Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 August 2017 to 24 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 421, Reproduction / Developmental Toxicity Screening Test, Organisation for Economic Co-Operation and Development, Paris, 29 July 2016
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
The subject was the Reproduction/Developmental Toxicity Screening Test in the Rat according to OECD guideline No. 421 and OECD guidance document No. 43. The guideline was designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.
The purpose of this study was to obtain information on the possible toxic effects of the test item NAUGARD® XL-1 when administered by oral gavage to Wistar rats at 3 dose levels.
The study was a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 13 post-partum.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Test Item Name: NAUGARD® XL-1
Laboratory Master Schedule No. at FumoPrep Ltd.: 4174
Manufacturer: Addivant USA LLC
Chemical Name: (1,2-dioxoethylene)bis(iminoethylene) bis[3-(3,5-di-tertbutyl-4-hydroxyphenyl)propionate]
Batch/Lot Number: 11608030
CAS Number: 70331-94-1
Appearance: White powder
Manufacture Date: 30 August 2016
Expiry Date: 29 August 2020
Purity: 99.4%
Storage: Condition Controlled room temperature (15-25 °C) protected from light
Safety Precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety.
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species and strain: Crl:WI (Wistar) rats.
Justification of species/strain: The rat is regarded as a suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Historical control data for the strain of rat used at the Test Facility demonstrated that sexual maturity is attained at 10 weeks of age.
Sex:
male/female
Details on test animals and environmental conditions:
Species and strain: Crl:WI (Wistar) rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Sandhofer Weg 7, D 97633, Sulzfeld, Germany) from SPF colony
Hygienic level: Standard laboratory conditions during the study
Number of animals: 48 male and 48 female rats, in 4 groups. Each group contained 12 animals/sex for treatment. Animals of the two sexes originated from different units, to avoid brother/sister mating. A sufficient number of spare animals were ordered for the study; those animals (including non-selected females) were allocated to the spare colony of the Test Facility after the final allocation to treatment had been finished.
Age of animals: Young adult rats, at least 10 weeks old at the start of the treatment and at least 12 weeks at the start of mating. The age range within the study was kept to the minimum practicable (within one week).
Body weight range: Males: 405 - 454 g, Females: 258 - 300 g at the start of the treatment; values did not exceed ± 20% of the mean weight for each sex.
Acclimatisation period: 6 days

Husbandry
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.
Cage type: Type II and/or III polycarbonate
Bedding: LIGNOCEL® ¾-S certified wooden chips (Batch number: 03018170329 / 03018170529, Expiry date: 29 March 2020 / 29 May 2020) produced by J. Rettenmaier & Söhne GmbH + Co. KG (Address: Holzmühle, D-73494 Rosenberg, Germany) were used in the study.
Nesting: ARBOCEL® crinklets natural (Batch number: 05072170228, Expiry date: 28 February 2020) produced by J. Rettenmaier & Söhne GmbH + Co. KG (Address: Holzmühle, D-73494 Rosenberg, Germany) were available to animals during the study.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.5 – 23.9°C (target range: 22±3°C)
Relative humidity: 39 – 70% (target range: 30-70%)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed (with pups), respectively. Males were caged individually after their mating had been finished (until the end of mating period). Group housing allowed social interaction, the deep wood sawdust bedding allowed digging and other normal rodent activities. Nest building material was also provided for the animals to allow normal nesting behaviour.
Environmental parameters (temperature and relative humidity) were continuously measured, minimum and maximum values were recorded twice a day during the study.

Food and water supply
Animals received ssniff® SM R/M+H “Autoclavable complete diet for rats and mice – breeding and maintenance” produced by ssniff Spezialdiäten GmbH (Address: Ferdinand-Gabriel-Weg 16, D-59494 Soest, Germany) ad libitum, and tap water from municipal supply, as for human consumption from 500 mL bottle ad libitum.
Food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Animal identification
Each parental/adult animal (P generation) was identified by a unique number within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at Citoxlab Hungary Ltd. During the pre-treatment period, animals got temporary identification number, the study animal numbers were allocated at randomisation (before the treatment started).
This number consisted of 4 digits, the first digit being the group number, the second digit was 0 for the males and 5 for the females, and the last 2 digits were the animal number within the group, as indicated in the Experimental design section.
The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery.

