Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test by Oral Administration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 June 2017 to 23 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 June 2017 to 23 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 422, Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, Organisation for Economic Co-Operation and Development, Paris, 29 July 2016
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
The Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rats was conducted according to OECD No. 422 guideline and OECD No. 43 guidance document. The guideline was designed for use with the rat, which is the preferred rodent species for reproduction toxicity testing.

The purpose of the study was to obtain information on the possible toxic effects of the test item 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] following repeated (daily) administration by oral gavage to Wistar rats at 3 dose levels. A control group received the vehicle only (corn oil).

The study included a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to post-natal day (PND) 13.
Specific details on test material used for the study:
No further details specified in the study report.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species and strain: Crl:WI (Wistar) rats
Justification of species/strain: The rat is regarded as a suitable species for toxicology and reproduction toxicology studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility. The same strain was used for the Dose Range Finding study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Species and strain: Crl:WI (Wistar) rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, Sulzfeld, D-97633, Germany), from SPF colony.
Housing conditions: Standard laboratory conditions
Number of animals: 49 male and 49 female rats used for treatment, at least 12 animals/sex/group, 4 groups. Additional females were also used in the pre-exposure period for oestrus cycle determination (those animals besides the spare animals were moved back to the spare colony of the Test Facility after the study). Animals originated from different units, to avoid brother/sister mating.
Age of animals: Young adult rats, approximately 10 weeks old at start and 12 weeks old at mating.
Body weight range: Males: 425-505 g, females: 223 – 288 g at the start of the treatment (did not exceed ± 20 % of the mean weight for each sex).
Acclimation period: 29 days

Husbandry
Animal health: Only healthy animals were used for the test, as certified by the clinical Veterinarian. Females were nulliparous and non-pregnant.
Room number: 243 (in-life phase), 505 (acclimatisation period)
Cage type: Type II polycarbonate
Bedding & nesting: LIGNOCEL® ¾ S certified wooden chips (Batch number: 03018170329 / 03018170529, Expiry date: 29 March 2020 / 29 May 2020) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, Rosenberg D-73494, Germany) were used in the study. ARBOCEL® nest building material (Batch number: 05072160415 / 05072170228, Expiry date: 15 April 2019 / 28 February 2020) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, Rosenberg D-73494, Germany).
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.0 – 28.7 °C (target range 22 ± 3 °C)
Relative humidity: 29 – 74 % (target range 30-70 %)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).
Environmental parameters (temperature and relative humidity) were continuously measured, minimum and maximum values were recorded twice a day during the study.

Food and water supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (Batch number: 285 17890 / 262 21592, Expiry date: 31 August 2017 / 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.

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.
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*.
*Note: Due to the incidental mortality of a Mid dose female (#3509), a replacement animal (#3609) was included in the study. In order to provide a mating pair with the same length of treatment before mating, a replacement male (#3109) was also included in the study.
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.

Randomisation
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the pre-exposure period (Day -14). 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.
The body weight ranges were additionally checked before the first exposure (Day 0) using Microsoft Excel to ensure that the individual values did not exceed ± 20% of the mean weight for each sex at the start of the treatment.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
The test item was formulated in the vehicle (as a visibly stable homogenous suspension) in the Pharmacy of CiToxLAB Hungary Ltd. Formulations were prepared daily at appropriate concentrations in the vehicle in the Pharmacy of CiToxLAB Hungary Ltd.
Formulations were prepared daily (on the day of treatment or on the previous afternoon) in the Pharmacy of CiToxLAB Hungary Ltd. as follows. The calculated volume of the vehicle was added into a beaker containing the calculated amount of test item, it was mixed vigorously by a magnetic stirrer to make a homogenous formulation. Based on the analytical method validation study (CiToxLAB Hungary Study code: 17/039-316AN), formulations in the 20 - 200 mg/mL concentration range were found to be stable for 7 days of storage at room temperature.

Vehicle
Based on the available information (trial formulations of the item as well as analytical method development trials and DRF study performed at the Test Facility), corn oil was selected as vehicle for this study in agreement with the Sponsor. The same vehicle was used in the Dose Range Finding (DRF) study.
Identification data of the chemical used in the study are as follows:

Name: Corn oil
Manufacturer: ACROS (Reanal)
Batch number: A0384372
Expiry date: 31 May 2019
Storage conditions: Room temperature
Details on mating procedure:
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 or 14 days. The female (#3501) that failed to mate (with #3001) within two weeks was re-mated with proven male (#3109) of the same group for 6 days.

A vaginal smear was prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope. The presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were housed individually.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and/or homogeneity was performed using a validated HPLC-UV (High Pressure Liquid Chromatography – Ultraviolet spectroscopy) method in the Analytical Laboratory of CiToxLAB Hungary Ltd. Duplicate samples were taken from the top, middle and bottom of the test item formulations four times during the study (during the first and last treatment weeks of the study and approximately midway during the treatment period), 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.
Duration of treatment / exposure:
Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating / post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13.
Frequency of treatment:
Daily.
Details on study schedule:
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 29 days of acclimatisation and 14 days of pre-exposure period, and it was performed for 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.
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 post–partum day (PPD) 0. Females showing no-evidence of copulation were sacrificed as practical, 25-28 days after the day of presumed mating.)
All F1 offspring were terminated on post-natal day (PND) 13 (F1 offspring selected for blood sampling on PND4 were terminated on that day). In order to allow for overnight fasting of dams prior to urine collection on PPD14, the offspring were euthanized on PPD/PND13 and the dams on PPD/PND14.
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 animal per group (11 male/11 female).
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose selection and route of administration
The dose levels were selected by the Sponsor in consultation with the Study Director based on available data including the results of an acute oral toxicity study in rats
(LD50 >2000 mg/kg bw for male and female rats in an OECD No. 401 study, data on file at the Sponsor) and a 14-day repeated dose Dose Range Finding (DRF) study in the rat performed at the Test Facility (CiToxLAB Hungary Study code: 17/039-220PE [3]). In the DRF study, there was no toxicity at 1000 mg/kg bw/day. The aim of this study was to use a maximum of 1000 mg/kg bw/day 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 these preliminary studies, doses of 100, 300 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.
Positive control:
Not specified
Parental animals: Observations and examinations:
Clinical observations
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. The principles and criteria summarized in the OECD Humane Endpoints Guidance Document No. 19 were taken into consideration. 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.
On gestation day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).
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.
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.


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.
Parental females were weighed on gestation days (GD) 0, 3, 7, 10, 14, 17 and 20 and on post-partum days (PPD) 0 (within 24 hours after parturition), 4, 7, 10, 13 and 14 (before termination). The body weight of the parental female animals measured on GD3, 10 and 17 as well as PPD7 and PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, but these data were not evaluated statistically.

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).

Functional observation battery (FOB) and SMART
Assessment of potential test item related neurotoxicity was performed during the last exposure week (males on Day 26; females on PPD8-9). 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, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or 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.

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.

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 at termination (PPD14) and up to two pups per litter on PND13,
-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 aliquots, if possible; any leftover material was also retained for safety reason) and stored in an ultrafreezer (-80 ± 10 °C) until shipping for analysis.

Samples (first aliquots) were shipped for hormone analysis on dry ice to the Principal Investigator (PI).
Samples of adult males and PND13 pups were assessed for T4 levels. The measurement of the T4 hormone levels in adult females and PND4 pups, or measurement of other thyroid hormone (TSH) were not performed as was not deemed necessary by the Study Director and Sponsor.

The thyroid hormone analysis was conducted using the validated method under the control of the Principal Investigator in compliance with the Test Site’s relevant SOPs. An audited study phase plan (Test Site phase code: 45039 ABR) was provided prior to any analysis.
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.


