 50, 300, or 600 mg/kg/day Naugalube APAN, once daily by oral gavage. The control article (vehicle) was Arachis oil BP, and formulations were administered at a dose volume of 2 mL/kg. Males were dosed for 42 consecutive days (2 weeks prior to pairing, during the pairing phase and approximately 2 weeks post-pairing) and sent to necropsy on Study Day 43 (Post-Pairing Day 14). Females were dosed for up to 57 days (2 weeks prior to pairing, during the pairing phase, and until LD 13) and sent to necropsy on LD 14.

Before dose initiation, all females were screened for regular estrous cycles; only females with regular estrous cycles were included in the study.

Assessment of adult toxicity was based on mortality/viability, clinical observations, body weights and food consumption, estrous cycles, mating, fertility and pregnancy indices, offspring development, and anatomic pathology. On the day of necropsy, blood samples were withdrawn for thyroid hormone assessment. Complete necropsies were performed on all animals, and any macroscopic abnormalities were noted. Organ weights were recorded, and a microscopic examination was performed on selected tissues/organs from all control and high-dose adults. In addition, seminal vesicles and prostates from low- and intermediate-dose adult males were also microscopically examined. Formulations were analysed twice during the study to assess accuracy (achieved concentration).

For offspring, clinical observations, litter size, sex, and body weight were recorded.

Ano-genital distance was recorded on Postnatal Day (PND) 4, and nipple retention was recorded for male pups on PND 13. On PND 4, litters were culled for uniformity of litters. Blood samples were taken for thyroid hormone assessment from surplus pups on PND 4. At scheduled sacrifice on PND 13, blood samples were withdrawn for thyroid hormone assessment, and the thyroid was retained from one pup/sex/litter from each dose group.

No adult mortality occurred, and no toxicologically significant post-dose observations were noted.

Administration of 600 mg/kg/day resulted in transient, occasional, instances of hunched posture and raised hair for females. Lower body weights and reduced food consumption, which continued into gestation and lactation, were also evident for females administered 600 mg/kg/day, compared with controls.

Estrous cycles, mating or fertility were unaffected by 600 mg/kg/day administration.

Gestation lengths of 24 days were noted for three females administered 600 mg/kg/day, compared with 23 days for remaining females from this dose group and all control females.

Fewer mean number of implantation sites, which resulted in smaller litter sizes, was evident following administration of 600 mg/kg/day, compared with controls. Higher incidences of stillborn pups and pup mortality were evident for pups from females administered 600 mg/kg/day, compared with controls. Body weights were lower for pups maternally administered 600 mg/kg/day, compared with controls, with body weight differences more noticeable with time. No nipples/areolae were present for male offspring, and ano-genital distance was not affected by maternal exposure of 600 mg/kg/day. Pup thyroid hormone levels were unaffected, and no test article-related macroscopic abnormalities were evident.

For males, lower total thyroxine levels were observed following administration of 600 mg/kg/day. At necropsy, lower seminal vesicle and prostate weights (unadjusted and body weight-relative) were recorded and test article-related macroscopic abnormalities were confined to a small prostate recorded for one male. No test article-related microscopic abnormalities were noted.

Lower body weight gains and reduced food consumption were evident for females following administration of 300 mg/kg/day.

Gestation lengths of 24 days were noted for three females administered 300 mg/kg/day, compared with 23 days for the remaining 300 mg/kg/day females and all control females. Fewer mean number of implantation sites were also evident, which resulted in smaller litter sizes, compared with controls; higher incidences of stillborn pups and pup mortality were also observed. From PND 4 (post-cull) onwards, body weights were marginally lower for pups maternally administered 300 mg/kg/day, compared with controls. Ano-genital distance and pup thyroid hormone levels were unaffected, and no test article-related macroscopic abnormalities were noted.

No adverse effect on body weight or food consumption was noted for males administered 300 mg/kg/day. Lower total thyroxine levels were evident, compared with controls; at necropsy, lower seminal vesicle and prostate weights (unadjusted and body weight-relative) were recorded for males administered 300 mg/kg/day, compared with controls. No macroscopic or microscopic abnormalities were detected.

Following administration of 50 mg/kg/day, test article-related effects consisted of lower overall body weight gain and food consumption for females. No adverse effect on body weight or food consumption was noted for males.

No test article-related effects were noted on mating, fertility, or gestation length; however, fewer mean number of implantation sites were evident following administration of 50 mg/kg/day, which resulted in a marginally smaller litter size, compared with controls, which was not statistically significant. No effects were noted on pup physical health or development, and pup thyroid hormone levels were unaffected by maternal exposure to 50 mg/kg/day. Furthermore, no test article-related macroscopic abnormalities were noted for pups.

No test article-related effects were noted on thyroid hormones or organ weights for adults administered 50 mg/kg/day, and no test article-related macroscopic or microscopic changes were evident at that dose.

In conclusion, once daily oral gavage administration of 50, 300, or 600 mg/kg/day Naugalube APAN to male rats for 42 consecutive days and to female rats for up to 57 days (pre-pairing, throughout gestation, and during the first 2 weeks of lactation) resulted in test article-related findings at all dose levels. Adverse findings were confined to 300 and 600 mg/kg/day Naugalube APAN in females.

Effects for males included lower thyroxine levels and lower prostate and seminal vesicle weights following administration of 300 or 600 mg/kg/day. Fertility was not affected in any of the dose levels used, therefore a ‘no observed adverse effect level’ (NOAEL) for male reproductive toxicity was established as 600 mg/kg/day.

For females, lower body weight gain and reduced food consumption may have affected pregnancy due to the smaller litter sizes along with pup mortality and lower pup weights following administration of 300 or 600 mg/kg/day; as such, the NOAEL for female reproductive toxicity and offspring development is considered 50 mg/kg/day.