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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 September 2017 - 16 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 September 2017 - 16 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See principles of method below.
Principles of method if other than guideline:
Due to technical reasons, temperature (maximum of 25.7 °C) and relative humidity (minimum of 27%) values outside the expected ranges of 22 ± 3 °C and 30-70 % were recorded during the study. However, these minor differences of the environmental parameters were considered not to adversely
affect the results of or integrity of the study as confirmed by the staff Veterinarian.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
N/A
Specific details on test material used for the study:
None other than above
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI Wistar rats
Details on species / strain selection:
The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for Dose Range Finding study (study code: 17/040-220PE
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.
Housing conditions: Standard laboratory conditions
Justification of species/strain: The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for Dose Range Finding study (study code: 17/040-220PE [3]).
Number of animals: 70 male, 70 female rats, 12 animals/sex/group, 5 groups, plus 5/sex/group non-mated recovery animals in the Control and High dose groups.
Age of animals: Young adult rats, approximately 12-13 weeks old at start of treatment and 14-15 weeks old at mating.
Body weight range: Males: 398 – 476 g, females: 235 – 309 g; did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
Acclimation period: 11 days
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.
Room number: 507
Cage type: Type II and III polycarbonate
Bedding & nesting: LIGNOCEL® ¾ S certified wooden chips (batch number: 03018170329, expiry date: 29 March 2020 and batch number: 03018170529, expiry date: 29 May 2020) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study. ARBOCEL® nest building material (batch number: 05072170228, expiry date: 28 February 2020) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Details of bedding and nest building material were archived with the raw data.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.2 – 25.7 °C (target range 22 ± 3 °C)
Relative humidity: 27 – 67 % (target range 30-70 %)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 3 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, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
Food and water supply: Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice –breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (batch number: 262 21592, expiry date: 31 January 2018), ad libitum, and tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided analytical certificate for the batch used, which is archived with the raw data. Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A. u. 36., Hungary). The quality control results are archived with the raw data at Citoxlab Hungary Ltd.
Animal identification: Each adult/parental 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-exposure period, animals will be identified with temporary numbers only. After this 2-week period, a randomisation will be performed based on the body weights and the selected animals will receive their final animal numbers, as follows. This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, 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. Identification of the new-borns (Offspring, F1 Generation) was performed by ink marking of the digit-tips up to one day after birth.
Randomisation: All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the first exposure (Day -1). There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The dose levels were selected by the Sponsor in consultation with the Study Director based on available acute oral toxicity data (LD50 > 5000 mg/kg bw in rats) and information from a Dose Range Finding study in the rat (Citoxlab study code 17/040-220PE [3]). In the DRF study hepatic effects were seen at 100, 300 and 1000 mg/kg bw but no severe toxicity was observed.

The aim of this study was to use a maximum of 1000 mg/kg bw/day or to induce toxic effects, but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Based on the results from the preliminary study, doses of 15, 60, 250 and 1000 mg/kg bw/day were selected for the main study.

The oral route was selected as it is one of the possible routes of human exposure.

The dosing solutions were administered to the test item or vehicle-treated (control) animals daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw were administered to all animals. The actual volume to be administered were calculated and adjusted based on each animal’s most recent body weight.

Dosing of both sexes began after 11 days of acclimatisation and pre-exposure period (14 days), and it was performed 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.

The first day of dosing of each animal was regarded as Day 0.

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating) and then euthanized and subjected to necropsy examination. The recovery male animals had a 14-day treatment free period after the 28-day dosing period, then subjected to necropsy similarly to the main animals. The recovery males were not included in the mating procedure.

Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13-day post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed as practical, on 24 days after the day of presumed mating. The recovery female animals were not mated. These animals were dosed daily for 59 days. After this, a 14-day treatment free period followed, then they were subjected to necropsy similarly to the main animals.

All F1 offspring were terminated on Day 13 post-partum (F1 offspring selected for blood sampling on PND4 were terminated on that day). In order to allow for overnight fasting of dams with urine collection on PPD14, the offspring were euthanized on PPD/PND13 and the dams on PPD/PND14.
Details on mating procedure:
The recovery animals were not mated. The following procedure only applies to the main animals.

Mating began after the animals had attained full sexual maturity, 2 weeks after the initiation of 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, except two animals (#1506, 3506) that were pseudopregnant during the mating period and therefore only produced positive mating sign on the twelfth/thirteenth day. 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:
Formulations were prepared on the day of treatment or on the day before (and stored refrigerated until use) Stability of the test item in the vehicle was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use) according to Test Site #1 method validation study.

Analysis of test item formulations for concentration and homogeneity was performed at Test Site #1 using a validated GC-FID method. Duplicate samples 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 a backup, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Formulation samples (both sets) were kept refrigerated (2-8 °C) until shipment. Samples were shipped on the day of collection for concentration and homogeneity measurement to the Principal Investigator (PI) #1:

János Török, Ph.D.
FumoPrep Ltd.
H-1044 Budapest, Ipari Park u. 10.
Hungary

The formulation analysis was conducted under the control of the Principal Investigator #1 in compliance with the Test Site #1 relevant SOPs. The results of the analysis in the form of a Phase Report is included in the study report as an appendix.

Any remaining samples (back-up set) were discarded following acceptance of the results of the formulation analysis by the Principal Investigator #1 and Study Director.

Description of the analytical method and the results of the formulation analysis are included in the Analytical phase report,
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating).
Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13-day post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.
Frequency of treatment:
Daily
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
70 male, 70 female rats, 12 animals/sex/group, 5 groups, plus 5/sex/group non-mated recovery animals in the Control and High dose groups.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available acute oral toxicity data (LD50 > 5000 mg/kg bw in rats) and information from a Dose Range Finding study in the rat (Citoxlab study code 17/040-220PE [3]). In the DRF study hepatic effects were seen at 100, 300 and 1000 mg/kg bw but no severe toxicity was observed.
The aim of this study was to use a maximum of 1000 mg/kg bw/day or to induce toxic effects, but id eally no death or suffering at the highest dose and a NOAEL at the lowest dose. Based on the results from the preliminary study, doses of 15, 60, 250 and 1000 mg/kg bw/day were selected for the main
study. The oral route was selected as it is one of the possible routes of human exposure.
- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the first exposure (Day -1). There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.
Positive control:
None
Parental animals: Observations and examinations:
All animals:
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment. More detailed examinations were performed once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were performed outside the home cage in a standard arena, at similar day times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and 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. During the recovery period, the detailed clinical observations of the recovery animals were performed weekly in the morning (am) similarly to the treatment period. All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable. On gestation day GD13 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).

Five males and five females/group will be selected from the main animals:

Assessment of potential test item related neurotoxicity was performed during the last exposure week (males on Day 24; females on PPD8-12). Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength. To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots for the hind limbs was measured. Fore/hind limb grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of the test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs and the appropriate grip support. The results were tabulated with individual and mean data. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex and vocalisation were evaluated using a scoring system, where score “0” was given when the behaviour or reaction of the animal was considered normal, and -1 or -2, or +1 and +2 was given if the response was less than or more than expected in an untreated animal.
Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for a 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. The data from all groups was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.

Body weight measurement:

All adult animals were weighed with an accuracy of 1 g for randomisation purposes, then at least weekly during the pre-exposure period, on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 3, 7, 10, 14, 17 and 20 and on post-partal Days PPD0 (within 24 hours after parturition), PPD4, 7, 10, 13 and 14 (before termination). The body weight of the female animals measured on gestational Days GD3, 10 and 17 as well as PPD7 and PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, thus these data were not evaluated statistically.

The recovery animals were weighed weekly during the study (pre-exposure period, treatment period and recovery period).

Food consumption measurement:

Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g weekly (on the days of body weight measurements).

Oestrus cycle monitoring:

Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments starts (for details see 4.4.5.). Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.

The oestrus cycles of the recovery animals were monitored by vaginal smears daily only during the pre-exposure period and in the first two weeks of the treatment period.

Additionally, vaginal smears were prepared and examined for each main and recovery female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.

Observation of the delivery process, offspring and nursing instinct

Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. All observations were recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition.

Dams were observed for signs of nest building with the bedding material and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect 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 (PND0) and on PND4 and PND13, with accuracy of 0.01g. All litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.

The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND0). Presence of nipples/areolae in male pups were recorded on PND13 (individual records were maintained).

All pups were examined externally at weighing on PND4. One male and one female pup (where possible) was allocated randomly for culling for blood sampling on
PND4.

All pups were culled on PND13. Dams were sacrificed on PND14 after fasting overnight.

Thyroid Hormone Analysis:

For thyroid hormone analysis, blood samples were taken by cardiac puncture or venepuncture (or decapitation in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
-from up to two pups per litter on PND4,
-from all dams and at least two pups per litter on PND14 (females) / PND13 (pups),
-from all adult males at termination.

Pup blood was pooled by litter.

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, 4ºC). The resulting plasma 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, if possible; any leftover material was also retained for safety reason) and stored in an ultrafreezer (-80 ± 10 °C) until shipping for analysis.

Samples for the Day 13 pups and adult males were assessed for T4 levels. An audited study phase plan (44305 ABR) was provided prior to any analysis.

Samples were shipped for hormone analysis on dry ice to the Principal Investigator (PI) #1:

Joachim Decorde
Citoxlab France,
RN13 Route de Pacy
27930 Miserey, France

The thyroid hormone analysis was conducted using the validated method [8] under the control of the Principal Investigator #1 in compliance with the Test Site #1’s relevant SOPs. The results of the evaluation in the form of a Phase Report is presented in Appendix 12.

Any sample not required for analysis will be discarded following acceptance of the results of the hormone analysis by the Principal Investigator and Study Director after the finalization of the study report.

There was no blood sampling for thyroid hormone analysis from the recovery animals.
Oestrous cyclicity (parental animals):
Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments starts (for details see 4.4.5.). Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.

The oestrus cycles of the recovery animals were monitored by vaginal smears daily only during the pre-exposure period and in the first two weeks of the treatment period.

Additionally, vaginal smears were prepared and examined for each main and recovery female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.
Sperm parameters (parental animals):
Not examined.
Litter observations:
Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. All observations were recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition.

Dams were observed for signs of nest building with the bedding material and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect 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 (PND0) and on PND4 and PND13, with accuracy of 0.01g. All litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.

The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND0). Presence of nipples/areolae in male pups were recorded on PND13 (individual records were maintained).

All pups were examined externally at weighing on PND4. One male and one female pup (where possible) was allocated randomly for culling for blood sampling on
PND4.

All pups were culled on PND13. Dams were sacrificed on PND14 after fasting overnight.

Postmortem examinations (parental animals):
At termination, the adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination.

Gross necropsy was performed on all animals, irrespective of the date of death. 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 of corpora lutea were recorded in the females as applicable.

At the time of termination, body weight and the weight of the following organs from all adult animals were determined:

-With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus

-With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids

Testes and epididymides were weighed individually. 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.
Postmortem examinations (offspring):
Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities. The anaesthetic product was diluted for pups’ euthanasia as required.
Statistics:
Data was recorded to provide information on parameters to include:
Parental Males
- Clinical observations and functional observation battery (FOB).
- Body weight and body weight gain.
- Food consumption.
- Number of pairings
- Number of fertile pairings.
- Number of infertile males.
- * Male mating index.
- * Male fertility index.
- Clinical pathology.
- Thyroid hormone analysis.
- Necropsy findings
- Organ weights (absolute and relative to the body and brain weights).
- Histopathology findings

Parental Females
- Clinical observations and functional observation battery (FOB).
- Body weight and body weight gain.
- Food consumption.
- Oestrus cycle data
- 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, 4 and 13.
- * Pre-implantation mortality.
- * Intrauterine mortality.
- * Total mortality (intra and extra uterine mortality).
- Clinical pathology.
- Necropsy findings
- Organ weights (absolute and relative to the body and brain weights).
- Histopathology findings

Offspring
- Mean pup body weight (per pup within the group and per litter) on PND0, 4 and 13.
- Mean pup body weight gain (per litter) between PND0-4, PND4-13 and PND0-13.
- Number of live births per litter, and number of viable pups per litter on postnatal Days 0, 4 and 13.
- * Survival Index of pups on postnatal Days 0, 4 and 13.
- F*Sex ratio % (on postnatal Days 0, 4 and 13).
- Thyroid hormone analysis.
- Anogenital distance, nipple retention.
- Necropsy findings (macroscopic)
- Organ weights (thyroid glands, absolute and relative to the body weight).
Reproductive indices:
Please see "Any Other Information on Materials and Methods incl Tables" below.
Offspring viability indices:
Please see "Any Other Information on Materials and Methods incl Tables" below.
Clinical signs:
no effects observed
Description (incidence and severity):
No test item related clinical signs were observed during the study. The following individual observations were not attributed to systemic effects of the test item.