CLINICAL PATHOLOGY
All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
For terminal blood sampling in all selected animals (5 males and 5 females/group), 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
The following parameters were evaluated for selected animals:
RBC Red Blood Cell (erythrocyte) count; WBC White Blood Cell (leukocyte) count; Hgb Haemoglobin concentration; Hct Haematocrit (relative volume of erythrocytes); MCV Mean Corpuscular (erythrocyte) Volume; MCH Mean Corpuscular (erythrocyte) Haemoglobin; MCHC Mean Corpuscular (erythrocyte) Haemoglobin Concentration; RDW Red Cell (erythrocyte) volume; Plt Platelet (thrombocyte) count; MPV Mean Platelet Thrombocyte volume; RETIC % Reticulocyte count; NE % Neutrophil; LY % Lymphocyte; MO % Monocyte; BA % Basophil; EO % Eosinophil; LUC % Large Unstained Cells; APTT Activated Partial Thromboplastin Time; PT Prothrombin Time.

Clinical chemistry
The following parameters were evaluated for selected animals:
Glucose Blood sugar concentration; T-BIL Total Bilirubin concentration; Urea Urea concentration; Chol. Cholesterol concentration; Creat. Creatinine concentration; Phos. Phosphorus concentration; Na+ Sodium concentration; K+ Potassium concentration; Ca++Calcium concentration; Cl- Chloride concentration; Tot. Prot. Total Protein concentration; Alb. Albumin concentration; A/G Alb/glob ration; AST/GOT Aspartate Aminotransferase activity; ALT/GPT Alanine Aminotransferase activity; GGT Gamma-Glutamyl transferase activity; ALKP Alkaline Phosphatase activity; Bile acids.

Urinalysis
Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours.
The evaluation of the urine samples was performed as indicated below:
LEU / Leukocyte; NIT / Nitrite; pH; PRO / Protein; GLU / Glucose; UBG / Urobilinogen; BIL / Bilirubin; KET / Ketones; BLD / ERY Blood/Erythrocytes; SG / Specific Gravity; SED / Sediment; VOL / Volume; Colour / Appearance.
Oestrous cyclicity (parental animals):
Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments starts. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.
Sperm parameters (parental animals):
Not specified.
Litter observations:
Observation of offspring
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 0.01 g. All litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized or autolyzed) 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 all 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 surviving pups were terminated on PND13.

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 at termination (PPD14) and up to two pups per litter on PND13,
-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 aliquots, if possible; any leftover material was also retained for safety reason) and stored in an ultrafreezer (-80 ± 10 °C) until shipping for analysis.

Samples (first aliquots) were shipped for hormone analysis on dry ice to the Principal Investigator (PI).
Samples of adult males and PND13 pups were assessed for T4 levels. The measurement of the T4 hormone levels in adult females and PND4 pups, or measurement of other thyroid hormone (TSH) were not performed as was not deemed necessary by the Study Director and Sponsor.
The thyroid hormone analysis was conducted using the validated method under the control of the Principal Investigator in compliance with the Test Site’s relevant SOPs. An audited study phase plan (Test Site phase code: 45039 ABR) was provided prior to any analysis.
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.
Postmortem examinations (parental animals):
PATHOLOGY
Terminal procedures and macroscopic evaluation
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.

Organ weight measurements
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 parathyroid.

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
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerves as well as testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.

In addition, on completion of the macroscopic examination the following tissues and organs were retained from all animals:
Adrenal gland; Animal identification; Aorta; Brain; Epididymis; Eye with the optic nerve; Oesophagus; Femur with marrow; Heart; Kidney; Large intestine; Extraorbital lachrymal gland; Harderian gland; Liver; Lungs with bronchi; Lymph node; Ovary; Oviduct; Pancreas; Pituitary; Prostate; Salivary gland (including mandibular, sublingual and parotid glands); Sciatic nerve; Seminal vesicle with coagulating gland; Skin, subcutis with mammary gland (inguinal); Skeletal muscle (quadriceps); Small intestine; Spinal cord; Spleen; Sternum with marrow; Stomach; Testis; Thymus; Thyroid with parathyroid gland; Tongue; Trachea; Urinary bladder; Uterus; Vagina.

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 selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group). Based on the histopathology findings (centrilobular hepatocellular hypertrophy) observed in the liver of the examined High dose group animals, slides of the liver samples of all Mid dose and Low dose animals, furthermore the rest of High dose group animals were processed and examined microscopically as agreed by the Sponsor.
-on the selected list of retained organs in one Mid dose female (#3509) and in one High dose female (#4508) which were found dead,
-the spleen of one Low dose male (#2012), the testes and epididymis of one Mid dose male (#3001), and the spleen of one Mid dose male (#3007) because macroscopic findings (abnormalities) were seen at necropsy,
-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 (#2003, #2007, #3007) and all females (#2503, #2507 and #3507) that failed to sire / conceive.

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.

Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes and bone marrow). Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.
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:
See "Any other information" for details.
Reproductive indices:
Number of pairings
Number of fertile pairings
Number of infertile males
* Male mating index
* Male fertility index
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)
Offspring viability indices:
Mean pup body weight (per pup 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 PND 0, 4 and 13
* Survival Index of pups on PND 0, 4 and 13
F*Sex ratio % (on PND 0, 4 and 13)
Thyroid hormone (T4) levels
Anogenital distance, nipple retention
Necropsy findings (macroscopic)
Organ weights (thyroid glands, absolute and relative to the body weight)
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related clinical signs were observed during the study.
Thin fur was recorded for two Mid dose males (#3011 on Days 11-26, and #3012 on Days 24-28) and for one Control female (#1501 on Days 29-50) and one Mid dose female (#3506 on Days 38-51). These minor observations were not related to the test item treatment.
In one Mid dose female (#3609) paleness was observed on forelimbs, hind limbs and both pinna on Days 39-41, piloerection and slight discharge from vagina was also recorded on Day 39. However, these findings were related to a difficult delivery process (delivery occurred on Day 40), not related to the test item administration.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item related mortality was observed in the study.
A Mid dose female (#3509) was found dead on Day 13 without any clinical symptoms. The cause of death, moderate erosion/ulcer of the oesophagus, moderate pleuritis and slight epicarditis recorded in this animal were indicated as consequences of previous technical gavage error.
A High dose female (#4508) was found dead on Day 18 after showing hunched back and slightly laboured respiration and piloerection in the previous two days. No test item-related changes were seen in this animal. The cause of death was identified by the pathologist as a possible gavage accident.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item effect on body weight or body weight gain was detected during the study.
There were no statistically significant body weight or body weight gain values in the test item treated groups of either sex when compared to the control at any occasion that could be ascribed to the test item. The measured values were within the range commonly recorded for this strain and age.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
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.
Occasional statistically significant differences were regarded as incidental and of no toxicological significance
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
When compared to the controls, there were no differences in males and females that could be considered toxicologically significant in the test item treated groups.
The relative amount of large unclassified cells was significantly lower (p<0.05) in Mid and High dose males when compared to control. But as all the individual values were within the historical control range, there was no clear dose response and no significant differences were noted in females, these differences were considered as animal variability, not being a test item related effect.
Significantly lower (p<0.01 or p<0.05) mean cell haemoglobin concentration was recorded for Mid and High dose females when compared to control. As the difference was small (less than 5%), the observed values were within the historical control range, there was no dose response and no similar trend in males, these differences were considered to not reflect any effect of the test item.
No statistically significant changes were recorded for any other haematology or coagulation parameters in the test item treated males and females.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant changes or adverse effects on the serum chemistry that could be ascribed to the test item administration.
Statistically significant increase (p<0.05) in total protein concentration was were seen in Low and High dose males when compared to the control. However, there was no dose response, and the observed values were within the historical control range. No significant change was seen in females; thus this difference was considered as not being related to test item treatment.
No statistically significant changes were recorded for any other parameters in the test item treated males and females.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test item related changes were recorded in any of the test item treated groups when compared to the control.
Significantly lower urine volume was collected from the Low (p<0.05) and Mid (p<0.01) dose males when compared to control. The collected urine volume is usually highly variable as indicated by the historical control data. All the individual values obtained in this study were considered to be normal, there was no dose response and no similar trend was seen in females, therefore this numerical difference was not considered to reflect a test item related effect. This fact was confirmed by the lack of any supporting evidence of any changes in these animals (clinical chemistry or other urinary analysis parameters).
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test item related effects in the neurological assessment as there were no observed differences in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test item treated groups.
No statistically significant or biologically relevant differences were noted during the assessment of landing foot splay test or grip strength.
In case of motor activity measurements (SMART), all dose groups of males and females had a normal locomotor activity; in all cases the initial activity was high, with generally a reduced activity in the 5-minute periods to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals and the relevant control groups when evaluating the total travelled distance (period of 0-60 minutes).
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):
TERMINAL / Parental Generation (Males: Days 28 / 42; Females: PPD14 or Day 42)
Test item-related findings (hepatocellular hypertrophy and microvesicular form of hepatocellular vacuolation) were observed. Hypertrophy was observed in males and females at Mid and High dose levels, and microvesicular vacuolation was occasionally noted only in High dose males. No degenerative/necrotic changes accompanied the hypertrophy and microvesicular vacuolation.
In the High dose group, minimal to slight hypertrophy* of the liver was described in 6/12 males and 7/11 females. In addition, microvesicular form of slight diffuse hepatocellular vacuolation** affected 2/12 males. No microvesicular vacuolation was observed in the females. In the Mid dose group, slight hypertrophy in the liver of 2/13 males and minimal to slight hypertrophy in 5/12 females was identified. There was no evidence of the test item-related microscopic findings in the liver of Low dose males and females.
*Note: Hypertrophy was characterized by enlargement of hepatocyte cytoplasm (granular) with centrilobular/periportal patterns of distribution.
**Note: Microvesicular vacuolation was diagnosed based on the presence of numerous small vacuoles within hepatocytes, without displaced nuclei.
Other changes were seen in control and/or treated animals without meaningful differences in microscopic feature, severity and incidence and were regarded as incidental or a common background.