Randomization
All adult/parental (P) male and female animals were assigned to their respective dose groups by randomisation based on their body weights at the start of the treatment period. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight (measured immediately before the first treatment); the software PROVANTIS v.9 was used in order to verify homogeneity/variation among/within groups. Males and females were randomized separately.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
Vehicle
Propylene glycol [CAS 57-55-6] was selected for vehicle of the study by the Sponsor based on the formulation and analytical trials. Identification data of the chemical used in the study are as follows:

Name (Abbreviation): Propylene glycol (PG)
Chemical name. 1,2-Propanediol
Manufacturer: ACROS / Merck
Batch number: 1692129 / K49089078
Expiry date: 30 April 2020 / 31 May 2022
Storage conditions: Room temperature

Formulation
The test item was formulated in the vehicle, as a visibly stable homogenous formulation at the appropriate concentrations according to the dose level and volume selected in the Pharmacy of Citoxlab Hungary Ltd.
Formulations were prepared daily (fresh prior to administration to animals) according to stability assessment results of the analytical method development and method validation studies of Test Site #1.
Details on mating procedure:
Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of the treatment, with one female and one male from the same dose group
(1:1 mating) in a single cage. Females remained with the same male until copulation occurred (for up to 5 days).
A vaginal smear was prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were housed individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and/or homogeneity was performed at the Test Site #1 for analytical work using a validated LC-MS (Liquid Chromatography - Mass Spectrometry) analytical method (FPBDOK-AN-366) [4]. Duplicate samples (0.5 mL/sample) were taken from the top, middle and bottom of the test item formulations three times during the study (during the first and last weeks and approximately midway during the treatment), one set to analyse and one set as back-up, if required for any confirmatory analyses. Similarly, duplicate samples (0.5 mL/sample) were taken from the middle of the vehicle control formulation for concentration measurement.
Formulation samples were kept at room temperature until shipment. Samples (both sets) were shipped on the same day as soon as possible after collection for concentration and/or homogeneity measurement to the Principal Investigator #1 (PI #1).
The number and date of shipments were agreed with the PI #1. After the receipt at Test Site #1, the samples were frozen and kept below -15°C until analysis.
The formulation analysis was conducted within the stability period determined during the validation (started from the time of formulation) under the control of the Principal Investigator #1 in compliance with the relevant SOPs of the Test Site #1 for analytical work. The results of the formulation analysis as well as documentation regarding stability of formulations was included in the study report as an appendix (in the form of a Phase Report).
Acceptance criteria of the concentration analysis were set according to the analytical method validation, expected to be at 100 ± 10% of the nominal concentration.
Acceptance criteria of the homogeneity was that the CV of replicates (top, middle and bottom of test item formulations) must be less than 10%.
Any sample not required for analysis were discarded following acceptance of the results of the formulation analysis by the Principal Investigator #1 and Study Director.
Duration of treatment / exposure:
Males were treated for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination.
Females were treated for 14 days pre-mating, for up to 5 days of mating period, through gestation and up to the day before necropsy (a total of 13 days of post-partum dosing). The day of birth (when parturition was complete) was defined as post-partum day 0 (abbreviated as PPD 0). Non-pregnant female were sacrificed on Day 50 (34 days after the start of mating).
Frequency of treatment:
Test item treated or control animals were treated continuously on a 7 days/week basis, by oral gavage.
Details on study schedule:
Treatment of both sexes began after the acclimatisation (6 days) plus a pre-exposure period (14 days), then treatment was made for 2 weeks before mating, during the mating, and was continued up to the day of necropsy.
The first day of treatment of each animal was regarded as Day 0.
See "Any other information" for further details.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
24 animals per dose (12 males/12 females)
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were selected by the Sponsor in consultation with the Study Director, based on the available data and information from previous experimental work conducted at different Test Facility (data on file at the Sponsor). The aim of dose selection is to use a maximum of at least 1000 mg/kg bw/day or to induce toxic effects, but ideally no death or suffering at the highest dose and NOAEL (No-observed adverse-effect level) at the lowest dose.
Based on the results from the available information, doses of 100, 300 and 1000 mg/kg bw/day were selected for this study.
The oral route was selected as it is one of the possible routes of human exposure.
Positive control:
Not required.