Slightly decreased activity, piloerection and wasted condition was observed in one Mid dose 2 male (#4010) on Day 27 (the last day of treatment).

A 1-2 cm width nodule was seen in one Control female (#1502) at the right axillary site from Day 29 until the end of the observation period.

Piloerection and wheal on both hindpaws was seen in one Control female (#1511) from Day 20, and then also hunched back from Day 30 until the end of the observation period.

Hunched back, piloerection and increased respiratory rate were seen in one Mid dose 1 female (#3509) from Day 8 until Day 20.

Alopecia of both fore paws was seen in one High dose female (#5504) from Day 30 until the end of the observation period.

Hunched back, piloerection and increased respiratory rate were seen in one High dose female (#5505) from Day 48 until the end of the observation period.

In the preterminally euthanized animal (#5508), ataxia, hunched back, piloerection, slightly increased respiratory rate and wheal on both hindpaws and fore paws were seen before the euthanasia.

In the found dead animal (#5511), slightly increased respiratory rate and hunched back were recorded on Day 30, the day before death.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No test item-related mortality was seen during the study.

One female (#5511) in the High dose group was found dead on Day 31. The cause of death could not be determined. No clear sign of treatment-relation was seen at necropsy.

One female (#5508) in the High dose group was preterminally euthanized on Day 38, because of animal welfare reasons. Based on the clinical signs and necropsy findings, it is considered that the symptoms were caused by a misgavage.
There was no other mortality during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the male main animals significantly (p<0.05) lower body weight gain was seen in the High dose during the first week of treatment. From the second week all body weight and body weight gain values were normal in all dose groups.

In females, the body weight and body weight gain values were normal during the pre-mating, mating and gestation periods, however during lactation period PPD4-13 the High dose body weight gain was lower than the Control.

Recovery male and female animals had normal values during the observation period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In males, there were no test item related differences in the mean daily food consumption in any test item treated group when compared to the controls. The measured values were within the normal range for this strain and age.

The food consumption of the females showed similar pattern as the body weight changes; there were consistently significantly lower values in the High dose during the lactation period.

Recovery male and female animals had normal values during the observation period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the haematology parameters. Statistically significant differences were considered to be incidental, there was no relationship with dose and/or all recorded values were near or within the historical control ranges. These differences were considered to not reflect an effect of the test item. These incidental statistical differences can be seen in Table 2 and 3.

It should be noted that the prothrombin time (PPT) and activated partial thromboplastin time (APTT) could not be measured in four Mid 2 dose females and in one High dose female, due to technical reasons (lipemic or clotted samples). No statistical evaluation could be performed on these parameters in the Mid 2 dose female group because of the low sample number.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically high urea levels were seen at 60, 250 and 1000 mg/kg bw/day in male dose groups, with no significant reversal in the recovery males. This change was considered to be test item-related. The difference at 60 mg/kg bw/day was generally within the historical control range. At 250 and 1000 mg/kg bw/day, there were no other indications of potentially adverse liver or kidney effects to support that the high urea was adverse, however the results were well outside the historical range, and there was no indication of recovery at 14 days post-exposure.

No other test-item related clinical chemistry findings were seen. The rest of the significant differences were considered to be incidental, there was no relationship with dose and/or all recorded values were near or within the historical control ranges. These differences were considered to not reflect an effect of the test item. These incidental statistical differences can be seen in Table 4 and 5.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the urinalysis parameters. Significant differences were considered to be incidental, there was no relationship with dose and all recorded values were within the historical control ranges. These differences were considered to not reflect an effect of the test item. These incidental statistical differences can be seen in Table 6 and 7.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.

There was no effect of treatment noted during the assessment of grip strength, foot splay or motor activity. Significantly (p<0.05) higher grip strength values were recorded in the Low and Mid dose males, these were considered as incidental and of no toxicological significance.

All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals and the Control when evaluating the overall distance travelled (0-60 min, cm). The test item did not increase or decrease the normal locomotor activity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
FOUND DEAD / Parental Generation

Microscopic Findings

Moderate microvesicular hepatocellular vacuolation of the periportal zone of the liver, slight multifocal pulmonary congestion of the lungs and slight multifocal haemorrhage of the thymus were recorded in the found dead animal.

PRETERMINAL EUTHANASIA / Parental Generation

Microscopic Findings

Slight bilateral extramedullary haematopoiesis of the adrenal glands, moderate increased cellularity of the marrow in the femur and sternum, moderate increased cellularity in the medulla and paracortex of the lung-associated lymph nodes with macrophages, slight focal or multifocal hepatocellular necrosis with haemorrhage in the liver, severe chronic bronchioloalveolar inflammation in the lungs, multiple severe abscesses in the lungs and moderate extramedullary haematopoiesis in the spleen were recorded in the preterminally euthanized animal. The macroscopic and microscopic changes in this animal were not considered to be related to systemic effects of the test item, but probably a consequence of local damage in the pulmonary system.

TERMINAL / Parental Generation (Males, Day 28, Females, PND14)

MAIN ANIMALS

Microscopic Findings

Test item-related findings were seen in the liver and kidneys of the following animals:

High dose group (1000 mg/kg bw/day): minimal to slight diffuse vacuolation in the cortex, medulla and tubule of the of the kidneys in 12/12 males and 6/10 females, minimal to moderate diffuse/multifocal/centrilobular/periportal hepatocellular vacuolation of the liver in 12/12 males and 9/10 females.

Mid 2 dose group (250 mg/kg bw/day): minimal to moderate diffuse/multifocal/ periportal hepatocellular vacuolation of the liver in 9/12 males and 3/12 females. No test-item related microscopic changes were seen in the kidney.

The vacuolation in the liver and kidney was considered to be a non-adverse finding.

Besides these, based on the low incidence and/or severity and/or distribution cross control and dosed animals the following observations were considered incidental or a common background: minimal focal acinal atrophy of the pancreas (#1001), minimal focal inflammatory cell infiltrate in the prostate (#1001, 5005), minimal extramedullary haematopoiesis in the spleen (evenly present in all groups), minimal multiple cysts at the pars distalis section of the pituary (#1002), minimal focal basophilia in the cortex of the left kidney (#1004), minimal casts in the kidneys (#1004, 1007, 2003, 4007, 5003, 5506, 5510), slight unilateral inflammatory cell infiltrate in the kidney (#1506), slight cysts in the kidneys (#3507, 3512, 4503), minimal to slight unilateral pyelonephritis in the kidney (#3507, 4506), minimal focal interstitial nephritis in the cortex of the kidneys (#3512).

In addition, the organs with a macroscopic lesion were also examined and the following findings were recorded: slight to moderate diffuse dilatation of the tubular section of the testes (#1011), sign of proestus in the uterine body and horns (#1505, 3505, 3506), slight hydrocephalus in the brain (#2506), slight inflammation in the pleura and granulomatous sections of the lungs with presence of foreign material (#3509), moderate abscess in the lungs (#3509, 5505), moderate chronic bronchioloalveolar inflammation in the lungs (#5505), slight unilateral atrophy in the seminal vesicles (#4010), slight multifocal erosion/ulcer in the forestomach (#4010). These were considered to be incidental changes.

RECOVERY ANIMALS

Microscopic Findings

Test item-related findings were seen in the liver and kidneys of the animals:

Minimal to slight diffuse vacuolation in the cortex, medulla and tubule of the of the kidneys in 3/5 males and 1/5 females, minimal to slight diffuse/ /centrilobular/periportal hepatocellular vacuolation of the liver in 5/5 males and 5/5 females. The severity and frequency of the kidney vacuolation decreased compared the main animals, indicating an ongoing resolution. The severity of the liver vacuolation also decreased, while the incidence remained the same.

Besides these, based on the low incidence and/or severity and/or distribution across control and dosed animals, the following observations were considered incidental or a common background: minimal extramedullary haematopoiesis in the spleen of 2/5 Control males, 2/5 Control females, 4/5 High dose recovery males and 3/5 High dose recovery females, minimal to slight multifocal eosinophilic droplets in the cortex and tubule of the kidneys in 2/5 High dose recovery males.

In addition, as macroscopic changes were seen in the uterus of 2 Control and 2 High dose recovery females (#1514, 1516, 5514, 5517) and in the lungs and lung-associated lymph nodes of one High dose recovery female (#5516), these organs were macroscopically examined and the following findings were recorded: sign of proestrus in the uterus of all these animals, moderate multifocal granulomatous inflammation of the pleura of the lungs with haemorrhage and moderate granulomatous inflammation with foreign material presence in the lung-associated lymph nodes. These changes were not considered to be related to systemic effects of the test item.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone levels (adult males)

Significantly lower values were recorded in the Mid 2 and High dose groups, possibly related to liver enzyme induction (as indicated by the liver weight increases).

However, all dose groups were within the historical control range, therefore these changes are not considered adverse.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Each female selected for the study showed acceptable cycles before starting the treatment period.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no differences between the control and test item treated groups with regard to reproductive ability, mating or gestation indices, and no effects considered adverse or toxicologically significant in correlation with the administration of NAUGARD® 412S. The mating indices were 100% in all groups. The male and female fertility indices were 67% in the Control, 92% in the Low dose group, 83% in the Mid 1 dose group, 100% in the Mid 2 dose group and 75% in the High dose group. The number of non-pregnant females were lower in all dose groups than the Control. The gestation index was 100% in all groups.

Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) occurred within 6 days of pairing (cohabitation) for all females.

The mean duration of mating was 2.3, 2.2, 2.0, 2.8 and 2.8 days in Control, Low, Mid 1, Mid 2 and High dose groups, respectively.

There was no effect of treatment noted during the pre-implantation and gestation periods.

The mean duration of pregnancy was comparable in the control and test item treated groups.

The number of corpora lutea and number of implantation sites was comparable to the control mean at all dose groups.

No test item-related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14 in the Low (15 mg/kg bw/day), Mid 1 (60 mg/kg bw/day) and Mid 2 (250 mg/kg bw/day) dose groups.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive performance
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
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
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
The number of implantations in pregnant females was similar in all groups, but the number of live born was slightly lower (p<0.05) in the High dose, indicating a slightly increased prenatal mortality. In the High dose group there were significantly lower pups born alive and the mortality of these pups were significantly higher until PND13. Most of the dead pups were autolysed or cannibalised therefore could not be examined at necropsy.

In the Mid 2 dose group a slight but similar trend was observed as in the High dose group. The mortality values in the Mid 2 dose group were within the historical control range and were not considered as an adverse effect.

In the Low and Mid 1 dose groups the number of viable pups on PND0, 4 and 13 as well as pup survival indices on PND0, 4 and 13 were comparable to control values.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight gain of pups was significantly lower in the High dose group during the lactation period by about 35%, with a similar trend in the Mid 2 group. The body weight and body weight gain values in the Mid 2 dose group were within the historical control range and were not considered as an adverse effect. The lower pup weight gain corresponded with the pup mortality data, and both correspond with the significantly lower food intake and weight gain of the High dose adults during lactation (where low food intake is likely to cause lower milk production, which would be linked to lower pup growth and increased mortality).

Low dose and Mid 1 had similar values to the control.
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:
no effects observed
Description (incidence and severity):
The few macroscopically examined pups did not show any test-item related findings at necropsy. These differences in the High dose group are considered to be test item-related and correlated with body weight loss of the dams during these the lactation period (it is noted that significant body weight loss in dams is likely to be associated with reduced milk production). The data appears to confirm that the principal effect was on the dam, including low body weight gain and low food consumption during the lactation period, with mortality of pups as a consequence, rather than a direct toxic effect on the pups.
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in the anogenital distance between the test item treated groups (males/females) and the control on PND0. There was no nipples/areolae presence seen in any of the male pups on PND13.