FOUND DEAD / Parental Generation
Female #3509: Changes unrelated to treatment included moderate ulcer/erosion extended with inflammation into surrounding tissue, diffuse moderate pleuritic of accessory lung lobe with adhesion to the surrounding tissues, diffuse moderate pulmonary congestion, diffuse slight subacute epicarditis, slight congestion and decrease of size/cellularity of the thymic cortex, corresponding with changes described at necropsy. A treatment related finding, corresponding to effects seen in terminal animals, was a slight mixed periportal vacuolation of the liver.
Female #4508: Changes unrelated to treatment included multifocal slight necrotizing pleuritis, diffuse slight subacute epicarditis, moderate decrease of size/cellularity of the thymic cortex corresponding with changes described at necropsy. A treatment related finding, corresponding to effects seen in terminal animals, was a slight mixed periportal vacuolation of the liver.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis
No endocrine disruptor effect of test item was observed in the study based on the results of thyroid hormone analysis and thyroid gland weights.
Compared to the relevant control values, there were no statistically significant differences in T4 thyroid hormone concentrations for parental males and PND13 pups (more details are shown in Table 7). The absolute and relative (to body) thyroid gland weights were line with those observations.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No effect of test item on oestrus cycles was noted.
Each female selected for the treatment showed acceptable cycles (mean cycle length was in the range of 4.00-4.03 days for each group) before starting the treatment period. There was no effect on test item on the oestrus cycle of females (mean cycle length was in the 4.01-4.13 days for each group after the treatment).
Reproductive function: sperm measures:
not specified
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, fertility or gestation.
The mating indices (males and females) were in the normal range (92-100%) in all groups (one male in the Mid dose group (#3001) did not mate). The fertility index was 100% for all males which mated. The fertility index values for females were in the normal range (83-100%) in all groups. A total of four females were non-pregnant: one female in the Control group (#1507), two females in the Low dose group (#2503 and #2507) and one female in the Mid dose group (#3507), but this incidence was considered as acceptable (in line with the data of earlier studies). 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 in the test item treated group (while prolonged mating of 10 days was observed for a Mid dose female, #3505). The mean duration of mating was 2.55, 2.50, 3.82 and 2.55 days in Control, Low, Mid and High dose groups, respectively.
Evaluation of the gestation, parturition and post-partum period
No effect of treatment was noted during the gestation, parturition and post-partum period.
The mean length of gestation was 22.36, 23.00, 22.73 and 22.73 days in Control, Low, Mid and High dose groups, respectively. The individual data (in the range of 21-24 days) were in line with the normal physiological range. These data indicated no effect of test item.
As far as it could be observed during the study, the parturition was normal for almost all animals, abnormal delivery was noted for two Low dose females (#2509 and #2510), one Mid dose female (#3609) and one High dose female (#4507).
The number of implantation sites was comparable to the control mean in all dose groups, no statistically significant differences were noted.
There were no statistically significant differences or effects that could be ascribed to treatment on pre-natal, post-natal or total mortality values (litter mean and %) in any dose group.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
The ratio of female pups was slightly lower in the Low dose group and slightly higher in the Mid and High dose group than in the Control group, but as there was no statistical significance and no dose response, this fact was considered as biological variability, not related to the test item.
Based on the external evaluation, almost all the pups were normal. The few clinical signs observed are detailed below.
Absent tail was recorded for a Low dose pup (#2501/14).
Absent tail, and hyperflexion on the left hind limb was noted for a Mid dose pup (#3502/17), this pup was found dead on the day of birth.
Malrotated right forelimb and left hind limb were noted for a High dose pup (#4502/1) at birth, this animal was found dead later (on PND1).
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
There was no test item effect on mortality or survival of the pups.
There were no significant differences or effects that could be ascribed to treatment on the pre-natal, post-natal or total mortality values (litter mean and %) in any of the dose groups.
Slightly higher number of autolyzed pups were noted in the Mid dose group, but all those 12 pups belonged to one dam with difficult delivery (#3609*). There was no dose response, and this fact was considered as animal variability not related to the treatment.
*Note: The two living pups of this litter was found dead by the next day (PND1).
The number of viable pups on PND0, 4 and 13 as well as the survival index of the pups at given time points were comparable to control value in each dose group, thus there was no treatment-related effects on the viability of pups at those time points.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no test item related differences in the offspring body weights or weight gains in any test item treated group when compared to the controls.
When evaluated per litter basis, the mean litter body weights on PND 0, PND 4 and PND 13 and body weight gain in the relevant periods showed no toxicologically significant differences compared to controls in the F1 generation. Occasionally, slightly higher values were observed in the Low, Mid and/or High dose groups, but without statistical significance and without dose response, thus those values were considered as not being test item related effect. In summary, there were no effects of treatment on pup weights or weight gains.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
TERMINAL / F1 Generation (PND13)
No test item-related macroscopic findings were recorded in F1 offspring generation euthanized and examined externally at scheduled termination on PND13.