Examinations

Parental animals: Observations and examinations:
Clinical observations
Animals were inspected for signs of morbidity and mortality at least twice daily, at the beginning and the end of the working day. The principles and criteria summarized in the Humane Endpoints Guidance Document were taken into consideration when necessary. Consultation with the staff veterinarian was made at appropriate frequency, if needed.
General clinical observations were made once a day*, during the treatment period in the afternoon (pm) at approximately the same time with minor variations as practical during the working day (no peak period of effects was noted after dosing during the first few days of treatment). When signs of toxicity are observed, animals were observed more frequently.
*Note: No general clinical observations were made on those days when the more extensive detailed clinical observations were made.

More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then weekly, in the morning (am) and on the day of necropsy. These observations were made outside the home cage in a standard arena, at similar times as practical. Signs evaluated included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded. Special attention was directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. Pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality were recorded including onset, degree and duration of signs as applicable.
On gestation day GD 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).

Body weight measurement
All adult animals were weighed with an accuracy of 1 g for selection purposes, then weekly during the pre-exposure period, on Day 0 (randomization), weekly thereafter and at termination.
Parental females were additionally weighed on gestation days (GD) 0, 3, 7, 10, 14, 17 and 20, and on post-partum days (PPD) 0 (within 24 hours after parturition), 4, 7, 10 and 14 (at termination).

Food consumption measurement
Food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g at least weekly (on body weight measurement days).

Thyroid Hormone Analysis
For thyroid hormone analysis, blood samples were taken after an overnight food deprivation at approximately the same time on each termination day; the group order for sampling was randomised.
Blood samples were taken by cardiac or venepuncture (in case of PND 4 pups blood was taken after decapitation) into tubes containing no anticoagulant as follows:
-from up to two pups per litter on PND 4*,
-from all dams on PPD14 and two pups per litter on PND 13,
-from all adult males at termination.
*Note: Based on the actual litter size, no PND 4 sample was taken for the #1505, #1508, #1510, #4501 and #4505 litters. As no pups survived until PND4 in case of the #2505 litter, no samples were generated for that litter.
To ensure sufficient blood for analysis, blood samples were pooled by litter in case of PND 4 and PND 13 samples, this fact was in harmony with the relevant OECD No. 421 guideline.
Blood samples were kept on ice from sampling until centrifugation (within 30 minutes of collection) then centrifuged rapidly (1600 g / approx. 3000 rpm, 10 minutes, 4°C). The resulting serum was divided in at least two aliquots (volume target of at least 125 µL for the first aliquot and at least 75 µL for the second aliquot) and stored in an ultrafreezer (-80±10°C) until shipping for analysis). Any leftover serum sample was kept as third aliquot for safety reason.
Samples were shipped for hormone analysis on dry ice to Test Site #2 (Citoxlab France) to the attention of the responsible Principal Investigator #2 (PI #2).
First, samples for the PND 13 pups and adult males were assessed for T4 levels using an LC-MS/MS (Liquid Chromatography-Tandem Mass Spectrometry) method. Based on the observed results, additional T4 analysis of female animals and PND 4 pups, and TSH analysis of parental males and females as well as PND 13 pups were not necessary. An audited Phase Plan (Test Site #2 code: 45503 ABR) was provided prior to any analysis.
Oestrous cyclicity (parental animals):
Oestrus cycles were monitored for each female (65 females in total) by vaginal smears daily during the pre-exposure period before the treatments started. Any females that failed to show a 4 or 5-day cycle were not included in the study, twelve females showing regular oestrus cycles were treated and used for mating in each group (the rest of the females was moved back to the spare colony after the mating process has been finished). Vaginal smears were also monitored for oestrus cyclicity daily from the beginning of the treatment period until evidence of mating.
Sperm parameters (parental animals):
Not examined
Litter observations:
Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded. Any evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy (GD 0) until post-partum day 0 (PPD 0), the day of completion of littering.
Dams were observed to record whether they formed a nest from the bedding material and covered their new-borns or not. The efficiency of suckling was verified by the presence of milk in the pups' stomach (by visual observation). All observations were recorded.
Each litter was examined as soon as possible after delivery (post-natal day 0, PND 0) to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities. Observations were reported individually for each adult animal. In addition to the observations on parent animals, any abnormal behaviour of the offspring was recorded.
Live pups were counted, sexed, weighed individually within 24 hours of parturition (PND 0) and on PND 4 and PND 13, with accuracy of 0.01 g. All the litters were checked and recorded daily for the number of viable and dead pups, any abnormal behaviour or appearance of the pups were also recorded.
The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND 0). Presence of nipples/areolae in all pups was recorded on PND 13 (individual records were maintained).
All pups were examined externally at weighing on PND 4. One male and one female pup (whenever it was possible based on the actual litter parameters) were allocated randomly for culling for blood sampling on PND 4.
All pups were terminated on PND 13.
Postmortem examinations (parental animals):
Terminal procedures and macroscopic evaluation
Gross necropsy was performed on each adult animal irrespective of the date of death. Terminally, animals were sacrificed under sodium pentobarbital anaesthesia (administered by intraperitoneal injection at 0.1 mL/100 g bw using 400 mg/mL sodium pentobarbital solution) by exsanguination; anaesthetic product was diluted by physiological saline for pups’ euthanasia as required.
After exsanguination, the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate. Special attention was paid to the organs of the reproductive system.
Vaginal smears were prepared and examined for each female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.
The number of implantation sites and corpora lutea was recorded in the females as applicable.