In summary, there were no effects on the anogenital distance on PND0 or on the presence of nipples/areolae on PND13 that were ascribed to the test item.
Gross pathological findings:
no effects observed
Description (incidence and severity):
During the lactation period 1 Control, 6 Low dose, 5 Mid 1 dose, 3 Mid 2 dose and 5 High dose pups were found dead that remained intact (not cannibalized or autolysed). Those were subjected to necropsy with macroscopic examination. No macroscopic findings were seen. No other pups that died during the study could be examined for test item-related macroscopic findings. The number of found dead but macroscopically not examined animals is presented in Table 13.
Histopathological findings:
no effects observed
Description (incidence and severity):
No macroscopic changes were seen in F1 offspring generation euthanized and examined externally at scheduled termination on PND13.
Other effects:
no effects observed
Description (incidence and severity):
The thyroid gland weights of the PND13 pups were statistically not different from the Control group. The slightly lower mean of the High dose group was calculated from only 3 litters, where 2 samples were in the normal range (49.5 ng/mL and 42.4 ng/mL) and one sample had very low T4 concentration (18.7 ng/mL). The low sample number in this group was not enough to adequately evaluate the T4 level. The measurement of the thyroid hormone levels in the PND4 pups and adult females was not performed as it was not deemed necessary by the Study Director.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Pup Development and Survival
Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
Treatment related:
no

Table 2:Significant differences in haematology parameters in males

Haematology (males)

Groups/Concentration (mg/kg bw/day)

Main animals

Recovery animals

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Control (0)

High (1000)

Haematocrit (%)

48.60

48.78

51.00 *

48.20

48.00

49.42

49.70

Differences from control

0%

5%

-1%

-1%

Differences from control

1%

Historical control data

40.90 – 50.20

Red cell distribution width (%)

12.50

12.64

12.58

13.40

12.88

12.44

13.08 *

Differences from control

1%

1%

7%

3%

Differences from control

5%

Historical control data

10.50 – 16.80

Reticulocytes (%)

2.22

2.42

2.66

3.06 *

3.10 *

1.84

2.16

Differences from control

9%

20%

38%

40%

Differences from control

17%

Historical control data

0.70 – 5.80

Neutrophils (%)

34.18

45.64

41.70

29.50

23.72 *

33.50

33.64

Differences from control

4%

22%

-14%

-31%

Differences from control

0%

Historical control data

11.80 – 46.50

Lymphocytes (%)

60.60

58.32

52.64

64.16

71.46 *

59.32

58.50

Differences from control

-4%

-13%

6%

18%

Differences from control

-1%

Historical control data

13.50 – 85.70

Basophils (%)

0.24

0.16

0.20

0.14

0.12

0.08

0.20 *

Differences from control

-33%

-17%

-42%

-50%

Differences from control

150%

Historical control data

 

Eosinophils (%)

1.10

1.80

1.26

2.20 *

0.80

1.74

2.22 *

Differences from control

64%

15%

100%

-27%

Differences from control

28%

Historical control data

0.00 – 1.30

APTT (sec)

12.84

13.04

14.38 **

13.92 *

12.82

12.30

12.32

Differences from control

2%

12%

8%

0%

Differences from control

0%

Historical control data

10.00 – 13.40

PTT (sec)

9.58

9.44

10.10

10.08

10.36 +

9.84

10.88 +

Differences from control

-2%

5%

5%

8%

Differences from control

11%

Historical control data

9.30 – 11.00

Notes: *= p<0.05; Dunnett two sided test.

                       += p<0.05,++= p<0.01; Dunn two sided test

 

Table 3:Significant differences in haematology parameters in females

Haematology (females)

Groups/Concentration (mg/kg bw/day)

Main animals

Recovery animals

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Control (0)

High (1000)

Haemoglobin concentration (g/dL)

14.74

14.90

14.98

14.72

15.18

15.66

14.74 *

Differences from control

1%

2%

0%

3%

Differences from control

-6%

Historical control data

11.50 – 16.40

Haematocrit (%)

46.46

46.94

47.10

44.20

47.38

47.06

42.88 *

Differences from control

1%

1%

-5%

2%

Differences from control

-9%

Historical control data

35.20 – 49.10

Mean Cell Volume

59.12

61.26

60.36

58.66

58.08

54.86

52.42 **

Differences from control

4%

2%

-1%

-2%

Differences from control

-4%

Historical control data

51.70 – 72.40

Mean Platelet Volume

8.12

8.72

8.42

8.98

8.98

7.28

8.28 **

Differences from control

7%

4%

11%

11%

Differences from control

14%

Historical control data

6.40 – 14.80

MCH concentration (g/dL)

31.74

31.74

31.78

33.30 *

32.08

33.26

34.40

Differences from control

0%

0%

5%

1%

Differences from control

3%

Historical control data

30.30 – 36.40

Neutrophils (%)

62.92

65.80

57.74

50.38

34.64++

33.88

41.58

Differences from control

5%

-8%

-20%

-45%

Differences from control

23%

Historical control data

14.60 – 76.80

Lymphocytes (%)

32.48

30.02

37.22

43.44

58.34+

60.14

51.22

Differences from control

-8%

15%

34%

80%

Differences from control

-15%

Historical control data

16.70 – 80.40

Basophils (%)

0.08

0.18

0.14

0.36

0.56++

0.26

0.86

Differences from control

125%

75%

350%

600%

Differences from control

231%

Historical control data

0.00 – 0.70

Eosinophils (%)

1.16

1.14

1.20

1.60

1.62

1.80

0.56 *

Differences from control

-2%

3%

38%

40%

Differences from control

-69%

Historical control data

0.30 – 9.30

Notes: *= p<0.05; Dunnett two sided test.

                       += p<0.05,++= p<0.01; Dunn two sided test

 

Table 4:Significant differences in clinical chemistry parameters in males

Clinical chemistry (males)

Groups/Concentration (mg/kg bw/day)

Main animals

Recovery animals

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Control (0)

High (1000)

Urea (mmol/L)

5.80

7.17

8.90 **

10.42 **

11.47 **

6.57

14.61 **

Differences from control

24%

53%

80%

98%

Differences from control

123%

Historical control data

4.01 – 9.89

Glucose (mmol/L)

8.56

10.03

8.97

8.84

8.76

8.04

9.71 *

Differences from control

17%

5%

3%

2%

Differences from control

21%

Historical control data

4.49 – 12.11

Cholesterol (mmol/L)

1.256

1.454

1.160

1.160

1.160

1.916

1.330 *

Differences from control

16%

-8%

-8%

-8%

Differences from control

-31%

Historical control data

1.16 – 2.17

Creatinine (µmol/L)

42.44

46.62

49.18

45.85

47.36

42.88

52.64+

Differences from control

10%

16%

8%

12%

Differences from control

23%

Historical control data

40.00 – 86.20

Phosphorus (mmol/L)

2.310

2.444

2.386

2.760 **

2.912 **

2.532

2.686

Differences from control

6%

3%

20%

26%

Differences from control

6%

Historical control data

1.96 – 3.16

Aspartate Aminotransferase (U/L)

159.8

221.0

605.6++

164.2

195.4

154.4

373.0 **

Differences from control

38%

279%

3%

22%

Differences from control

142%

Historical control data

93.00 – 628.00

Alanine Aminotranferase (U/L)

54.8

55.6

106.0++

58.2

73.2

41.0

84.8+

Differences from control

2%

93%

6%

34%

Differences from control

107%

Historical control data

28.00 – 150.00

Alkaline Phosphatase (U/L)

88.4

90.6

87.2

105.4

150.2 **

91.4

98.2

Differences from control

3%

-1%

19%

70%

Differences from control

7%

Historical control data

60.00 – 171.00

Notes: *= p<0.05; Dunnett two sided test.

                       += p<0.05,++= p<0.01; Dunn two sided test

 

Table 5:Significant differences in clinical chemistry parameters in females

Clinical chemistry (males)

Groups/Concentration (mg/kg bw/day)

Main animals

Recovery animals

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Control (0)

High (1000)

Urea (mmol/L)

12.83

13.93

13.49

13.87

13.59

7.47

10.86 **

Differences from control

9%

5%

8%

6%

Differences from control

45%

Historical control data

4.44 – 11.18

Glucose (mmol/L)

8.24

8.80

7.25

7.56

7.64

9.46

4.65 **

Differences from control

7%

-12%

-8%

-7%

Differences from control

-51%

Historical control data

5.17 – 11.84

Total bilirubin (µmol/L)

4.08

5.68

4.74

6.40 *

4.14

5.78

6.12

Differences from control

39%

16%

57%

2%

Differences from control

6%

Historical control data

1.70 – 13.70

Total protein (g/L)

54.12

67.56

54.36

58.44

54.62 *

59.00

60.94

Differences from control

6%

0%

8%

1%

Differences from control

3%

Historical control data

49.50 – 68.70

Albumin (g/L)

30.44

33.10

30.84

33.50

32.04

34.98

34.08

Differences from control

9%

1%

10%

5%

Differences from control

-3%

Historical control data

 

Cholesterol (mmol/L)

2.260

2.260

2.488

2.550

4.018

2.632

1.610 **

Differences from control

10%

13%

78%

-21%

Differences from control

-39%

Historical control data

25.70 – 42.70

Phosphorus (mmol/L)

3.040

3.046

2.822

3.050

2.780

1.742

2.358 **

Differences from control

0%

-7%

0%

-9%

Differences from control

35%

Historical control data

1.60 – 3.78

Chloride (mmol/L)

97.40

96.76

97.16

94.74

99.64

101.26

96.10+

Differences from control

-1%

0%

-3%

2%

Differences from control

-5%

Historical control data

90.70 – 106.60

Alkaline Phosphatase (U/L)

94.0

83.4

92.8

85.0

89.2

42.4

94.6 *

Differences from control

-11%

-1%

-10%

-5%

Differences from control

123%

Historical control data

37.00 – 137.00

Notes: *= p<0.05; Dunnett two sided test.

                       += p<0.05,++= p<0.01; Dunn two sided test

 

 

Table 6:Significant differences in urinalysis parameters in males

Urinalysis (males)

Groups/Concentration (mg/kg bw/day)

Main animals

Recovery animals

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Control (0)

High (1000)

pH

7.20

7.20

7.00

7.00

6.80

7.80

7.00+

Differences from control

0%

-3%

-3%

-6%

Differences from control

-10%

Historical control data

6.00 – 8.00

Specific gravity

1.0060

1.0070

1.0100

1.0100

1.0120

1.0070

1.0130+

Differences from control

0%

0%

0%

1%

Differences from control

1%

Historical control data

1.01 – 1.04

 

 

Notes: *= p<0.05; Dunnett two sided test.

                       += p<0.05,++= p<0.01; Dunn two sided test

 

Table 7:Significant differences in urinalysis parameters in females

Urinalysis (females)

Groups/Concentration (mg/kg bw/day)

Main animals

Recovery animals

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Control (0)

High (1000)

pH

6.00

7.00

6.40

6.40

6.20

8.00

6.80++

Differences from control

17%

7%

7%

3%

Differences from control

-15%

Historical control data

6.00 – 8.00

Notes: *= p<0.05; Dunnett two sided test.

                       += p<0.05,++= p<0.01; Dunn two sided test

 

Table 8:Parental male thyroid hormone concentration levels

(adult males)

Group /Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (100)

T4 concentration

(ng/mL)

43.83

41.59

38.13

36.78 *

35.81 **

Differences from control

-5%

-13%

-16%

-18%

Historical control data

34.3 – 60.7

Note: *= p<0.05; Dunnett two sided test, **= p<0.01; Dunnett two sided test.