FOUND DEAD / F1 Generation
No test item-related macroscopic findings were seen in unscheduled deaths of F1 pups.
There were stillborn pups on PND 0 including 5 Control, 9 Low dose, 1 Mid dose and 5 High dose pups. Cannibalization of 1 Control, 5 Low dose and 2 High dose pups was observed at necropsy. There were 2, 12 and 3 autolysed pups from the Low, Mid and High dose groups.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Anogenital distance, nipple retention
No test item effect was observed on anogenital distance or nipple retention during the study.
No statistically significant changes in the anogenital distance measured on PND 0 were noted for test item treated male and female pups when compared to control.
There were no effects on the nipple retention: no nipples/areolae were counted for any male pups on PND 13.
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Summary of reproductive parameters (males)

Parameters

Dose groups

Control

Low dose

Mid dose

High dose

Number of treated animals

12

12

13

12

Number of pre-terminal death

0

0

0

0

Number of males used for mating

12

12

12

12

Number of successful mating

12

12

11§

12

Number of fertile males

12

12

11

12

Male mating index (%)

100

100

92

100

Male fertility index (%)

100

100

100

100

Notes: One animal (#3009) was not used for mating due to the death of its mating pair (#3509).

§: Mating of one Mid dose male (#3001) was unsuccessful, this animal was included in the mating evaluation, but excluded from the fertility evaluation. It was replaced by #3109 later, this male produced successful mating with #3501 female.

 

Summary of reproductive parameters (females)

Parameters

Dose groups

Control

Low dose

Mid dose

High dose

Number of treated animals

12

12

13

12

Number of pre-terminal death

0

0

1

0

Number of females used for mating

12

12

12

12

Number of sperm-positive females

12

12

12

12

Number of females with no implantation sites

1

2

1

0

Number of pregnant females

11

10

11

11

Pregnant females with live born(s)

11

10

11

11

Pregnant females not delivered living pups

0

0

0

0

Female mating index (%)

100

100

100§

100

Female fertility index (%)

92

83

92

100§§

Female gestation index (%)

100

100

100

100§§

Notes: One Mid dose female (#3509) was found dead in the pre-mating period, but was replaced by another female (#3609) for mating. Four females (#1507, #2503, #2507 and #3507) were not pregnant.

§: Female #3501 was unsuccessfully mated with male #3001 for 14 days. Later the male mating pair was switched to #3109 and a successful mating was observed within 6 days. Parameters from the second mating are included in the table.

§§: One High dose female (#4508) was found dead soon after showing a sperm-positive smear (it was used for mating index calculation, but excluded from fertility and gestation index calculation).

 

Summary of the pregnancy evaluation

Parameters

Dose groups

 

Control

Low

Mid

High

 

Number of evaluated females

11

10

11

11

 

Number of implantations, mean

15.00

14.60

16.18

15.00

NS

Pre-natal mortality, mean

2.27

2.80

1.91

2.64

NS

Pre-natal mortality (%), mean

19.46

20.00

12.13

16.84

NS

Number of pups born, mean

13.27

13.40

15.45

13.27

NS

Number of live born pups, mean

12.73

11.80

14.27

12.36

NS

Number of living pups on PND13, mean

10.18

9.70

11.64

9.27

NS

Post-natal mortality, mean

0.91

0.70

0.82

1.27

NS

Post-natal mortality (%), mean

7.21

7.90

13.16

13.11

NS

Total mortality, mean

3.18

3.50

2.73

3.91

NS

Total mortality (%), mean

24.99

24.94

17.18

26.73

NS

Duration of pregnancy (days)

22.36

23.00

22.73

22.73

NS

Notes: Data (group mean values) were rounded to two decimal places. Pre-natal mortality was also called “intrauterine mortality” in the Study Plan. Post-natal mortality PND0-13 and total mortality on PND13 are shown in the table. Four females (#1507, #2503, #2507 and #3507) had no implantation sites. they were excluded from calculation.

NS: Statistically not significant compared to control

 

Summary of survival (offspring)

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated litters

11

10

11

11

 

Number of implantations, mean

15.00

14.60

16.18

15.00

NS

Number of pups born, mean

13.27

13.40

15.45

13.27

NS

Number of live born pups, mean

12.73

11.80

14.27

12.36

NS

Post-natal mortality on PND0-4, mean

0.82

0.60

0.64

1.27

NS

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

6.64

7.34

12.06

13.11

NS

Total mortality on PND4, mean

3.09

3.40

2.55

3.91

NS

Total mortality on PND4 (%), mean

24.46

24.39

16.17

26.73

NS

Post-natal mortality on PND0-13, mean

0.91

0.70

0.82

1.27

NS

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

7.21

7.90

13.16

13.11

NS

Total mortality on PND13, mean

3.18

3.50

2.73

3.91

NS

Total mortality on PND13 (%), mean

24.99

24.94

17.18

26.73

NS

Survival index on PND0

96.43

87.34

91.14

90.62

NS

Sex ratio (%) on PND0

47.09

46.56

50.22

54.65

NS

Survival index on PND4

93.36

92.66

88.48

87.50

NS

Sex ratio (%) on PND4

48.23

46.14

50.31

54.09

NS

Survival index on PND13

99.35

99.33

98.56

100.00

NS

Sex ratio (%) on PND13

47.80

45.56

49.44

51.96

NS

Notes: Data (group mean values) were rounded to two decimal places Sex ratio means shows the ratio of females. Survival index was calculated in comparison with the end of previous period (on PND0 was compared to the number of pups born, on PND4 it was compared to the number of live born pups, on PND13 it was compared to the number of pups after culling on PND4).

NS: Statistically not significant compared to control

 

Summary of mortality (offspring)

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated litters

11

10

11

11

 

Number of pups born

146

134

170*

146

CH

Number of cannibalized pups

1

5

0

2

NS

Number of autolyzed pups

0

2

12**

3

CH

Number of stillborn pups

5

9

1

5

NS

Number of live born pups

140

118*

157

136

CH

Number of found dead pups (born alive)

0

0

1

1

NS

Number of living pups on PND0

140

118

156

135

NS

Number of cannibalized pups (PND0-13)

9

6

7

11

NA

Number of autolyzed pups (PND0-13)

1

1

1

2

NA

Number of found dead, intact pups (PND0-13)

0

0

0

0

NA

Total number of pups died (born alive)

10

7

9

14

NS

Culled for blood sampling on PND4

18

14

20

20

NS

Number of viable pups on PND13

112

97

128

102

NS

Notes: Mortality numbers mean number of pups / number of affected litters.

PND0-13 means the lactation period, counted from the end of working day on PND0.

There were no living pups in litter #3609 by PND1.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

NA: Not applicable, CH: Chi square test, NS: Statistically not significant compared to control

 

Body weight data (offspring)

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Number of evaluated litters

11

10

11

11

 

Number of viable pups (PND0)

140

118

156

135

NS

Mean litter body weight (PND0), g

6.633

6.617

6.649

6.615

NS

Number of viable pups (PND4)

131

112

150

122

NS

Mean litter body weight (PND4), g

10.064

11.058

10.364

10.608

NS

Mean litter body weight gain (PND0-4), g

3.400

4.419

3.605

3.938

NS

Number of viable pups (PND13)

112

97

128

102

NS

Mean litter body weight (PND13), g

26.922

30.633

28.299

29.204

NS

Mean litter body weight gain (PND4-13), g

16.850

19.548

17.890

18.531

NS

Mean litter body weight gain (PND0-13), g

20.251

23.994

21.527

22.491

NS

Notes: Body weight / body weight gain data (litter mean values) were rounded to three decimal places. All living pups of a Mid dose female (#3609) died by PND1, therefore PND4 and PND13 results for Mid dose group were calculated from 10 litters.

NS: Statistically not significant

 

Anogenital distance

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male pups

 

Number of evaluated male pups

67

64

78

63

NS

Anogenital distance, litter mean of males (mm)

3.35

3.44

3.41

3.38

NS

Minimum / Maximum value, litter mean (mm)

2.26 / 4.41

2.52 / 4.79

2.24 / 4.34

2.35 / 4.24

 

Female pups

 

Number of evaluated female pups

73

54

78

72

NS

Anogenital distance, litter mean of females (mm)

1.63

1.63

1.68

1.61

NS

Minimum / Maximum value, litter mean (mm)

1.10 / 2.30

1.07 / 2.33

1.22 / 2.34

1.12 / 2.40

 

Notes: Data (group mean or litter mean values) were rounded to two decimal places.