Organ weight measurements
At the time of termination, body weight and the weight of the following organs from all euthanized adult animals were determined as applicable:
- With a precision of at least 0.01 g: uterus (with cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, levator ani plus bulbocavernosus muscle complex, Cowper’s glands and glans penis, brain
- With a precision of at least 0.001 g: ovaries, thyroid gland (with parathyroid glands)
Individual and/or paired absolute organ weight was reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported.

Tissue preservation and microscopic evaluation
The ovaries, testes, accessory sex organs (oviduct, uterus, cervix (with body and horn) and vagina for females, epididymides, prostate, seminal vesicles plus coagulating glands for males), thyroid and all organs showing macroscopic lesions of all adult animals were preserved. Testes with epididymides were retained in modified Davidson’s fixative*, and all other organs in 10% buffered formalin solution.
*Note: The eyes with the optic nerves would have been preserved also in modified Davidson’s fixative, but no such a case was required.
Additionally, thyroid glands from at least one male and one female PND 13 pup from each litter (wherever it was possible based on the actual litter parameters)* were preserved in 10% buffered formalin solution wherever possible. In this case, the thyroid weight was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage.
*Note: In case of #1505 litter with prolonged parturition, the thyroids of the only living male pups of the second delivery day was preserved additionally (thus thyroids of a total of three pups, two males and one female, were preserved). In case of #4505 litter, there was no living male pups at the time of necropsy, thus only a sample of a female pup was preserved.
Detailed histological examination was performed on ovaries, testes and epididymides in the Control and High dose groups (parental generation), and all macroscopic findings (abnormalities) from all animals, and additional reproductive organs (prostate / seminal vesicle with coagulating gland or oviduct / uterus / cervix+body+horn / vagina) for High dose mating pair (#4507 / #4007) where no pregnancy was achieved.
Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.
In case of histopathology, tissues or organs were embedded in paraffin wax; sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.
Postmortem examinations (offspring):
The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. Furthermore, pups terminated on PND 13 were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals (no signs of altered development was recorded). All observed abnormalities were recorded.
Additionally, the sex of each pup was confirmed by an internal examination performed at the time of removal (after death, if the body remained intact, or at scheduled termination on PND 13).
Statistics:
For pathology data (macroscopic and microscopic data) first a Chi-squared test was used to check for overall similarity of the relative frequencies, the system then checked the significance of the result against a user-defined value (0.05) and where suitable, also performed pairwise tests of the treatment groups versus the control group. The Fisher’s Exact Test was performed replacing the Chi-squared test as the final evaluation method if the group size was <5.
Reproductive indices:
Parental Males
Number of pairings; Number of fertile pairings; Number of infertile males; Male mating index; Male fertility index.

Parental Females
Number of pairings; Number of pregnant females; Number of sperm positive, but non-pregnant females; Number of non-mated females; Female mating index; Female fertility index; Gestation index; Duration of pregnancy (days); Number of Corpora lutea / dams; Number of implantations / dams; Number of dams with live pups Day 0 and 4 and 13; Pre-implantation mortality; Intrauterine mortality; Total mortality (intra and extra uterine mortality).
Offspring viability indices:
Mean pup body weight (per pup within the group and per litter) on PND 0, 4 and 13; Mean pup body weight gain (per litter) between post-natal days 0-4, 4-13 and 0-13; Number of live births per litter, and number of viable pups per litter on post-natal days 0, 4 and 13; Survival Index of pups on post-natal days 0, 4 and 13; Sex ratio % (on post-natal days 0, 4 and 13).

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related adverse effect was observed during clinical observation in any dose groups.