 

 

Table 9:Summary of reproductive parameters (males)

Parameters

Group /Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Number of treated animals

12

12

12

12

12

Number of pre-terminal death

0

0

0

0

0

Number of males used for mating

12

12

12

12

12

Number of successful mating

12

12

12

10

10

Number of infertile animals

4

1

2

0

3

Male mating index (%)

100

100

100

100

100

Male fertility index (%)

67

92

83

100

75

Table 10:Summary of reproductive parameters (females)

Parameters

 

Group /Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Number of treated animals

12

12

12

12

12

Number of death during mating or gestation

0

0

0

0

0

Number of females used for mating

12

12

12

12

12

Number of sperm positive females

12

12

12

12

12

Number of females with no implantation sites

4

1

2

0

3

Number of pregnant females

8

11

10

12

9

Number of pregnant females with live born(s)

8

11

10

12

9

Female mating index (%)

100

100

100

100

100

Female fertility index (%)

67

92

83

100

75

Gestation index (%)

100

100

100

100

100

 

Table 11:Summary of the pre-implantation and gestation periods

Parameters

Group /Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Number of females examined

12 (8)¥

12 (11)¥

12 (10)¥

12

12 (9)¥

Mean number ofcorpora lutea

11.17
(16.75)¥

14.92
(16.27)¥

14.17
(17.00)¥

16.58

10.83
(14.44)¥

Mean number of implantations

10.25
(15.38)¥

13.75
(15.00)¥

12.83
(15.40)¥

15.00

9.75
(13.00)¥

Gestation length, mean (days)

22.3

22.6

22.5

22.5

22.4

¥ = Excluding the non-pregnant females

 

Table 12:Summary of the pre-natal and post-natal periods with survival indices

Parameters

Group /Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Mean number of implantations

10.25
(15.38)

13.75
(15.00)

12.83
(15.40)

15.00

9.75
(13.00)

Number of pups born, mean

14.63

14.82

15.30

13.83

11.78

Number of live born pups, mean

14.38

14.36

14.90

13.50

10.44 *

Pre-natal mortality¥, mean

1.00

0.64

0.50

1.50

2.56

Pre-natal mortality¥(%), mean

6.61

3.63

3.16

9.78

18.11

Post-natal mortality on PND0-4, mean

0.13

1.45

0.30

2.08

4.11

Post-natal mortality on PND0-4 (%), mean

0.83

10.39

2.11

15.74+

47.47+

Total mortality on PND4, mean

1.13

2.09

0.80

3.58

6.67

Total mortality on PND4 (%), mean

7.40

12.47

5.20

22.94+

49.63

Post-natal mortality on PND0-13, mean

0.38

1.45

0.40

2.58

6.11++

Post-natal mortality on PND0-13 (%), mean

2.40

10.39

2.73

19.21

70.10++

Total mortality on PND13, mean

1.38

2.09

0.90

4.08+

8.67++

Total mortality on PND13 (%), mean

8.96

12.47

5.75

26.04

70.63++

Survival index on PND0 (pups born)

97.83

96.06

97.51

96.31

85.20

Sex ratio (%) on PND0

53.45

48.85

52.68

55.76

45.47

Survival index on PND4

99.17

90.37

97.89

85.32

52.53

Sex ratio (%) on PND4

53.69

47.82

52.21

55.48

46.93

Survival index on PND13 (from PND4 after culling)

98.21

100.00

99.23

94.34

48.59++

Sex ratio (% of females) (PND13)

52.46

47.39

51.99

54.43

59.92

Notes:*= p<0.05; **= p<0.01; Duncan's multiple range test
                += p<0.05;++= p<0.01;Mann-Whitney U-test

                      ¥=The pre-natal mortality parameter is also called “intrauterine mortality” in the study plan and report.

            Numbers in brackets = Excluding the non-pregnant females

 

Table 13: Details of the offspring mortality and viability

Parameters

Groups/Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Number of evaluated litters

8

11

10

12

9

Number of pups born

117

163

153

166

106

Number of liveborns

115

158

149

162

94

Evaluation of PND0

Number of found dead / stillborn pups on PND0

2

7

4

6

12 **

Number of cannibalised pups (PND0)

1

0

0

1

0

Number of autolysed pups (PND0)

0

1

0

2

7 **

Number of found dead, intact pups, FT - (PND0)

1

4

4

1

5

Number of found dead, intact pups, FT + (PND0)

0

2

0

2

0

Number of viable pups (PND0)

115

156

149

160

94

Evaluation of PND1-4+

Number of dead pups on PND1-4

1

14 **

3

23 **

37 **

Number of cannibalized pups (PND1-4)

1

10 *

3

22 **

23 **

Number of autolysed pups (PND1-4)

0

4

0

1

14 **

Number of found dead, intact pups
(PND1-4)

0

0

0

0

0

Number of viable pups (PND4)

114

142 **

146

137 **

57 **

Number of viable pups after culling (PND4)

98

120

126

115

49

Evaluation of PND5-13++

Number of dead pups on PND5-13

2

0

1

6

18 **

Number of cannibalized pups (PND5-13)

2

0

1

6

10 **

Number of autolysed pups (PND5-13)

0

0

0

0

8 **

Number of found dead, intact pups
(PND5-13)

0

0

0

0

0

Number of viable pups (PND13)

96

120

125

109

31 **

Notes:*= p<0.05; **= p<0.01; Chi-squared test.

            +Counted from end of working day PND0.

            ++Counted from end of working day PND4.

Abbreviations: FT - : Floating test negative, FT + : Floating test positive

 

Table 14:Body weight data (offspring)

Parameters

 

Groups/Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (300)

Number of evaluated litters

8

11

10

12

9

Number of viable pups (PND0)

115

156

149

160

94

Mean pup bodyweight per litter (PND0), g

6.28

6.39

6.32

5.98

5.64

Historical data (mean ± SD):

6.418 ± 0.66

Number of viable pups (PND4)

114

142

146

137

57

Mean pup bodyweight per litter (PND4), g

10.18

10.50

10.48

9.04

8.40 *

Historical data (mean ± SD):

10.236 ± 1.46

Mean pup bodyweight gain per litter (PND0-4), g

3.94

4.16

4.26

2.78 *

2.13 **

Historical data (mean ± SD):

3.793 ± 1.126

Number of viable pups (PND13)

96

120

125

109

31

Mean pup bodyweight per litter (PND13), g

27.97

30.37

29.45

25.66

20.55 *

Historical data (mean ± SD):

27.802 ± 3.77

Mean pup bodyweight gain per litter (PND4-13), g

17.77

19.83

18.93

16.58

11.77 *

Historical data (mean ± SD):

17.564 ±2.814

Mean pup bodyweight gain per litter (PND0-13), g

21.24

23.71

22.96

19.60

14.48 *

Historical data (mean ± SD):

21.356 ± 3.491

Notes:*= p<0.05; **= p<0.01; Duncan's multiple range test.

 

Table 15:PND13 pup thyroid hormone concentration levels

(pups PND13)

Group /Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (100)

T4 concentration

(ng/mL)

44.61

49.66

45.17

39.24

36.87

Differences from control

11%

1%

-12%

-17%

Historical control data

26.3 – 61.6

Note: *= p<0.05; Dunnett two sided test, **= p<0.01; Dunnett two sided test.

 

Table 16:Kidney and liver organ weights of the male main animals

Organ weights (males)

Groups/Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Terminal bodyweight (g)

494

501

487

462

470

Differences from control

1%

-2%

-6%

-5%

Historical control data

368 – 668

Kidneys (g)

2.93

3.10

3.05

3.06

3.23

Differences from control

6%

4%

5%

10%

Historical control data

2.31 – 3.98

Kidneys / Bodyweight (%)

0.59

0.62

0.63

0.66 **

0.69 **

Differences from control

5%

6%

12%

16%

Historical control data

0.51 – 0.80

Kidneys / Brain weight (%)

136.91

144.26

139.16

141.42

152.68 *

Differences from control

5%

2%

3%

12%

Historical control data

104.52 – 177.68

Liver (g)

13.90

14.69

15.38

17.40 **

18.99 **

Differences from control

6%

11%

25%

37%

Historical control data

9.66 – 17.44

Liver / Bodyweight (%)

2.81

2.93

3.16

3.77 **

4.04 **

Differences from control

4%

13%

34%

44%

Historical control data

2.12 – 3.48

Liver / Brain weight (%)

651.0

683.6

701.5

803.4 **

898.9 **

Differences from control

5%

8%

23%

38%

Historical control data

439.1 – 766.0

Note: *= p<0.05; **= p<0.01; Dunnett two sided test.

 

Table 17:Kidney and liver organ weights of the male recovery animals

Organ weights (males)

Groups/Concentration (mg/kg bw/day)

Control (0)

High (1000)

Terminal bodyweight (g)

515

516

Differences from control

0%

Historical control data

368 – 668

Kidneys (g)

3.18

3.15

Differences from control

-1%

Historical control data

2.31 – 3.98

Kidneys / Bodyweight (%)

0.62

0.61

Differences from control

-1%

Historical control data

0.51 – 0.80

Kidneys / Brain weight (%)

139.52

146.41

Differences from control

5%

Historical control data

104.52 – 177.68

Liver (g)

14.00

16.91 **

Differences from control

21%

Historical control data

9.66 – 17.44

Liver / Bodyweight (%)

2.72

3.27 **

Differences from control

21%

Historical control data

2.12 – 3.48

Liver / Brain weight (%)

613.4

784.4 **

Differences from control

28%

Historical control data

439.1 – 766.0

Note: *= p<0.05; **= p<0.01; Dunnett two sided test.

 

Table 18:Kidney and liver organ weights of the female main animals

Organ weights (females)

Groups/Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Terminal bodyweight (g)

335

347

347

355

316

Differences from control

4%

3%

6%

-6%

Historical control data

236 – 409

Kidneys (g)

2.08

2.07

2.04

2.11

2.10

Differences from control

0%

-2%

2%

1%

Historical control data

1.57 – 2.64

Kidneys / Bodyweight (%)

0.62

0.60

0.59

0.60

0.67

Differences from control

-4%

-5%

-4%

7%

Historical control data

0.50 – 0.80

Kidneys / Brain weight (%)

102.22

103.21

100.88

104.46

103.76

Differences from control

1%

-1%

2%

2%

Historical control data

72.52 – 125.12

Liver (g)

12.71

13.73

14.06

14.93 *

14.58

Differences from control

8%

11%

17%

15%

Historical control data

6.39 – 19.52

Liver / Bodyweight (%)

3.80

3.97

4.06

4.21 *

4.60 **

Differences from control

4%

7%

11%

21%

Historical control data

2.46 – 6.06

Liver / Brain weight (%)

625.0

685.5

695.5

740.3

720.8

Differences from control

10%

11%

19%

15%

Historical control data

304.3 – 985.9

Note: *= p<0.05; **= p<0.01; Dunnett two sided test.

 

Table 19:Kidney and liver organ weights of the female recovery animals

Organ weights (females)

Groups/Concentration (mg/kg bw/day)

Control (0)

High (1000)

Terminal bodyweight (g)

299

312

Differences from control

5%

Historical control data

236 – 409

Kidneys (g)

1.71

1.94

Differences from control

14%

Historical control data

1.57 – 2.64

Kidneys / Bodyweight (%)

0.57

0.63

Differences from control

9%

Historical control data

0.50 – 0.80

Kidneys / Brain weight (%)

82.94

94.31 *

Differences from control

14%

Historical control data

72.52 – 125.12

Liver (g)

8.51

10.80 **

Differences from control

27%

Historical control data

6.39 – 19.52

Liver / Bodyweight (%)

2.85

3.49 **

Differences from control

27%

Historical control data

2.46 – 6.06

Liver / Brain weight (%)

413.3

525.1

Differences from control

27%

Historical control data

304.3 – 985.9

Note: *= p<0.05; **= p<0.01; Dunnett two sided test.

 

 

Conclusions:
No test item-related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14 in the Low (15 mg/kg bw/day), Mid 1 (60 mg/kg bw/day) and Mid 2 (250 mg/kg bw/day) dose groups. However, the pup mortality was increased in the High dose (1000 mg/kg bw/day) and lower growth of developing pups was seen. These data correlated with dam low body weight gain and low food consumption during the lactation period; the effect was considered as probably secondary to maternal toxicity. No developmental or endocrine changes were seen in the pups at any of the dose levels (anogenital distance, thyroid gland weights, thyroid hormone level, etc.).

The NOAEL for Reproductive effects was considered to be 1000 mg/kg bw/day.

The NOAEL for Pup development and survival was considered to be 250 mg/kg bw/day.
Executive summary:

No test item-related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14 in the Low (15 mg/kg bw/day), Mid 1 (60 mg/kg bw/day) and Mid 2 (250 mg/kg bw/day) dose groups. However, the pup mortality was increased in the High dose (1000 mg/kg bw/day) and lower growth of developing pups was seen. These data correlated with dam low body weight gain and low food consumption during the lactation period; the effect was considered as probably secondary to maternal toxicity. No developmental or endocrine changes were seen in the pups at any of the dose levels (anogenital distance, thyroid gland weights, thyroid hormone level, etc.).

 

The NOAEL for Reproductive effects was considered to be 1000 mg/kg bw/day.

 

The NOAEL for Pup development and survival was considered to be 250 mg/kg bw/day.