NS: Statistically not significant compared to control

Conclusions:
In summary, daily administration of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] test item by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) during the treatment period of this study did not result in test item related mortality, clinical signs, or significant changes in body weight / body weight gain, food consumption, haematology, coagulation, clinical chemistry or urinalysis parameters.

No test item related effect was detected during neurotoxicity assessment.
No test item effect on oestrus cycle of parental females were noted.
No test item related changes were noted in the reproductive parameters, gestation, parturition and lactation.
There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding were recorded for F1 pups at necropsy.
No test item-related macroscopic findings were recorded in any of the dose groups at necropsy. Increased liver weights were observed in all treated groups, associated with test item-related microscopic findings (centrilobular/periportal hepatocellular hypertrophy) in the Mid and High dose groups (males and females), the intensity ranged from minimal to slight. Hypertrophy was considered to be an adaptive, non-adverse change. A slight microvesicular vacuolation in the liver seen at a low incidence in High dose males was regarded as a non-adverse finding. There were no other organ weight or histopathological findings related to treatment.
No endocrine disruptor effect of test item was observed in the study.

In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was considered to be 1000 mg/kg bw/day for the female and male parental (adult) generation, and also for the F1 generation (pups).
Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rats was to obtain information on the toxicity of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] test item following repeated daily administration by oral gavage to Wistar rats. The study included a reproductive / developmental toxicity-screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to post-natal day (PND) 13.

 

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating / post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13. The Experimental design was as follows:

 

Gr. No.

Group designation

Dose level
(mg/kg bw/day)

Concentration (mg/mL)

Dose volume

(mL/kg bw)

Animal numbers

Male

Female

1

Control

0

0

5

1001-1012

1501-1512

2

Low dose

100

20

2001-2012

2501-2512

3

Mid dose

300

60

3001-3012, 3109

3501-3512, 3609

4

High dose

1000

200

4001-4012

4501-4512

 

Parameters measured during the study included signs of morbidity and mortality twice daily, daily or weekly detailed observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment of functional observation battery (including the measurements of the landing foot splay and grip strength) and measurement of motor activity was performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND 13.

At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues / organs were sampled and preserved in appropriate fixatives from the adult animals and offspring. The thyroxine (T4) levels in the adult males and PND 13 pups were also assessed. For the adult animals, a detailed histological examination was performed on the selected list of retained organs of 5 Control and High dose animals per sex as well as reproduction organs of all animals in the Low and Mid dose groups which failed to sire / conceive. Additionally, livers were examined histologically on all High, Mid dose and Low dose animals.

 

All test item formulations were within the range of 95-109% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.

 

In summary, daily administration of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] test item by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) during the treatment period of this study did not result in test item related mortality, clinical signs, or significant changes in body weight / body weight gain, food consumption, haematology, coagulation, clinical chemistry or urinalysis parameters.

 

No test item related effect was detected during neurotoxicity assessment.

 

No test item effect on oestrus cycle of parental females were noted.

 

No test item related changes were noted in the reproductive parameters, gestation, parturition and lactation.

 

There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding were recorded for F1 pups at necropsy.

No test item-related macroscopic findings were recorded in any of the dose groups at necropsy. Increased liver weights were observed in all treated groups, associated with test item-related microscopic findings (centrilobular/periportal hepatocellular hypertrophy) in the Mid and High dose groups (males and females), the intensity ranged from minimal to slight. Hypertrophy was considered to be an adaptive, non-adverse change. A slight microvesicular vacuolation in the liver seen at a low incidence in High dose males was regarded as a non-adverse finding. There were no other organ weight or histopathological findings related to treatment.

 

No endocrine disruptor effect of test item was observed in the study.

 

In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was considered to be 1000 mg/kg bw/day for the female and male parental (adult) generation, and also for the F1 generation (pups).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals No. 422, Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, Organisation for Economic Co-Operation and Development, Paris, 29 July 2016
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
EC Number:
201-044-5
EC Name:
2,2'-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
Cas Number:
77-62-3
Molecular formula:
C29H40O2
IUPAC Name:
2-{[2-hydroxy-5-methyl-3-(1-methylcyclohexyl)phenyl]methyl}-4-methyl-6-(1-methylcyclohexyl)phenol
Test material form:
solid: particulate/powder
Details on test material:
Name: 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol]
CAS Number: 77-62-3
Batch/Lot number: WWP4J0001
Appearance: White to cream powder
Purity: Considered as 100% (>95%)
Manufacture date: 04 September 2014
Expiry date: 03 September 2018
Storage conditions: Controlled room temperature (15-25 °C, below 70 RH%)
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species and strain: Crl:WI (Wistar) rats
Source: Charles River Laboratories, Research Models and Services, Germany GmbH (Address: Sandhofer Weg 7, Sulzfeld, D-97633, Germany), from SPF colony.
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. The same strain was used for the Dose Range Finding study.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Housing conditions: Standard laboratory conditions
Number of animals: 49 male and 49 female rats used for treatment, at least 12 animals/sex/group, 4 groups. Additional females were also used in the pre-exposure period for oestrus cycle determination (those animals besides the spare animals were moved back to the spare colony of the Test Facility after the study). Animals originated from different units, to avoid brother/sister mating.
Age of animals: Young adult rats, approximately 10 weeks old at start and 12 weeks old at mating.
Body weight range: Males: 425-505 g, females: 223 – 288 g at the start of the treatment (did not exceed ± 20 % of the mean weight for each sex).
Acclimation period: 29 days

Husbandry
Animal health: Only healthy animals were used for the test, as certified by the clinical Veterinarian. Females were nulliparous and non-pregnant.
Room number: 243 (in-life phase), 505 (acclimatisation period)
Cage type: Type II polycarbonate
Bedding & nesting: LIGNOCEL® ¾ S certified wooden chips (Batch number: 03018170329 / 03018170529, Expiry date: 29 March 2020 / 29 May 2020) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, Rosenberg D-73494, Germany) were used in the study. ARBOCEL® nest building material (Batch number: 05072160415 / 05072170228, Expiry date: 15 April 2019 / 28 February 2020) produced by J. Rettenmaier & Söhne GmbH+Co.KG (Address: Holzmühle 1, Rosenberg D-73494, Germany).
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 20.0 – 28.7 °C (target range 22 ± 3 °C)
Relative humidity: 29 – 74 % (target range 30-70 %)
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 2 animals of the same sex and dose group/cage with the exception of the mating and gestation/delivery period when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).

Environmental parameters (temperature and relative humidity) were continuously measured, minimum and maximum values were recorded twice a day during the study

Food and water supply
Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (Batch number: 285 17890 / 262 21592, Expiry date: 31 August 2017 / 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.
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).

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.
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*.
*Note: Due to the incidental mortality of a Mid dose female (#3509), a replacement animal (#3609) was included in the study. In order to provide a mating pair with the same length of treatment before mating, a replacement male (#3109) was also included in the study.
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.

Randomisation
All adult/parental (P) male and female animals were sorted according to body weight by computer and divided into weight ranges before the pre-exposure period
(Day -14). 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.
The body weight ranges were additionally checked before the first exposure (Day 0) using Microsoft Excel to ensure that the individual values did not exceed ± 20% of the mean weight for each sex at the start of the treatment.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as it is one of the possible routes of human exposure.
Vehicle:
corn oil
Details on oral exposure:
The test item was formulated in the vehicle (as a visibly stable homogenous suspension) in the Pharmacy of CiToxLAB Hungary Ltd. Formulations were prepared daily at appropriate concentrations in the vehicle in the Pharmacy of CiToxLAB Hungary Ltd.