Male animals
No clinical signs were recorded for male animals except of thin fur which was recorded for one Low dose male (#2001) and two Mid dose males (#3008 and #3009) from Day 8 or Day 19 until the necropsy; but this finding was not related to the test item treatment.

Female animals
No clinical signs were recorded for any Control or High dose females.
Crust / nodule in the ventral area of thorax was recorded for a Low dose female (#2505) from Day 22 (the animal was under close veterinary control after that point), but these findings were considered as not related to the test item. Piloerection was also recorded for this animal during Days 38-40 (around the end of gestation period). However, this finding was most probably related to a difficult delivery process.
Thin fur was recorded for one Mid dose female (#3510) from Day 29 until termination, but this finding was not related to the test item treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There was no mortality in the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item effect was seen in the body weight and body weight gain values of the Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) and High (1000 mg/kg bw/day) dose animals (males and females).
No statistically significant differences were observed in the body weight or body weight gain values of the Low, Mid and High dose animals (males and females) during the treatment period when compared to the relevant control values.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no test item related effects in the mean daily food consumption of the Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) and High (1000 mg/kg bw/day) dose animals (males and females).
The mean daily food consumption in the pre-treatment and treatment period was similar in the Low, Mid and High dose males and females when compared to the Control group. Occasional statistically significant difference was regarded as incidental, without dose response and of no toxicological significance.
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
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item related effect was observed on the organ weight of any dose groups in the study.
Terminal body weights of animals (both sexes) were not statistically significantly different between the groups.
There were no treatment-related statistically significant differences among groups in the weights of organs measured (including thyroid glands with parathyroid) when compared to controls (absolute and relative to body). Occasional statistically significant difference without dose response (Mid dose male brain weight) was considered as incidental, not related to the treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test item-related macroscopic findings were observed at any dose level.
A firm, subcutaneous nodule (approximate size of 1 cm x 1 cm) found on the chest of a Low dose female (#2505) was regarded as an incidental finding.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test item-related microscopic findings were seen by light microscope at the High dose level (1000 mg/kg bw/day) or in any organs showing macroscopic lesions.
Minimal focal tubular degeneration/atrophy of the right testis in 1/12 High dose male (#4010), and spontaneous adenocarcinoma of the mammary gland in one Low dose female (#2505, examined due to firm nodule) were considered to be incidental.
Histopathological evaluation of the male gonads as well as testicular interstitial cell structure; the spermatogenic cells representing different phases of the development and differentiation of the spermatozoons were similar in Control and High dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma had a similar histological structure in both Control and High dose females.
Furthermore, reproductive organs of the only mating pair of the High dose groups where no pregnancy was achieved (#4007 / #4507) were also examined (testes, epididymides, prostate, seminal vesicles with coagulation gland for male; uterus, cervix, ovary, oviduct and vagina for female). No test item-related microscopic changes or any specific abnormalities was detected that could be causal for the lack of litter in that case.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
TYHROID HORMONE ANALYSIS
No test item related effect was observed in the thyroid hormone analysis in the Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) and High (1000 mg/kg bw/day) dose groups.

Evaluation of the gestation, parturition and post-partum period
There was no effect of treatment noted during gestation, parturition or the post-partum period.
The mean duration of pregnancy was 22.7, 22.5, 22.4 and 22.4 days in Control, Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) and High (1000 mg/kg bw/day) dose groups, respectively, indicating that the process was comparable between control and test item treated groups. The individual data (in the range of 22-24 days) were also in line with the normal physiological range.
As far it could be observed in the study, parturition was normal in all but two females. In case of a control animal (#1505), the delivery process lasted for three days* in total. However, this fact was not related to the test item administration. Furthermore, in case of a Low dose female (#2505) the parturition was noted as normal, but five dead pups were recorded during the delivery process. However, this fact was considered to be incidental, and not related to the treatment.
*Note: After delivering 8 pups (7 living and 1 dead pups) on 07 October 2017, this female (#1505) delivered additional 4 pups (1 living and 3 dead pups) on 09 October 2017.