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 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
See principles of method below.
Principles of method if other than guideline:
Due to technical reasons, temperature (maximum of 25.7 °C) and relative humidity (minimum of 27%) values outside the expected ranges of 22 ± 3 °C and 30-70 % were recorded during the study. However, these minor differences of the environmental parameters were considered not to adversely affect the results of or integrity of the study as confirmed by the staff Veterinarian.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: pellets
Details on test material:
Name: NAUGARD® 412S
CAS Number: 29598-76-3
Batch/Lot number: 1005151109
Appearance: White to off-white pellet
Purity: Considered as 100%
Manufacture date: 13 November 2015
Expiry date: 12 November 2019
Storage conditions: Controlled room temperature (15-25 °C, below 70 RH%)
Safety precautions: Routine safety precautions for unknown materials (lab coat, gloves, safety glasses and face mask) were applied to assure personnel health and safety.
Specific details on test material used for the study:
None other than above.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI Wistar rats
Details on species / strain selection:
The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for Dose Range Finding study (study code: 17/040-220PE [3]).
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.
Housing conditions: Standard laboratory conditions
Justification of species/strain: The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. Crl:WI rats were used for Dose Range Finding study (study code: 17/040-220PE [3]).
Number of animals: 70 male, 70 female rats, 12 animals/sex/group, 5 groups, plus 5/sex/group non-mated recovery animals in the Control and High dose groups. Additional 10 spare animals/sex were ordered and excluded during the randomisation.
Age of animals: Young adult rats, approximately 11-12 weeks old at start of treatment and 13-14 weeks old at mating.
Body weight range: Males: 398 – 476 g, females: 235 – 309 g; did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
Acclimation period: 11 days
Animal health: Only healthy animals were used for the test, as certified by the staff Veterinarian. Females were nulliparous and non-pregnant.
Room number: 507
Cage type: Type II and III polycarbonate
Bedding & nesting: LIGNOCEL® ¾ S certified wooden chips (batch number: 03018170329, expiry date: 29 March 2020 and batch number: 03018170529, expiry date: 29 May 2020) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany) were used in the study. ARBOCEL® nest building material (batch number: 05072170228, expiry date: 28 February 2020) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Details of bedding and nest building material were archived with the raw data.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.2 – 25.7 °C (target range 22 ± 3 °C)
Relative humidity: 27 – 67 % (target range 30-70 %)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 3 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, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
Food and water supply: Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice –breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (batch number: 262 21592, expiry date: 31 January 2018), ad libitum, and tap water from the municipal supply, as for human consumption from a 500 mL bottle, ad libitum. The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided analytical certificate for the batch used, which is archived with the raw data. Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A. u. 36., Hungary). The quality control results are archived with the raw data at Citoxlab Hungary Ltd.
Animal identification: Each adult/parental 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-exposure period, animals will be identified with temporary numbers only. After this 2-week period, a randomisation will be performed based on the body weights and the selected animals will receive their final animal numbers, as follows. This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, 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. Identification of the new-borns (Offspring, F1 Generation) was performed by ink marking of the digit-tips up to one day after birth.
Randomisation: All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the first exposure (Day -1). There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The dosing solutions were administered to the test item or vehicle-treated (control) animals daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw were administered to all animals. The actual volume to be administered were calculated and adjusted based on each animal’s most recent body weight.
Vehicle:
corn oil
Details on oral exposure:
The dose levels were selected by the Sponsor in consultation with the Study Director based on available acute oral toxicity data (LD50 > 5000 mg/kg bw in rats) and information from a Dose Range Finding study in the rat (Citoxlab study code 17/040-220PE [3]). In the DRF study hepatic effects were seen at 100, 300 and 1000 mg/kg bw but no severe toxicity was observed.

The aim of this study was to use a maximum of 1000 mg/kg bw/day or to induce toxic effects, but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Based on the results from the preliminary study, doses of 15, 60, 250 and 1000 mg/kg bw/day were selected for the main study.

The oral route was selected as it is one of the possible routes of human exposure.

The dosing solutions were administered to the test item or vehicle-treated (control) animals daily on a 7 days/week basis, by oral gavage using a tipped gavage needle attached to a syringe. A constant volume of 5 mL/kg bw were administered to all animals. The actual volume to be administered were calculated and adjusted based on each animal’s most recent body weight.

Dosing of both sexes began after 11 days of acclimatisation and pre-exposure period (14 days), and it was performed 2 weeks before mating, during the mating, and was continued up to and including the day before the necropsy.

The first day of dosing of each animal was regarded as Day 0.

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating) and then euthanized and subjected to necropsy examination. The recovery male animals had a 14-day treatment free period after the 28-day dosing period, then subjected to necropsy similarly to the main animals. The recovery males were not included in the mating procedure.

Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13-day post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed as practical, on 24 days after the day of presumed mating. The recovery female animals were not mated. These animals were dosed daily for 59 days. After this, a 14-day treatment free period followed, then they were subjected to necropsy similarly to the main animals.

All F1 offspring were terminated on Day 13 post-partum (F1 offspring selected for blood sampling on PND4 were terminated on that day). In order to allow for overnight fasting of dams with urine collection on PPD14, the offspring were euthanized on PPD/PND13 and the dams on PPD/PND14.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Formulations were prepared on the day of treatment or on the day before (and stored refrigerated until use) Stability of the test item in the vehicle was assessed in the conditions employed on the study (concentration range and storage conditions of the dose formulations pending use) according to Test Site #1 method validation study.

Analysis of test item formulations for concentration and homogeneity was performed at Test Site #1 using a validated GC-FID method. Duplicate samples 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 a backup, if required for any confirmatory analyses. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.

Formulation samples (both sets) were kept refrigerated (2-8 °C) until shipment. Samples were shipped on the day of collection for concentration and homogeneity measurement to the Principal Investigator (PI) #1:

János Török, Ph.D.
FumoPrep Ltd.
H-1044 Budapest, Ipari Park u. 10.
Hungary

The formulation analysis was conducted under the control of the Principal Investigator #1 in compliance with the Test Site #1 relevant SOPs. The results of the analysis in the form of a Phase Report is included in the study report as an appendix.

Any remaining samples (back-up set) were discarded following acceptance of the results of the formulation analysis by the Principal Investigator #1 and Study Director.

Description of the analytical method and the results of the formulation analysis are included in the Analytical phase report,
Duration of treatment / exposure:
Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating).
Females were dosed for 14 days pre-mating, during the mating period, through gestation and until the day before the necropsy (13-day post-partum dosing). The day of birth (when parturition was complete) was defined as Day 0 post-partum.
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
70 male, 70 female rats, 12 animals/sex/group, 5 groups, plus 5/sex/group non-mated recovery animals in the Control and High dose groups.
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor in consultation with the Study Director based on available acute oral toxicity data (LD50 > 5000 mg/kg bw in rats) and information from a Dose Range Finding study in the rat (Citoxlab study code 17/040-220PE [3]). In the DRF study hepatic effects were seen at 100, 300 and 1000 mg/kg bw but no severe toxicity was observed. The aim of this study was to use a maximum of 1000 mg/kg bw/day or to induce toxic effects, but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. Based on the results from the preliminary study, doses of 15, 60, 250 and 1000 mg/kg bw/day were selected for the main study. The oral route was selected as it is one of the possible routes of human exposure.

- Rationale for animal assignment (if not random): All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the first exposure (Day -1). There were an equal number of animals from each weight group randomly assigned to each dose group to ensure that animals of all test groups were as nearly as practicable of a uniform weight. This process was controlled by the software PROVANTIS v.9, to verify the homogeneity/variability between/within the groups. Males and females were randomised separately.

- Rationale for selecting satellite groups: Not used.

- Post-exposure recovery period in satellite groups: Not used.

- Section schedule rationale (if not random): In accordance with OECD 422 guideline.
Positive control:
None.

Examinations

Observations and examinations performed and frequency:
All animals:
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon as practical during the working day, as no peak period of effects was noted after dosing during the first few days of treatment. More detailed examinations were performed once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning or before treatment. These observations were performed outside the home cage in a standard arena, at similar day times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, and 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. During the recovery period, the detailed clinical observations of the recovery animals were performed weekly in the morning (am) similarly to the treatment period. All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including their onset, degree and duration as applicable. On gestation day GD13 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).

Five males and five females/group will be selected from the main animals:

Assessment of potential test item related neurotoxicity was performed during the last exposure week (males on Day 24; females on PPD8-12). Selected animals were subjected to the functional observation battery including quantitative assessment of grip strength and measurement of landing foot splay and fore/hind limb grip strength. To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots for the hind limbs was measured. Fore/hind limb grip strength was measured using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of the test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs and the appropriate grip support. The results were tabulated with individual and mean data. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity was conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. Parameters including body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex and vocalisation were evaluated using a scoring system, where score “0” was given when the behaviour or reaction of the animal was considered normal, and -1 or -2, or +1 and +2 was given if the response was less than or more than expected in an untreated animal.
Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for a 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 60 minutes, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. The data from all groups was evaluated for distance travelled in 5-minute segments. The data from the 5-minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.

Body weight measurement:

All adult animals were weighed with an accuracy of 1 g for randomisation purposes, then at least weekly during the pre-exposure period, on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 3, 7, 10, 14, 17 and 20 and on post-partal Days PPD0 (within 24 hours after parturition), PPD4, 7, 10, 13 and 14 (before termination). The body weight of the female animals measured on gestational Days GD3, 10 and 17 as well as PPD7 and PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, thus these data were not evaluated statistically.

The recovery animals were weighed weekly during the study (pre-exposure period, treatment period and recovery period).

Food consumption measurement:

Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g weekly (on the days of body weight measurements).

Oestrus cycle monitoring:

Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments starts (for details see 4.4.5.). Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.

The oestrus cycles of the recovery animals were monitored by vaginal smears daily only during the pre-exposure period and in the first two weeks of the treatment period.

Additionally, vaginal smears were prepared and examined for each main and recovery female on the day of necropsy to determine the stage of oestrus cycle and allow correlation with histopathology of the reproductive organs.

Observation of the delivery process, offspring and nursing instinct

Females were allowed to litter and rear their offspring. The delivery process was observed as carefully as possible. All observations were recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy until the completion of parturition.

Dams were observed for signs of nest building with the bedding material and for covering their new-borns. Evidence of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded.

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are apparently smaller than normal pups) and to detect 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 (PND0) and on PND4 and PND13, with accuracy of 0.01g. All litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination and the cause of death was identified if possible. All observed abnormalities were recorded.

The anogenital distance (AGD) of each pup was measured at the time of the first weighing (PND0). Presence of nipples/areolae in male pups were recorded on PND13 (individual records were maintained).

All pups were examined externally at weighing on PND4. One male and one female pup (where possible) was allocated randomly for culling for blood sampling on
PND4.

All pups were culled on PND13. Dams were sacrificed on PND14 after fasting overnight.

Thyroid Hormone Analysis:

For thyroid hormone analysis, blood samples were taken by cardiac puncture or venepuncture (or decapitation in case of pups) into tubes containing K3-EDTA as anticoagulant as follows:
-from up to two pups per litter on PND4,
-from all dams and at least two pups per litter on PND14 (females) / PND13 (pups),
-from all adult males at termination.

Pup blood was pooled by litter.

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, 4ºC). The resulting plasma 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, if possible; any leftover material was also retained for safety reason) and stored in an ultrafreezer (-80 ± 10 °C) until shipping for analysis.

Samples of the first aliquot for the PND13 pups and adult males were assessed for T4 levels. An audited study phase plan (45040 ABR) was provided prior to any analysis.

Samples were shipped for hormone analysis on dry ice to the Principal Investigator (PI) #1:

Joachim Decorde
Citoxlab France,
RN13 Route de Pacy
27930 Miserey, France

The thyroid hormone analysis was conducted using the validated method [8] under the control of the Principal Investigator #1 in compliance with the Test Site #1’s relevant SOPs. The results of the evaluation in the form of a Phase Report is presented in Appendix 12.

Any sample not required for analysis will be discarded following acceptance of the results of the hormone analysis by the Principal Investigator and Study Director after the finalization of the study report.

There was no blood sampling for thyroid hormone analysis from the recovery animals.
Sacrifice and pathology:
CLINICAL PATHOLOGY
All animals were fasted (overnight period of food deprivation, after the litter had been culled). From the selected animals blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.

For urine collection and terminal blood sampling in all selected animals (5 male and 5 female main animals/group plus all recovery animals), 3 samples were taken from each animal: one for haematology (in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (in tubes with sodium citrate as anticoagulant) and one to obtain serum (in tubes with no anticoagulant) for clinical chemistry.

Haematology and blood clotting times
Please refer to “Any Other Information on Materials and Methods inc Tables” below for full details. Blood smears were prepared for all selected animals but not examined. The smears are stored/archived at Citoxlab Hungary Ltd.

Clinical chemistry
Please refer to “Any Other Information on Materials and Methods inc Tables” below for full details.

Urinalysis
Please refer to “Any Other Information on Materials and Methods inc Tables” below for full details. Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.

PATHOLOGY
Terminal procedures and macroscopic evaluation
At termination, the surviving adult rats were euthanized under pentobarbital anaesthesia, followed by exsanguination.
Gross necropsy was performed on all animals, irrespective of the date of death. 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.

The preterminally euthanized and the found dead animals were examined similarly to the terminal animals.

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 of corpora lutea were recorded in the females as applicable.
Dead pups and pups killed on PND4 and/or PND13 were carefully examined externally for gross abnormalities, where possible. The anaesthetic product was diluted for pups’ euthanasia as required.