Formulations were prepared daily (on the day of treatment or on the previous afternoon) in the Pharmacy of CiToxLAB Hungary Ltd. as follows. The calculated volume of the vehicle was added into a beaker containing the calculated amount of test item, it was mixed vigorously by a magnetic stirrer to make a homogenous formulation. Based on the analytical method validation study (CiToxLAB Hungary Study code: 17/039-316AN), formulations in the 20 - 200 mg/mL concentration range were found to be stable for 7 days of storage at room temperature.

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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of test item formulations for concentration and/or homogeneity was performed using a validated HPLC-UV (High Pressure Liquid Chromatography – Ultraviolet spectroscopy) method in the Analytical Laboratory of CiToxLAB Hungary Ltd. Duplicate samples were taken from the top, middle and bottom of the test item formulations four times during the study (during the first and last treatment weeks of the study and approximately midway during the treatment period), 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.
Duration of treatment / exposure:
Dosing of both sexes began after 29 days of acclimatisation and 14 days of pre-exposure period, and it was performed for 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.
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 post–partum day (PPD) 0. Females showing no-evidence of copulation were sacrificed as practical, 25-28 days after the day of presumed mating.)
All F1 offspring were terminated on post-natal day (PND) 13 (F1 offspring selected for blood sampling on PND4 were terminated on that day). In order to allow for overnight fasting of dams prior to urine collection on PPD14, the offspring were euthanized on PPD/PND13 and the dams on PPD/PND14.
Frequency of treatment:
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.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
22 animals per dosing group (11 male/11 female)
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose selection and route of administration
The dose levels were selected by the Sponsor in consultation with the Study Director based on available data including the results of an acute oral toxicity study in rats
(LD50 >2000 mg/kg bw for male and female rats in an OECD No. 401 study, data on file at the Sponsor) and a 14-day repeated dose Dose Range Finding (DRF) study in the rat performed at the Test Facility (CiToxLAB Hungary Study code: 17/039-220PE [3]). In the DRF study, there was no toxicity at 1000 mg/kg bw/day. The aim of this study was to use a maximum of 1000 mg/kg bw/day 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 these preliminary studies, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study
Positive control:
Not specified.

Examinations

Observations and examinations performed and frequency:
Clinical observations
Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and the end of the working day. The principles and criteria summarized in the OECD Humane Endpoints Guidance Document No. 19 were taken into consideration. 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.
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.
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.

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.
Parental females were weighed on gestation days (GD) 0, 3, 7, 10, 14, 17 and 20 and on post-partum days (PPD) 0 (within 24 hours after parturition), 4, 7, 10, 13 and 14 (before termination). The body weight of the parental female animals measured on GD3, 10 and 17 as well as PPD7 and PPD10 were only additional measurements as aid for the calculation of accurate treatment volumes, but these data were not evaluated statistically.

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).

Functional observation battery (FOB) and SMART
Assessment of potential test item related neurotoxicity was performed during the last exposure week (males on Day 26; females on PPD8-9). 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, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or 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.

CLINICAL PATHOLOGY
All animals selected for blood sampling were fasted (overnight period of food deprivation, after the litter had been culled). Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.
For terminal blood sampling in all selected animals (5 males and 5 females/group), 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
The following parameters were evaluated for selected animals:
RBC (Red Blood Cell [erythrocyte]); WBC (White Blood Cell [leukocyte]); Hgb (Haemoglobin concentration); Hct (Haematocrit [relative volume of erythrocytes]); MCV (Mean Corpuscular [erythrocyte] Volume); MCH (Mean Corpuscular [erythrocyte] Haemoglobin); MCHC (Mean Corpuscular [erythrocyte] Haemoglobin Concentration); RDW (Red Cell [erythrocyte] Volume); Plt (Platelet [thrombocyte] Count); MPV (Mean Platelet Thrombocyte Volume); RETIC % (Reticulocyte count); NE% (Neutrophil); LY% (Lymphocyte); MO% (Monocyte); BA% (Basophil); EO% (Eosinophil); LUC% (Large Unstained Cells); APTT (Activated Partial Thromboplastin Time); PT (Prothrombin Time).

Clinical chemistry
The following parameters were evaluated for selected animals:
Glucose (Blood sugar concentration); T_BIL (Total Nilirubin concentration); Urea (Urea concentration); Chol. (Cholesterol concentration); Creat. (Creatinine concentration); Phos. (Phosphorus concentration); Na+ (Sodium concentration); K+ (Potassium concentration); Ca++ (Calcium concentration); Cl- (Chloride concentration); Tot. Prot. (Total Protein concentration); Alb. (Albumin concentration); A/G (Alb/glob ration); AST/GOT (Aspartate Aminotransferase activity); ALT/GPT (Alanine Aminotransferase activity); GGT (Gamma-Glutamyl tranferase activity); ALKP (Alkaline Phosphatase activity); Bile acids.

Urinalysis
Urine sampling was performed prior to necropsy by placing the selected animals in metabolic cages for approximately 16 hours. The evaluation of the urine samples was performed as indicated below:
LEU (Leukocyte); NOT (Nitrite); pH; PRO (Protein); GLU (Glucose); UBG (Urobilinogen); BIL (Bilirubin); KET (Ketones); BLD/ERY (Blood/Erythrocytes); SG (Specific Gravity); SED (Sediment); VOL (Volume); Colour / Appearance.
Sacrifice and pathology:
PATHOLOGY
Terminal procedures and macroscopic evaluation
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.
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.
Organ weight measurements
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.

Tissue preservation and microscopic evaluation
The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerves as well as testes with epididymides were retained in modified Davidson’s fixative, all other organs in 10% buffered formalin solution.

In addition, on completion of the macroscopic examination the following tissues and organs were retained from all animals:
Gross findings; Adrenal gland; Animal identification; Aorta; Brain; Epididymis; Eye with optic nerve; Oesophagus; Femur with marrow; Heart; Kidney; Large intestine; Extraorbital lachrymal gland; Harderian gland; Liver; Lungs with bronchi; Lymph node; Ovary; Oviduct; Pancreas; Pituitary; Prostate; Salivary gland (including mandibular, sublingual and parotid glands); Sciatic nerve; Seminal vesicle with coagulating gland; Skin, subcutis with mammary gland (inguinal); Skeletal muscle; Small intestine; Spinal cord; Spleen; Sternum with marrow; Stomach; Testis; Thymus; Thyroid with parathyroid gland; Tongue; Trachea; Urinary bladder; Uterus; Vagina;

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 selected list of retained organs in the Control and High dose groups (selected 5 animals/sex/group). Based on the histopathology findings (centrilobular hepatocellular hypertrophy) observed in the liver of the examined High dose group animals, slides of the liver samples of all Mid dose and Low dose animals, furthermore the rest of High dose group animals were processed and examined microscopically as agreed by the Sponsor.
-on the selected list of retained organs in one Mid dose female (#3509) and in one High dose female (#4508) which were found dead,
-the spleen of one Low dose male (#2012), the testes and epididymis of one Mid dose male (#3001), and the spleen of one Mid dose male (#3007) because macroscopic findings (abnormalities) were seen at necropsy,
-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 (#2003, #2007, #3007) and all females (#2503, #2507 and #3507) that failed to sire / conceive.

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.

Special attention was paid to the organ weight, appearance and histopathology of immune-system tissues for any evidence of immunotoxicity (spleen, thymus, lymph nodes and bone marrow). Special attention was paid to the central and peripheral nervous system tissues for any evidence of neurotoxicity.
Other examinations:
Clinical observations
On gestation day (GD) 13 and/or 14 the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat).