The number of implantation sites was comparable to the control mean in all dose groups, no statistically significant differences were noted.
There were no statistically significant differences or effects that could be ascribed to treatment on pre-natal, post-natal or total mortality values (litter mean and %) in any dose group

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No effect of the test item was detected on the oestrus cycle of the female animals.
Each female selected for the treatment showed acceptable cycles (mean cycle length was 4.00 days for each female) before starting the treatment period. There was no effect on test item on the oestrus cycle of females (mean cycle length was in the range of 4.00-4.06 days for each group after the treatment) as no statistically significant or biologically relevant changes were observed.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no biologically relevant differences between the Control and test item treated groups with regard to reproductive ability, mating fertility or gestation. Data from all groups were in the normal expected range.
The mating index was 100% in all test item treated groups for both males and females. The fertility indices were also in the normal range (92-100%) for all test item treated males and females. The gestation index was 100% in all test item treated groups.
Successful coitus (sperm positive vaginal smear and/or vaginal plug) occurred within up to 5 days of pairing (cohabitation) in all test item treated groups. The mean duration of mating was 2.50, 3.00, 2.50 and 2.08 days in Control, Low (100 mg/kg bw/day), Mid (300 mg/kg bw/day) and High (1000 mg/kg bw/day) dose groups, respectively. The length of mating did not differ significantly in the test item treated groups when compared to control.

Details on results (P0)

No mortality or test item related clinical signs were observed in the study.
No test item effect was seen in the body weight, body weight gain and food consumption values in any dose groups.
There were no biologically relevant differences between the Control and test item treated groups with regard to reproductive ability, mating fertility or gestation.
No effect of the test item was detected on the oestrus cycle of the parental female animals.
No test item related effect was observed on the organ weights of any test item treated groups in the study.
Test item administration was not associated with gross test item-related changes or microscopic findings in any animals.
There were no test item-related microscopic findings in the reproductive organs examined in the High dose parental animals.
No test item related effect was observed in the thyroid hormone analysis or thyroid weights in any test item treated parental male or female animals.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
There was no test item effect on the survival of the pups.
There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %).
The number of viable pups on PND0, 4 and 13 as well as the survival index of the pups at given time points were comparable to control value in each dose group, thus there was no treatment-related effects on the viability of pups at those time points.
The sex ratio of pups in the test item treated groups was similar to the control, no test item effect was noted.

Increased number of autolyzed pups was seen during the lactation period in the Low dose group when compared to control, but this fact was mostly caused by one litter (all the 12 live born pups of litter #2505 were found dead (autolyzed) by PND 2, while additional 5 pups died during the delivery). Due to the lack of dose response as well as the dam’s condition, this fact was considered as incidental finding, not being a treatment related effect.
Based on the external evaluation, almost all the pups were normal, the few clinical signs observed are detailed below.
Some pups (1510/7,8,9,10,11, 3509/16, 3511/1 and 4505/1) were cyanotic on PND0, they were cannibalized or found dead / autolyzed on PND1
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test item effect on mortality.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was no effect of treatment on the offspring body weight or body weight gain on PND0, PND4 or PND13 following test item administration at 100 mg/kg bw/day (Low dose), 300 mg/kg bw/day (Mid dose) or 1000 mg/kg bw/day (High dose).
When evaluated per litter basis, no relevant, statistically significant changes were seen in the litter mean body weight values on PND 0, PND 4 and PND 13 in any test item treated groups. There was no effect of the test item on the body weight gain values in the periods of PND 0-4, PND 4-13 or PND 0-13. Occasionally, slightly higher or lower values were observed in the Low, Mid and/or High dose groups, but without statistical significance and without dose response, thus those values were considered as not being test item related effect. In summary, there were no effects of treatment on pup weights or weight gains.
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:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
No test item effect was observed on anogenital distance or nipple retention during the study.
No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when compared to control, thus no effect of test item was concluded.
There were no effects on the nipple retention: no nipples/areolae were counted for any male pups on PND 13.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test item effect was seen on the thyroid weight of the pups.
No statistically significant differences were observed on the thyroid weight (absolute and relative to body) of pups of Low, Mid and High dose groups (100, 300 and 1000 mg/kg bw/day, respectively) when compared to control data (combined for male and female pups).
Gross pathological findings:
no effects observed
Description (incidence and severity):
FOUND DEAD / F1 Generation
There were unscheduled deaths in control and treated groups including:
-9, 6, 5 and 2 found dead / still born / cannibalized or autolyzed animals on PND 0 from the Control, Low, Mid and High dose groups, respectively;
-13, 18, 6 and 16 cannibalized or autolyzed pups during the lactation period from the Control, Low, Mid and High dose groups, respectively.
No test item-related macroscopic findings were noted in any of those cases.