Organ weight measurements

At the time of termination, body weight and the weight of the following organs from all surviving adult animals were determined:
With a precision of 0.01 g: uterus (including cervix), testes, epididymides, prostate, seminal vesicles with coagulating glands, brain, heart, kidneys, liver, spleen and thymus

With a precision of 0.001 g: adrenals, ovaries, thyroids with parathyroids

Testes and epididymides were weighed individually. 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
Please refer to “Any Other Information on Materials and Methods inc Tables” below for full details. The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerves were retained in modified Davidson’s fixative, testes with epididymides in Bouin’s solution, and all other organs in 10% buffered formalin solution.

Additionally, thyroid glands from one male and one female PND 13 pup selected by the Study Director from each litter were preserved in 10% buffered formalin solution. In this case, the thyroid weight (pooled) was determined after fixation. Trimming was done very carefully and only after fixation to avoid tissue damage.

In case microscopic examination, the retained tissues and 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.

For the adult animals, a detailed histological examination was performed as follows:

• on the retained livers, kidneys and spleens from all main and recovery animals of all dose groups,
• on the selected list of retained organs in the Control and High dose groups (selected 5 main animals/sex/group),
• on the selected list of retained organs in one High dose female (#5511) that was found dead and in in one High dose female (#5508)) that was preterminally euthanised.
• all organs where macroscopic findings (abnormalities) were seen,
• retained reproductive organs (testes, epididymides, prostate gland, seminal vesicles with coagulation gland for males and uterus, cervix, ovary, oviduct and vagina for females) of all animals of the Control and High dose groups and of all males that failed to sire and all females that failed to deliver healthy pups.

Special attention was paid to the 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.
Other examinations:
None other than above.
Statistics:
Data was recorded to provide information on parameters to include:
Parental Males
- Clinical observations and functional observation battery (FOB).
- Body weight and body weight gain.
- Food consumption.
- Number of pairings
- Number of fertile pairings.
- Number of infertile males.
- * Male mating index.
- * Male fertility index.
- Clinical pathology.
- Thyroid hormone analysis.
- Necropsy findings
- Organ weights (absolute and relative to the body and brain weights).
- Histopathology findings

Parental Females
- Clinical observations and functional observation battery (FOB).
- Body weight and body weight gain.
- Food consumption.
- Oestrus cycle data
- 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, 4 and 13.
- * Pre-implantation mortality.
- * Intrauterine mortality.
- * Total mortality (intra and extra uterine mortality).
- Clinical pathology.
- Necropsy findings
- Organ weights (absolute and relative to the body and brain weights).
- Histopathology findings

Offspring
- Mean pup body weight (per pup within the group and per litter) on PND0, 4 and 13.
- Mean pup body weight gain (per litter) between PND0-4, PND4-13 and PND0-13.
- Number of live births per litter, and number of viable pups per litter on postnatal Days 0, 4 and 13.
- * Survival Index of pups on postnatal Days 0, 4 and 13.
- F*Sex ratio % (on postnatal Days 0, 4 and 13).
- Thyroid hormone analysis.
- Anogenital distance, nipple retention.
- Necropsy findings (macroscopic)
- Organ weights (thyroid glands, absolute and relative to the body weight).

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related clinical signs were observed during the study. The following individual observations were not attributed to systemic effects of the test item.

Slightly decreased activity, piloerection and wasted condition was observed in one Mid dose 2 male (#4010) on Day 27 (the last day of treatment) and Day 28 (before necropsy).

A 1-2 cm width nodule was seen in one Control female (#1502) at the right axillary site from Day 29 until the end of the observation period.

Piloerection and wheal on both hindpaws was seen in one Control female (#1511) from Day 20, and then also hunched back from Day 30 until the end of the observation period.

Hunched back, piloerection and slightly to moderately increased respiratory rate were seen in one Mid dose 1 female (#3509) from Day 8 until Day 20.

Alopecia of both fore paws was seen in one High dose female (#5504) from Day 30 until the end of the observation period.

Hunched back, piloerection and slightly increased respiratory rate were seen in one High dose female (#5505) from Day 48 until the end of the observation period.

In the preterminally euthanized animal (#5508), from Day 27 ataxia, hunched back, piloerection, slightly increased respiratory rate and wheal on both hindpaws and fore paws were seen before the euthanasia (Day 38).

In the found dead animal (#5511), slightly increased respiratory rate and hunched back were recorded on Day 30, the day before death.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item-related mortality was seen during the study.

One female (#5511) in the High dose group was found dead on Day 31. The cause of death could not be determined. No clear sign of treatment-relation was seen at necropsy.

One female (#5508) in the High dose group was preterminally euthanized on Day 38, because of animal welfare reasons. Based on the clinical signs and necropsy findings, it is considered that the symptoms were caused by a misgavage.
There was no other mortality during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the male main animals significantly (p<0.05) lower body weight gain was seen in the High dose during the first week of treatment. From the second week all body weight and body weight gain values were normal in all dose groups.

In females, the body weight and body weight gain values were normal during the pre-mating, mating and gestation periods, however during lactation period PPD4-13 the High dose body weight gain was lower than the Control. The lower body weight gain during the lactation period was considered to be test item-related and adverse to the dams

Recovery male and female animals had normal values during the observation period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
In males, there were no test item related differences in the mean daily food consumption in any test item treated group when compared to the controls. The measured values were within the normal range for this strain and age.

The food consumption of the females showed similar pattern as the body weight changes; there were consistently significantly lower values in the High dose during the lactation period.

Recovery male and female animals had normal values during the observation period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the haematology parameters. Statistically significant differences were considered to be incidental, there was no relationship with dose and/or all recorded values were near or within the historical control ranges. These differences were considered to not reflect an effect of the test item. These incidental statistical differences can be seen in Table 2 and 3.

It should be noted that the prothrombin time (PPT) and activated partial thromboplastin time (APTT) could not be measured in four Mid 2 dose females and in one High dose female, due to technical reasons (lipemic or clotted samples). No statistical evaluation could be performed on these parameters in the Mid 2 dose female group because of the low sample number.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically high urea levels were seen at 60, 250 and 1000 mg/kg bw/day in male dose groups, with no significant reversal in the recovery males. This change was considered to be test item-related. The difference at 60 mg/kg bw/day was generally within the historical control range. At 250 and 1000 mg/kg bw/day, there were no other indications of potentially adverse liver or kidney effects to support that the high urea was adverse, however the results were well outside the historical range, and there was no indication of recovery at 14 days post-exposure.

No other test-item related clinical chemistry findings were seen. The rest of the significant differences were considered to be incidental, there was no relationship with dose and/or all recorded values were near or within the historical control ranges. These differences were considered to not reflect an effect of the test item. These incidental statistical differences can be seen in Table 4 and 5.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item-related changes were observed in the urinalysis parameters. Significant differences were considered to be incidental, there was no relationship with dose and all recorded values were within the historical control ranges. These differences were considered to not reflect an effect of the test item. These incidental statistical differences can be seen in Table 6 and 7.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no changes in animal behaviour, general physical condition or in the reactions to different type of stimuli in the control or test groups.

There was no effect of treatment noted during the assessment of grip strength, foot splay or motor activity.

All dose groups of males and females had a normal locomotor activity; in all cases, the initial activity was high, with reduced activity in each 5-minute period to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals and the Control when evaluating the overall distance travelled (0-60 min, cm). The test item did not increase or decrease the normal locomotor activity.

Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the male main animals, significantly higher absolute and/or relative kidney weights were recorded in the Mid 2 (250 mg/kg bw/day) and High dose (1000 mg/kg bw/day) groups. The absolute and relative liver weights were also higher in the Mid 2 and High dose groups, compared to the control. In the female main animals, significantly higher absolute and relative liver weights were recorded in the Mid 2 and High dose groups. The kidney weights in the female dose groups were comparable to the control. These liver and kidney weight changes were considered to be test item related, the toxicological significance of the differences is discussed in the histopathology section of the report (see 9.4.2).

After the 14-day recovery period, the kidney weights of the male recovery High dose animals became comparable to the control, however, the liver weights were still significantly higher than the control, but this difference decreased compared to the main animals. In the female recovery High dose animals, the liver weights were still significantly higher than the control, but the organ weights started to decrease compared to the main animals. (The female recovery control animals had smaller organ weights than the female main control animals, therefore the relative liver weight differences were the same between the main females and between the recovery females. However, when looking at the absolute weights, it can be seen that the liver weights decreased in the High dose during the recovery period.)

Besides these, the following significant values were recorded, but considered to be incidental and not test item-related (and thus not presented in Tables 16, 17, 18, 19): significantly higher (p<0.05) relative brain weights in the male Mid 2 dose group, significantly higher (p<0.01) absolute brain weights in the male recovery group, significantly higher (p<0.01 or 0.05) absolute and relative thymus weights in the female main High dose, significantly higher (p<0.01) absolute ovary weights in the female main and recovery High doses, significantly higher (p<0.01 or 0.05) absolute and relative adrenal gland weights in the female recovery group. As the observed values were near the middle of the historical control range and there were no clear dose responses observed in these parameters, these statistical differences were considered to have no toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
FOUND DEAD / Parental Generation

Macroscopic Findings

The following findings were seen in the found dead animal (#5511): Pale diffuse discoloration of all lobes of the liver, dark red diffuse discoloration and enlargement of all lobes of the lungs and foamy white material in the trachea. These findings are considered as agonal or post-mortem changes.

PRETERMINAL EUTHANASIA / Parental Generation

Macroscopic Findings

The following findings were seen in the found dead animal (#5508): Bilateral enlargement of the adrenal glands, red diffuse discoloration of the lungs, small size of all lobes of the lungs, enlargement of the spleen, multiple abscesses in the thoracic cavity and enlargement of the lung-associated lymph nodes.

TERMINAL / Parental Generation (Males, Day 28, Females, PND14)

MAIN ANIMALS

Macroscopic Findings

Treatment related diffuse pale discoloration of the liver was observed in the Mid 2 (250 mg/kg bw/day) and High dose (1000 mg/kg bw/day) groups. This discoloration correlated with the microscopically observed liver vacuolation and was attributed to an effect of the test item.

Based on the low incidence and the lack of correlation with the histopathology, sporadic liver discolorations observed in the Low and Mid 1 dose groups were considered to be not test item-related.

Besides these, the following incidental or background findings were seen in the terminally euthanized animals: enlargement of the right testis (#1011), dilatation of the uterine body and horns with clear fluid (#1505, 3505, 3506, 5507), dilatation of the cerebrum and both hemispheres in the brain (#2506), dark red focal discoloration of the glandular mucosa in the stomach (#4010), bilateral enlargement of the adrenal glands (#5001), mucoid yellowish material in the digestive content (#4010), small seminal vesicles (#4010), diffuse thickness of the non-glandular region of the stomach wall and many ulcers in the mucosa of the non-glandular region (#4010), dark red multifocal discoloration of all lobes of the lungs (#3509, 5505), abscess in the thoracic cavity (#3509, 5505), and enlargement of the right, caudal and the left lobes of the lungs (#5505).

RECOVERY ANIMALS

Macroscopic Findings

Treatment related diffuse pale discoloration of the liver was observed in 3/5 males and in 4/5 females of the High dose recovery group (1000 mg/kg bw/day). This discoloration correlated with the microscopically observed liver vacuolation.

Besides these, the following incidental or background findings were seen in the terminally euthanized animals: dilatation of the uterine body and horns with clear fluid (#1514, 1516, 5514, 5517), single firm nodule on the wall of the uterine body with creamy yellow/white material on the surface (#5514), dilatation of the right pelvis of the kidney (#5015), adhesion of all lobes of the lungs to the thoracic cavity wall and diaphragm with few yellowish firm lung masses and enlargement of the lung-associated lymph nodes (#5516).
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
FOUND DEAD / Parental Generation

Microscopic Findings

Moderate microvesicular hepatocellular vacuolation of the periportal zone of the liver, slight multifocal pulmonary congestion of the lungs and slight multifocal haemorrhage of the thymus were recorded in the found dead animal.

PRETERMINAL EUTHANASIA / Parental Generation

Microscopic Findings

Slight bilateral extramedullary haematopoiesis of the adrenal glands, moderate increased cellularity of the marrow in the femur and sternum, moderate increased cellularity in the medulla and paracortex of the lung-associated lymph nodes with macrophages, slight focal or multifocal hepatocellular necrosis with haemorrhage in the liver, severe chronic bronchioloalveolar inflammation in the lungs, multiple severe abscesses in the lungs and moderate extramedullary haematopoiesis in the spleen were recorded in the preterminally euthanized animal. The macroscopic and microscopic changes in this animal were not considered to be related to systemic effects of the test item, but probably a consequence of local damage in the pulmonary system.