Oestrus cycle monitoring
Oestrus cycles were monitored by vaginal smears daily during the pre-exposure period before the treatments starts. Vaginal smears were also checked daily from the beginning of the treatment period until evidence of mating.

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 0.01 g. All litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized or autolyzed) 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 all 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 surviving pups were terminated on PND13.

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 at termination (PPD14) and up to two pups per litter on PND13,
-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 aliquots, if possible; any leftover material was also retained for safety reason) and stored in an ultrafreezer (-80 ± 10 °C) until shipping for analysis.
Samples (first aliquots) were shipped for hormone analysis on dry ice to the Principal Investigator (PI):
Joachim Decorde
CiToxLAB France
RN13 Route de Pacy
27930 Miserey, France
Samples of adult males and PND13 pups were assessed for T4 levels. The measurement of the T4 hormone levels in adult females and PND4 pups, or measurement of other thyroid hormone (TSH) were not performed as was not deemed necessary by the Study Director and Sponsor.
Statistics:
See "Any other information" for full details.

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.
Thin fur was recorded for two Mid dose males (#3011 on Days 11-26, and #3012 on Days 24-28) and for one Control female (#1501 on Days 29-50) and one Mid dose female (#3506 on Days 38-51). These minor observations were not related to the test item treatment.
In one Mid dose female (#3609) paleness was observed on forelimbs, hind limbs and both pinna on Days 39-41, piloerection and slight discharge from vagina was also recorded on Day 39. However, these findings were related to a difficult delivery process (delivery occurred on Day 40), not related to the test item administration.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test item related mortality was observed in the study.
A Mid dose female (#3509) was found dead on Day 13 without any clinical symptoms. The cause of death, moderate erosion/ulcer of the oesophagus, moderate pleuritis and slight epicarditis recorded in this animal were indicated as consequences of previous technical gavage error.
A High dose female (#4508) was found dead on Day 18 after showing hunched back and slightly laboured respiration and piloerection in the previous two days. No test item-related changes were seen in this animal. The cause of death was identified by the pathologist as a possible gavage accident.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test item effect on body weight or body weight gain was detected during the study.
There were no statistically significant body weight or body weight gain values in the test item treated groups of either sex when compared to the control at any occasion that could be ascribed to the test item. The measured values were within the range commonly recorded for this strain and age
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
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.
Occasional statistically significant differences were regarded as incidental and of no toxicological significance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
When compared to the controls, there were no differences in males and females that could be considered toxicologically significant in the test item treated groups.
The relative amount of large unclassified cells was significantly lower (p<0.05) in Mid and High dose males when compared to control. But as all the individual values were within the historical control range, there was no clear dose response and no significant differences were noted in females, these differences were considered as animal variability, not being a test item related effect.
Significantly lower (p<0.01 or p<0.05) mean cell haemoglobin concentration was recorded for Mid and High dose females when compared to control. As the difference was small (less than 5%), the observed values were within the historical control range, there was no dose response and no similar trend in males, these differences were considered to not reflect any effect of the test item.
No statistically significant changes were recorded for any other haematology or coagulation parameters in the test item treated males and females.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no significant changes or adverse effects on the serum chemistry that could be ascribed to the test item administration.
Statistically significant increase (p<0.05) in total protein concentration was were seen in Low and High dose males when compared to the control. However, there was no dose response, and the observed values were within the historical control range. No significant change was seen in females; thus this difference was considered as not being related to test item treatment.
No statistically significant changes were recorded for any other parameters in the test item treated males and females.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No test item related changes were recorded in any of the test item treated groups when compared to the control.
Significantly lower urine volume was collected from the Low (p<0.05) and Mid (p<0.01) dose males when compared to control. The collected urine volume is usually highly variable as indicated by the historical control data. All the individual values obtained in this study were considered to be normal, there was no dose response and no similar trend was seen in females, therefore this numerical difference was not considered to reflect a test item related effect. This fact was confirmed by the lack of any supporting evidence of any changes in these animals (clinical chemistry or other urinary analysis parameters).
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test item related effects in the neurological assessment as there were no observed differences in animal behaviour, general physical condition or in the reactions to different types of stimuli in the control or test item treated groups.
No statistically significant or biologically relevant differences were noted during the assessment of landing foot splay test or grip strength.
In case of motor activity measurements (SMART), all dose groups of males and females had a normal locomotor activity; in all cases the initial activity was high, with generally a reduced activity in the 5-minute periods to an approximate plateau by about 20-30 minutes. There was no statistical significance between the test item treated animals and the relevant control groups when evaluating the total travelled distance (period of 0-60 minutes).
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Test item related increased liver weights were observed in the Mid and High dose groups (males and females); these finding were in line with the observed microscopic changes of the affected animals, and were considered as not being an adverse effect.
Terminal body weights of test item treated animals (males and females) were not significantly different from control animals.
The liver weights were statistically significantly higher than control in Low, Mid and High dose groups of both sexes; the relatively small liver weight increase in the Low dose was not consistently statistically significant in absolute and adjusted weights in both sexes. These changes were considered to be an effect of the treatment with the test item, corresponding with hepatic hypertrophy observed in Mid and High dose animals; the relatively small increase in the Low dose would not be expected to be detectable by histopathology.
In the males, absolute liver weights were larger by 10.1% (p<0.05), 16.1% (p<0.01) and 19.9% (p<0.01) in the Low, Mid and High dose rats, respectively. Similar differences were seen when adjusted for body and brain weight, although the Low dose was not consistently statistically significant. In the females, absolute liver weights were larger by 19% (not statistically significant), 31.0% (p<0.01) and 29.4% (p<0.01) in the Low, Mid and High dose animals, respectively. Similar differences were seen when adjusted for body and brain weight, although the Low dose was sometimes statistically significant.
There was a statistically significance in female thyroid weights of the High dose group, but only at p<0.05 for absolute and brain adjusted, and not significant when body weight adjusted. There were no statistically significant differences in male thyroid weights between test item treated and control groups. The thyroid hormone levels and histopathology show no treatment related effects in this organ (all organs were normal). Additionally, the female control range (14-40 mg) was comparable with the and the range of the High dose females (15-32 mg), the maximum value of the High dose group was even lower than the current study control maximum. Taking into account all the available information, it was concluded that the statistical difference in High dose female thyroid weight was not an adverse effect of treatment.
There were no other treatment-related statistically significant differences among groups in the weights of organs measured when compared to controls
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
TERMINAL / Parental Generation (Males: Days 28 / 42; Females: PPD14 or Day 42)
No test item-related macroscopic findings were noted at necropsy.
The following incidental or background findings were seen in the terminally euthanized animals: many pale foci (bilateral) in the adrenal glands (#2012), enlarged spleen (#2012 and #3007), small testis and small epididymis (#3001), thin fur (#3012), dilatation of cervical body and horn with clear fluid (#4504), and dark red spots on the glandular mucosa of the stomach (#4506).