TERMINAL / F1 Generation (PND 13)
No test item-related macroscopic changes were recorded in the F1 generation pups of any dose groups at termination.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
TYHROID HORMONE ANALYSIS
No statistically significant differences were detected in the thyroxine (T4) hormone levels of parental males and PND13 pup samples of any dose groups when compared to control data.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No test item related changes were noted in the F1 offspring viability, clinical signs, body weight or sexual development in any dose groups.
No test item related effect was observed on the organ weights of any test item treated groups in the study.
Test item administration was not associated with gross test item-related changes or microscopic findings in any animals.
No test item related effect was observed in the thyroid hormone analysis or thyroid weights in any F1 generation pups.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
developmental neurotoxicity

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
In summary, daily administration of NAUGARD® XL-1 by oral gavage to Wistar rats at 100, 300 and 1000 mg/kg bw/day dose (Low, Mid and High dose groups, respectively) resulted the following findings:

No mortality or test item related clinical signs were observed in the study.
No test item effect was seen in the body weight, body weight gain and food consumption values in any dose groups.
There were no biologically relevant differences between the Control and test item treated groups with regard to reproductive ability, mating fertility or gestation.
No effect of the test item was detected on the oestrus cycle of the parental female animals.
No test item related changes were noted in the F1 offspring viability, clinical signs, body weight or sexual development in any dose groups.
No test item related effect was observed on the organ weights of any test item treated groups in the study (parental and F1 generation).
Test item administration was not associated with gross test item-related changes or microscopic findings in any animals (parental or F1 offspring).
There were no test item-related microscopic findings in the reproductive organs examined in the High dose parental animals.
No test item related effect was observed in the thyroid hormone analysis or thyroid weights in any test item treated parental male or female animals or F1 generation pups.

In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for NAUGARD® XL-1 was considered to be:
NOAEL for general systemic toxicity: 1000 mg/kg bw/day
NOAEL for reproductive toxicity: 1000 mg/kg bw/day
NOAEL for developmental toxicity: 1000 mg/kg bw/day
Executive summary:

The purpose of the Reproduction/Developmental Toxicity Screening Test in the Rats was to obtain initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to Day 13 post-partum.

 

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating/post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including post-partum/lactation Day PPD13. The Experimental Design was as follows:

 

Group No.

Group Designation

Dose Level
(mg/kg bw/day)

Concentration

(mg/mL)

Dose volume

(mL/kg bw)

Animal Numbers
(proposed)

Male

Female

1

Control

0

0

5

1001-1012

1501-1512

2

Low dose

100

20

2001-2012

2501-2512

3

Mid dose

300

60

3001-3012

3501-3512

4

High dose

1000

200

4001-4012

4501-4512

Note: Only females with proper oestrus cycles were selected for treatment and used for mating.

 

Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, at least weekly body weight and food consumption. In addition, the reproductive performance, pregnancy, parturition and post-partum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND13. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. Blood samples for thyroid hormone measurement were also collected for parental males and females at termination (Day 28 and PPD14), and for PND4 and PND13 pups.

 

For the adult animals, a detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups as well as on the reproductive organs of High dose animals with possible impaired reproductive capacity.

 

In summary, daily administration of NAUGARD® XL-1 by oral gavage to Wistar rats at 100, 300 and 1000 mg/kg bw/day did not result in mortality or adverse changes in clinical signs.

 

No test item effect was seen in the body weight, body weight gain and food consumption values in any dose groups.

 

There were no biologically relevant differences between the Control and test item treated groups with regard to reproductive ability, mating fertility or gestation.

 

No effect of the test item was detected on the oestrus cycle of the parental female animals.

 

No test item related changes were noted in the F1 offspring viability, clinical signs, body weight or sexual development in any dose groups.

 

No test item related effect was observed on the organ weights of any test item treated groups in the study (parental and F1 generation).

 

Test item administration was not associated with gross test item-related changes or microscopic findings in any animals (parental or F1 offspring).

 

There were no test item-related microscopic findings in the reproductive organs examined in the High dose parental animals.

 

No test item related effect was observed in the thyroid hormone analysis or thyroid weights in any test item treated parental male or female animals or F1 generation pups.

 

In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for NAUGARD® XL-1 was considered to be:

 

NOAEL for general systemic toxicity:           1000 mg/kg bw/day

NOAEL for reproductive toxicity:                  1000 mg/kg bw/day

NOAEL for developmental toxicity:               1000 mg/kg bw/day