TERMINAL / Parental Generation (Males, Day 28, Females, PND14)

MAIN ANIMALS

Microscopic Findings

Test item-related findings were seen in the liver and kidneys of the following animals:

High dose group (1000 mg/kg bw/day): minimal to slight diffuse vacuolation in the cortex, medulla and tubule of the of the kidneys in 12/12 males and 6/10 females, minimal to moderate diffuse/multifocal/centrilobular/periportal hepatocellular vacuolation of the liver in 12/12 males and 9/10 females.

Mid 2 dose group (250 mg/kg bw/day): minimal to moderate diffuse/ centrilobular/ periportal hepatocellular vacuolation of the liver in 9/12 males and 3/12 females. (No test-item related microscopic changes were seen in the kidney of the Mid 2 dose group.)

The vacuolation in the liver and kidney was considered to be a non-adverse finding.

Besides these, based on the low incidence and/or severity and/or distribution cross control and dosed animals the following observations were considered incidental or a common background: minimal focal acinar atrophy of the pancreas (#1001), minimal focal/multifocal inflammatory cell infiltrate in the prostate (#1001, 5005), minimal extramedullary haematopoiesis in the spleen (evenly present in all groups), minimal multiple cysts at the pars distalis section of the pituary (#1002), minimal focal basophilia in the cortex of the left kidney (#1004), minimal casts in the kidneys (#1004, 1007, 2003, 4007, 5003, 5506, 5510), slight unilateral inflammatory cell infiltrate in the kidney (#1506), slight cysts in the kidneys (#3507, 3512, 4503), minimal to slight unilateral pyelonephritis in the kidney (#3507, 4506), minimal focal interstitial nephritis in the cortex of the kidneys (#3512), slight multifocal eosinophilic droplets in the cortex and tubule of the kidneys (#3008).

In addition, the organs with a macroscopic lesion were also examined and the following findings were recorded: slight to moderate diffuse dilatation of the tubular section of the testes and slightly reduced sperm in the epididymis (#1011), sign of proestrus in the uterine body and horns (#1505, 3505, 3506), slight hydrocephalus in the brain (#2506), slight inflammation in the pleura and granulomatous sections of the lungs with presence of foreign material (#3509), moderate abscess in the lungs (#3509, 5505), moderate chronic bronchioloalveolar inflammation in the lungs (#5505), slight unilateral atrophy in the seminal vesicles (#4010), slight multifocal erosion/ulcer in the forestomach (#4010). These were considered to be incidental changes.

RECOVERY ANIMALS

Microscopic Findings

Test item-related findings were seen in the liver and kidneys of the animals:

Minimal to slight diffuse vacuolation in the cortex, medulla and tubule of the of the kidneys in 3/5 males and 1/5 females, minimal to slight diffuse/ /centrilobular/periportal hepatocellular vacuolation of the liver in 5/5 males and 5/5 females. The severity and frequency of the kidney vacuolation decreased compared the main animals, indicating an ongoing resolution. The severity of the liver vacuolation also decreased, while the incidence remained the same.

Besides these, based on the low incidence and/or severity and/or distribution across control and dosed animals, the following observations were considered incidental or a common background: minimal extramedullary haematopoiesis in the spleen of 2/5 Control males, 2/5 Control females, 4/5 High dose recovery males and 3/5 High dose recovery females, minimal to slight multifocal eosinophilic droplets in the cortex and tubule of the kidneys in 2/5 High dose recovery males.

In addition, as macroscopic changes were seen in the uterus of 2 Control and 2 High dose recovery females (#1514, 1516, 5514, 5517) and in the lungs and lung-associated lymph nodes of one High dose recovery female (#5516), these organs were macroscopically examined and the following findings were recorded: sign of proestrus in the uterus of all these animals, moderate multifocal granulomatous inflammation of the pleura of the lungs with haemorrhage and moderate granulomatous inflammation with foreign material presence in the lung-associated lymph nodes. These changes were not considered to be related to systemic effects of the test item.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone levels (adult males)

Significantly lower values were recorded in the Mid 2 and High dose groups, possibly related to liver enzyme induction (as indicated by the liver weight increases).

However, all dose groups were within the historical control range, therefore these changes are not considered adverse.
Details on results:
In summary, daily administration of NAUGARD® 412S by oral gavage to Wistar rats at dose levels of 15, 60, 250 or 1000 mg/kg bw/day during the treatment period under the conditions of this study did not result in test item related mortality, clinical adverse effects, or changes in neurological assessment. Significant adverse effects on body weight were seen in the dams of the High dose group (1000 mg/kg bw/day) during the lactation period, with similar changes in food intake. Significantly higher blood urea levels were seen in the Mid 2 (250 mg/kg bw/day) and High dose (1000 mg/kg bw/day) groups with no significant reversal in the recovery animals. There were no other test item-related effects on the clinical pathology parameters (haematology, coagulation, clinical chemistry or urinalysis).

At necropsy, test item-related diffuse pale discoloration was observed in the liver of the Mid 2 (250 mg/kg bw/day) and High dose (1000 mg/kg bw/day) animals. The organ weights of the liver and kidney were also increased at 250 and 1000 mg/kg bw/day. At histopathology vacuolation in the liver at 250 and 1000 mg/kg bw/day and in the kidney at the High dose were seen. The hepatic and kidney vacuolation did not fully reverse at the end of the recovery period. The histological findings were not considered to be adverse effects. The high urea which did not reverse may indicate an adverse systemic effect of the test item.

No test item-related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14 in the Low (15 mg/kg bw/day), Mid 1 (60 mg/kg bw/day) and Mid 2 (250 mg/kg bw/day) dose groups. However, the pup mortality was increased in the High dose (1000 mg/kg bw/day) and lower growth of developing pups was seen. These data correlated with dam low body weight gain and low food consumption during the lactation period; the effect was considered as probably secondary to maternal toxicity. No developmental or endocrine changes were seen in the pups at any of the dose levels (anogenital distance, thyroid gland weights, thyroid hormone level, etc.).

The NOAEL for Reproductive effects was considered to be 1000 mg/kg bw/day.

The NOAEL for Pup development and survival was considered to be 250 mg/kg bw/day.

The NOAEL for Systemic toxicity for the adults was considered to be 60 mg/kg bw/day.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic

Target system / organ toxicity

open allclose all
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
250 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

DOSE FORMULATION ANALYSIS

The measured active ingredient concentrations of NAUGARD®412S evaluated for each test item-dose group varied between 92 % and 110 % of the nominal concentrations. The relative standard deviation (RSD) was below 10% in each case. No test item was detected in the control samples. These results were within the acceptable ranges (85% - 115%) and were considered suitable for the study purposes.

 

Table 1: Summary of analytical results

 

Nominal
concentration
(mg/mL)

Measured concentration
(mg/mL)

Percentage of the nominal concentration
(%)

Analytical sampling #1(Sampling: 20 September 2017)

Control

not detectable

-

3

2.90 ± 0.18

96.5

12

10.98 ± 0.26

91.5

50

51.66 ± 0.21

103.3

200

192.97 ± 3.77

96.5

Analytical sampling #2(Sampling: 11 October 2017)

Control

not detectable

-

3

2.90 ± 0.14

96.7

12

11.05 ± 0.42

92.1

50

51.68 ± 3.17

103.4

200

196.27 ± 11.24

98.1

Analytical sampling #3(Sampling: 02 November 2017)

Control

not detectable

-

3

3.28 ± 0.18

109.3

12

12.98 ± 0.44

108.2

50

55.07 ± 2.08

110.1

200

208.59 ± 8.82

104.3

 

Table 2:Significant differences in haematology parameters in males

 

Haematology (males)

Groups/Concentration (mg/kg bw/day)

Main animals

Recovery animals

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Control (0)

High (1000)

Haematocrit (%)

48.60

48.78

51.00 *

48.20

48.00

49.42

49.70

Differences from control

0%

5%

-1%

-1%

Differences from control

1%

Historical control data

40.90 – 50.20

Red cell distribution width (%)

12.50

12.64

12.58

13.40

12.88

12.44

13.08 *

Differences from control

1%

1%

7%

3%

Differences from control

5%

Historical control data

10.50 – 16.80

Reticulocytes (%)

2.22

2.42

2.66

3.06 *

3.10 *

1.84

2.16

Differences from control

9%

20%

38%

40%

Differences from control

17%

Historical control data

0.70 – 5.80

Neutrophils (%)

34.18

45.64

41.70

29.50

23.72 *

33.50

33.64

Differences from control

4%

22%

-14%

-31%

Differences from control

0%

Historical control data

11.80 – 46.50

Lymphocytes (%)

60.60

58.32

52.64

64.16

71.46 *

59.32

58.50

Differences from control

-4%

-13%

6%

18%

Differences from control

-1%

Historical control data

13.50 – 85.70

Basophils (%)

0.24

0.16

0.20

0.14

0.12

0.08

0.20 *

Differences from control

-33%

-17%

-42%

-50%

Differences from control

150%

Historical control data

 

Eosinophils (%)

1.10

1.80

1.26

2.20 *

0.80

1.74

2.22 *

Differences from control

64%

15%

100%

-27%

Differences from control

28%

Historical control data

0.00 – 1.30

APTT (sec)

12.84

13.04

14.38 **

13.92 *

12.82

12.30

12.32

Differences from control

2%

12%

8%

0%

Differences from control

0%

Historical control data

10.00 – 13.40

PTT (sec)

9.58

9.44

10.10

10.08

10.36 +

9.84

10.88 +

Differences from control

-2%

5%

5%

8%

Differences from control

11%

Historical control data

9.30 – 11.00

Notes: *= p<0.05; Dunnett two sided test.

                       += p<0.05,++= p<0.01; Dunn two sided test

 

Table 3:Significant differences in haematology parameters in females

 

Haematology (females)

Groups/Concentration (mg/kg bw/day)

Main animals

Recovery animals

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Control (0)

High (1000)

Haemoglobin concentration (g/dL)

14.74

14.90

14.98

14.72

15.18

15.66

14.74 *

Differences from control

1%

2%

0%

3%

Differences from control

-6%

Historical control data

11.50 – 16.40

Haematocrit (%)

46.46

46.94

47.10

44.20

47.38

47.06

42.88 *

Differences from control

1%

1%

-5%

2%

Differences from control

-9%

Historical control data

35.20 – 49.10

Mean Cell Volume

59.12

61.26

60.36

58.66

58.08

54.86

52.42 **

Differences from control

4%

2%

-1%

-2%

Differences from control

-4%

Historical control data

51.70 – 72.40

Mean Platelet Volume

8.12

8.72

8.42

8.98

8.98

7.28

8.28 **

Differences from control

7%

4%

11%

11%

Differences from control

14%

Historical control data

6.40 – 14.80

MCH concentration (g/dL)

31.74

31.74

31.78

33.30 *

32.08

33.26

34.40

Differences from control

0%

0%

5%

1%

Differences from control

3%

Historical control data

30.30 – 36.40

Neutrophils (%)

62.92

65.80

57.74

50.38

34.64++

33.88

41.58

Differences from control

5%

-8%

-20%

-45%

Differences from control

23%

Historical control data

14.60 – 76.80

Lymphocytes (%)

32.48

30.02

37.22

43.44

58.34+

60.14

51.22

Differences from control

-8%

15%

34%

80%

Differences from control

-15%

Historical control data

16.70 – 80.40

Basophils (%)

0.08

0.18

0.14

0.36

0.56++

0.26

0.86

Differences from control

125%

75%

350%

600%

Differences from control

231%

Historical control data

0.00 – 0.70

Eosinophils (%)

1.16

1.14

1.20

1.60

1.62

1.80

0.56 *

Differences from control

-2%

3%

38%

40%

Differences from control

-69%

Historical control data

0.30 – 9.30

Notes: *= p<0.05; Dunnett two sided test.

                       += p<0.05,++= p<0.01; Dunn two sided test

 

Table 4:Significant differences in clinical chemistry parameters in males

 

Clinical chemistry (males)

Groups/Concentration (mg/kg bw/day)

Main animals

Recovery animals

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Control (0)

High (1000)

Urea (mmol/L)

5.80

7.17

8.90 **

10.42 **

11.47 **

6.57

14.61 **

Differences from control

24%

53%

80%

98%

Differences from control

123%

Historical control data

4.01 – 9.89

Glucose (mmol/L)

8.56

10.03

8.97

8.84

8.76

8.04

9.71 *

Differences from control

17%

5%

3%

2%

Differences from control

21%

Historical control data

4.49 – 12.11

Cholesterol (mmol/L)

1.256

1.454

1.160

1.160

1.160

1.916

1.330 *

Differences from control

16%

-8%

-8%

-8%

Differences from control

-31%

Historical control data

1.16 – 2.17

Creatinine (µmol/L)

42.44

46.62

49.18

45.85

47.36

42.88

52.64+

Differences from control

10%

16%

8%

12%

Differences from control

23%

Historical control data

40.00 – 86.20

Phosphorus (mmol/L)

2.310

2.444

2.386

2.760 **

2.912 **

2.532

2.686

Differences from control

6%

3%

20%

26%

Differences from control

6%

Historical control data

1.96 – 3.16

Aspartate Aminotransferase (U/L)

159.8

221.0

605.6++

164.2

195.4

154.4

373.0 **

Differences from control

38%

279%

3%

22%

Differences from control

142%

Historical control data

93.00 – 628.00

Alanine Aminotranferase (U/L)

54.8

55.6

106.0++

58.2

73.2

41.0

84.8+

Differences from control

2%

93%

6%

34%

Differences from control

107%

Historical control data

28.00 – 150.00

Alkaline Phosphatase (U/L)

88.4

90.6

87.2

105.4

150.2 **

91.4

98.2

Differences from control

3%

-1%

19%

70%

Differences from control

7%

Historical control data

60.00 – 171.00

Notes: *= p<0.05; Dunnett two sided test.

                       += p<0.05,++= p<0.01; Dunn two sided test

 

Table 5:Significant differences in clinical chemistry parameters in females

 

Clinical chemistry (males)

Groups/Concentration (mg/kg bw/day)

Main animals

Recovery animals

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Control (0)

High (1000)

Urea (mmol/L)

12.83

13.93

13.49

13.87

13.59

7.47

10.86 **

Differences from control

9%

5%

8%

6%

Differences from control

45%

Historical control data

4.44 – 11.18

Glucose (mmol/L)

8.24

8.80

7.25

7.56

7.64

9.46

4.65 **

Differences from control

7%

-12%

-8%

-7%

Differences from control

-51%

Historical control data

5.17 – 11.84

Total bilirubin (µmol/L)

4.08

5.68

4.74

6.40 *

4.14

5.78

6.12

Differences from control

39%

16%

57%

2%

Differences from control

6%

Historical control data

1.70 – 13.70

Total protein (g/L)

54.12

67.56

54.36

58.44

54.62 *

59.00

60.94

Differences from control

6%

0%

8%

1%

Differences from control

3%

Historical control data

49.50 – 68.70

Albumin (g/L)

30.44

33.10

30.84

33.50

32.04

34.98

34.08

Differences from control

9%

1%

10%

5%

Differences from control

-3%

Historical control data

 

Cholesterol (mmol/L)

2.260

2.260

2.488

2.550

4.018

2.632

1.610 **

Differences from control

10%

13%

78%

-21%

Differences from control

-39%

Historical control data

25.70 – 42.70

Phosphorus (mmol/L)

3.040

3.046

2.822

3.050

2.780

1.742

2.358 **

Differences from control

0%

-7%

0%

-9%

Differences from control

35%

Historical control data

1.60 – 3.78

Chloride (mmol/L)

97.40

96.76

97.16

94.74

99.64

101.26

96.10+

Differences from control

-1%

0%

-3%

2%

Differences from control

-5%

Historical control data

90.70 – 106.60

Alkaline Phosphatase (U/L)

94.0

83.4

92.8

85.0

89.2

42.4

94.6 *

Differences from control

-11%

-1%

-10%

-5%

Differences from control

123%

Historical control data

37.00 – 137.00

Notes: *= p<0.05; Dunnett two sided test.

                       += p<0.05,++= p<0.01; Dunn two sided test

 

Table 6:Significant differences in urinalysis parameters in males

 

Urinalysis (males)

Groups/Concentration (mg/kg bw/day)

Main animals

Recovery animals

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Control (0)

High (1000)

pH

7.20

7.20

7.00

7.00

6.80

7.80

7.00+

Differences from control

0%

-3%

-3%

-6%

Differences from control

-10%

Historical control data

6.00 – 8.00

Specific gravity

1.0060

1.0070

1.0100

1.0100

1.0120

1.0070

1.0130+

Differences from control

0%

0%

0%

1%

Differences from control

1%

Historical control data

1.01 – 1.04

 

 

Notes: *= p<0.05; Dunnett two sided test.

                       += p<0.05,++= p<0.01; Dunn two sided test

 

Table 7:Significant differences in urinalysis parameters in females

 

Urinalysis (females)

Groups/Concentration (mg/kg bw/day)

Main animals

Recovery animals

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Control (0)

High (1000)

pH

6.00

7.00

6.40

6.40

6.20

8.00

6.80++

Differences from control

17%

7%

7%

3%

Differences from control

-15%

Historical control data

6.00 – 8.00

Notes: *= p<0.05; Dunnett two sided test.

                       += p<0.05,++= p<0.01; Dunn two sided test

 

Table 8:Parental male thyroid hormone concentration levels

 

(adult males)

Group /Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (100)

T4 concentration

(ng/mL)

43.83

41.59

38.13

36.78 *

35.81 **

Differences from control

-5%

-13%

-16%

-18%

Historical control data

34.3 – 60.7

Note: *= p<0.05; Dunnett two sided test, **= p<0.01; Dunnett two sided test.

 

Table 16:Kidney and liver organ weights of the male main animals

 

Organ weights (males)

Groups/Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Terminal bodyweight (g)

494

501

487

462

470

Differences from control

1%

-2%

-6%

-5%

Historical control data

368 – 668

Kidneys (g)

2.93

3.10

3.05

3.06

3.23

Differences from control

6%

4%

5%

10%

Historical control data

2.31 – 3.98

Kidneys / Bodyweight (%)

0.59

0.62

0.63

0.66 **

0.69 **

Differences from control

5%

6%

12%

16%

Historical control data

0.51 – 0.80

Kidneys / Brain weight (%)

136.91

144.26

139.16

141.42

152.68 *

Differences from control

5%

2%

3%

12%

Historical control data

104.52 – 177.68

Liver (g)

13.90

14.69

15.38

17.40 **

18.99 **

Differences from control

6%

11%

25%

37%

Historical control data

9.66 – 17.44

Liver / Bodyweight (%)

2.81

2.93

3.16

3.77 **

4.04 **

Differences from control

4%

13%

34%

44%

Historical control data

2.12 – 3.48

Liver / Brain weight (%)

651.0

683.6

701.5

803.4 **

898.9 **

Differences from control

5%

8%

23%

38%

Historical control data

439.1 – 766.0

Note: *= p<0.05; **= p<0.01; Dunnett two sided test.

 

Table 17:Kidney and liver organ weights of the male recovery animals

 

Organ weights (males)

Groups/Concentration (mg/kg bw/day)

Control (0)

High (1000)

Terminal bodyweight (g)

515

516

Differences from control

0%

Historical control data

368 – 668

Kidneys (g)

3.18

3.15

Differences from control

-1%

Historical control data

2.31 – 3.98

Kidneys / Bodyweight (%)

0.62

0.61

Differences from control

-1%

Historical control data

0.51 – 0.80

Kidneys / Brain weight (%)

139.52

146.41

Differences from control

5%

Historical control data

104.52 – 177.68

Liver (g)

14.00

16.91 **

Differences from control

21%

Historical control data

9.66 – 17.44

Liver / Bodyweight (%)

2.72

3.27 **

Differences from control

21%

Historical control data

2.12 – 3.48

Liver / Brain weight (%)

613.4

784.4 **

Differences from control

28%

Historical control data

439.1 – 766.0

Note: *= p<0.05; **= p<0.01; Dunnett two sided test.

 

Table 18:Kidney and liver organ weights of the female main animals

 

Organ weights (females)

Groups/Concentration (mg/kg bw/day)

Control (0)

Low (15)

Mid 1 (60)

Mid 2 (250)

High (1000)

Terminal bodyweight (g)

335

347

347

355

316

Differences from control

4%

3%

6%

-6%

Historical control data

236 – 409

Kidneys (g)

2.08

2.07

2.04

2.11

2.10

Differences from control

0%

-2%

2%

1%

Historical control data

1.57 – 2.64

Kidneys / Bodyweight (%)

0.62

0.60

0.59

0.60

0.67

Differences from control

-4%

-5%

-4%

7%

Historical control data

0.50 – 0.80

Kidneys / Brain weight (%)

102.22

103.21

100.88

104.46

103.76

Differences from control

1%

-1%

2%

2%

Historical control data

72.52 – 125.12

Liver (g)

12.71

13.73

14.06

14.93 *

14.58

Differences from control

8%

11%

17%

15%

Historical control data

6.39 – 19.52

Liver / Bodyweight (%)

3.80

3.97

4.06

4.21 *

4.60 **

Differences from control

4%

7%

11%

21%

Historical control data

2.46 – 6.06

Liver / Brain weight (%)

625.0

685.5

695.5

740.3

720.8

Differences from control

10%

11%

19%

15%

Historical control data

304.3 – 985.9

Note: *= p<0.05; **= p<0.01; Dunnett two sided test.

 

Table 19:Kidney and liver organ weights of the female recovery animals

 

Organ weights (females)

Groups/Concentration (mg/kg bw/day)

Control (0)

High (1000)

Terminal bodyweight (g)

299

312

Differences from control

5%

Historical control data

236 – 409

Kidneys (g)

1.71

1.94

Differences from control

14%

Historical control data

1.57 – 2.64

Kidneys / Bodyweight (%)

0.57

0.63

Differences from control

9%

Historical control data

0.50 – 0.80

Kidneys / Brain weight (%)

82.94

94.31 *

Differences from control

14%

Historical control data

72.52 – 125.12

Liver (g)

8.51

10.80 **

Differences from control

27%

Historical control data

6.39 – 19.52

Liver / Bodyweight (%)

2.85

3.49 **

Differences from control

27%

Historical control data

2.46 – 6.06

Liver / Brain weight (%)

413.3

525.1

Differences from control

27%

Historical control data

304.3 – 985.9

Note: *= p<0.05; **= p<0.01; Dunnett two sided test.

 

Applicant's summary and conclusion

Conclusions:
The NOAEL for Reproductive effects was considered to be 1000 mg/kg bw/day.

The NOAEL for Pup development and survival was considered to be 250 mg/kg bw/day.

The NOAEL for Systemic toxicity for the adults was considered to be 60 mg/kg bw/day.
Executive summary:

In summary, daily administration of NAUGARD®412S by oral gavage to Wistar rats at dose levels of 15, 60, 250 or 1000 mg/kg bw/day during the treatment period under the conditions of this study did not result in test item related mortality, clinical adverse effects, or changes in neurological assessment. Significant adverse effects on body weight were seen in the dams of the High dose group (1000 mg/kg bw/day) during the lactation period, with similar changes in food intake. Significantly higher blood urea levels were seen, out of the historical control range in the Mid 2 (250 mg/kg bw/day) and High dose (1000 mg/kg bw/day) groups with no significant reversal in the recovery animals. There were no other test item-related effects on the clinical pathology parameters (haematology, coagulation, clinical chemistry or urinalysis).

 

At necropsy, test item-related diffuse pale discoloration was observed in the liver of the Mid 2 (250 mg/kg bw/day) and High dose (1000 mg/kg bw/day) animals. The organ weights of the liver and kidney were also increased at 250 and 1000 mg/kg bw/day. At histopathology vacuolation in the liver at 250 and 1000 mg/kg bw/day and in the kidney at the High dose group were seen. The hepatic and kidney vacuolation did not fully reverse at the end of the recovery period. The histological findings were not considered to be adverse effects. The high urea which did not reverse may indicate an adverse systemic effect of the test item in the Mid 2 and High dose groups.

 

No test item-related changes were noted in the reproductive parameters during mating and gestation, delivery and post-partum/lactation period until PPD14 in the Low (15 mg/kg bw/day), Mid 1 (60 mg/kg bw/day) and Mid 2 (250 mg/kg bw/day) dose groups. However, the pup mortality was increased in the High dose (1000 mg/kg bw/day) and lower growth of developing pups was seen. These data correlated with the dams’ low body weight gain and low food consumption during the lactation period; the effect was considered as probably secondary to maternal toxicity. No developmental or endocrine changes were seen in the pups at any of the dose levels (anogenital distance, thyroid gland weights, thyroid hormone level, etc.).

 

The NOAEL for Reproductive effects was considered to be 1000 mg/kg bw/day.

 

The NOAEL for Pup development and survival was considered to be 250 mg/kg bw/day.

 

The NOAEL for Systemic toxicity for the adults was considered to be 60 mg/kg bw/day.