FOUND DEAD / Parental Generation
Two females were found dead during the study, unrelated to test item treatment. A Mid dose female (#3509) was found dead on Day 13, with some cannibalisation. The cause of death, moderate erosion/ulcer of the oesophagus, moderate pleuritis and slight epicarditis recorded in this animal were indicated as consequences of previous technical gavage error. A High dose female (#4508) was found dead probably due to misgavage on Day 18 followed by slight pleuritis and slight epicarditis. No test item-related changes were seen in these animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
TERMINAL / Parental Generation (Males: Days 28 / 42; Females: PPD14 or Day 42)
Test item-related findings (hepatocellular hypertrophy and microvesicular form of hepatocellular vacuolation) were observed. Hypertrophy was observed in males and females at Mid and High dose levels, and microvesicular vacuolation was occasionally noted only in High dose males. No degenerative/necrotic changes accompanied the hypertrophy and microvesicular vacuolation.
In the High dose group, minimal to slight hypertrophy* of the liver was described in 6/12 males and 7/11 females. In addition, microvesicular form of slight diffuse hepatocellular vacuolation** affected 2/12 males. No microvesicular vacuolation was observed in the females. In the Mid dose group, slight hypertrophy in the liver of 2/13 males and minimal to slight hypertrophy in 5/12 females was identified. There was no evidence of the test item-related microscopic findings in the liver of Low dose males and females.
*Note: Hypertrophy was characterized by enlargement of hepatocyte cytoplasm (granular) with centrilobular/periportal patterns of distribution.
**Note: Microvesicular vacuolation was diagnosed based on the presence of numerous small vacuoles within hepatocytes, without displaced nuclei.
Other changes were seen in control and/or treated animals without meaningful differences in microscopic feature, severity and incidence and were regarded as incidental or a common background.

FOUND DEAD / Parental Generation
Female #3509: Changes unrelated to treatment included moderate ulcer/erosion extended with inflammation into surrounding tissue, diffuse moderate pleuritic of accessory lung lobe with adhesion to the surrounding tissues, diffuse moderate pulmonary congestion, diffuse slight subacute epicarditis, slight congestion and decrease of size/cellularity of the thymic cortex, corresponding with changes described at necropsy. A treatment related finding, corresponding to effects seen in terminal animals, was a slight mixed periportal vacuolation of the liver.
Female #4508: Changes unrelated to treatment included multifocal slight necrotizing pleuritis, diffuse slight subacute epicarditis, moderate decrease of size/cellularity of the thymic cortex corresponding with changes described at necropsy. A treatment related finding, corresponding to effects seen in terminal animals, was a slight mixed periportal vacuolation of the liver.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone analysis
No endocrine disruptor effect of test item was observed in the study based on the results of thyroid hormone analysis and thyroid gland weights.
Compared to the relevant control values, there were no statistically significant differences in T4 thyroid hormone concentrations for parental males and PND13 pups. The absolute and relative (to body) thyroid gland weights were line with those observations.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Selected body weight parameters of parental animals

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Male, Body weight on Day 27 (g)

523.3

522.0

528.7

527.8

NS

difference (%)

-0.2

1.0

0.9

 

Male, Body weight gain Day 0-27 (g)

57.9

56.9

64.8

61.7

NS

difference (%)

-1.7

11.8

6.5

 

Female, Body weight on GD20 (g)

413.0

422.6

447.3

443.2

NS

difference (%)

2.3

8.3

7.3

 

Female, Body weight on PPD13 (g)

367.1

382.2

385.5

383.5

NS

difference (%)

4.1

5.0

4.5

 

Female, Body weight gain Day 0- PPD13 (g)

107.0

124.6

128.2

124.3

NS

difference (%)

16.4

19.8

16.1

 

Notes: Data (group mean values, n=10-13) were rounded to one decimal place.

NS: Statistically not significant when compared to control

 

Summary of selected FOB and SMART parameters

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males, Landing foot splay, mm (hind paws)

84.5

104.0

94.9

92.0

NS

Males, Grip-strength, g (forelimbs)

1873

2011

1752

2000

NS

Males, Grip-strength, g (hind limbs)

772

794

825

790

NS

Males, Total travelled distance (cm)

6824

7527

8391

7640

NS

Females, Landing foot splay, mm (hind paws)

85.5

82.8

98.9

80.9

NS

Females, Grip-strength, g (forelimbs)

1313

1414

1518

1522

NS

Females, Grip-strength, g (hind limbs)

6825

570

628

673

NS

Females, Total travelled distance (cm)

5869

6849

6385

7188

NS

Notes: Data (group mean values, n=5) are rounded to one digit or to whole number.

Total travelled distance of 0-60 minutes was calculated.

NS: Statistically not significant when compared to the control.

 

Summary of selected haematology parameters

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

 

Relative amount of LUC (%)

0.88

0.50

0.26*

0.26*

DN

HC range: 0.00-5.00

difference (%)

-43.2

-70.5

-70.5

 

MCHC (g/dL)

34.56

34.20

34.34

33.32

NS

HC range: 31.90-36.80

difference (%)

-1.0

-0.6

-3.6

 

Females

 

Relative amount of LUC (%)

0.42

0.34

0.38

0.26

NS

HC range: 0.00-2.30

difference (%)

-19.0

-9.5

-38.1

 

MCHC (g/dL)

34.10

33.48

32.92**

33.18*

DN

HC range: 30.30-36.40

difference (%)

-1.8

-3.5

-2.7

 

Notes: Data (group mean values, n=5) were rounded to two decimal places.

LUC: Large Unclassified Cells, MCHC: Mean cell haemoglobin concentration

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s Multiple Range Test, NS: Statistically not significant compared to control

 

Summary of selected clinical chemistry parameters

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

 

Total protein (g/L)

56.98

60.14*

56.78

60.82*

DN

HC range: 48.10-63.20

difference (%)

5.5

-0.4

6.7

 

Females

 

Total protein (g/L)

55.34

57.92

59.20

58.40

NS

HC range: 49.50-68.70

difference (%)

4.7

7.0

5.5

 

Notes: Data (group mean values, n=5) were rounded to two decimal places.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s Multiple Range Test, NS: Statistically not significant compared to control

 

Summary of selected urinary analysis parameters

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Males

 

Volume (mL)

12.10

7.20*

6.20**

9.60

DN

HC range: 1.0-51.0

difference (%)

-40.5

-48.8

-20.7

 

Females

 

Volume (mL)

9.80

10.50

9.30

11.80

NS

HC range: 0.8-32.0

difference (%)

7.1

-5.1

20.4

 

Notes: Data (group mean values, n=5) were rounded to two decimal places.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s Multiple Range Test, NS: Statistically not significant compared to control

 

Selected parameters related to thyroid hormone levels

Parameters

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Parental males

 

Number of evaluated males

12

12

13

12

 

T4 concentration (ng/mL)

42.02

41.04

39.55

39.01

NS

Thyroid gland weights (g)

0.0258

0.0269

0.0281

0.0305

NS

Thyroid gland / body weight (%)

0.0051

0.0053

0.0055

0.0060

NS

PND13 pups

 

Number of evaluated litters

10

10

10

10

 

T4 concentration (ng/mL)

47.35

44.75

47.77

52.23

NS

Thyroid gland weights (g)

0.0069

0.0067

0.0067

0.0069

NS

Thyroid gland / body weight (%)

2.6933

2.2306

2.3682

2.3601

NS

Notes: Data (group mean values) were rounded to two or four decimal places. Thyroid and parathyroid weights were measured together.

T4: thyroxin

NS: Statistically not significant compared to control

 

Organ weight data (males)

Organ weight

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Terminal body weight, g

505.8

507.2

508.5

509.2

NS

(difference %)

0.3

0.6

0.7

 

Liver (absolute), g

14.056

15.474*

16.325**

16.856**

DN

(difference %)

10.1

16.1

19.9

 

Liver (relative to body), %

2.779

3.048**

3.212**

3.314**

DN

(difference %)

9.7

15.6

19.3

 

Liver (relative to brain), %

641.15

691.03

737.91**

771.33**

DN

(difference %)

7.8

15.1

20.3

 

Thyroid (absolute), g

0.0258

0.0269

0.0281

0.0305

NS

(difference %)

4.5

9.0

18.4

 

Thyroid (relative to body), %

0.0051

0.0053

0.0055

0.0060

NS

(difference %)

4.2

8.2

17.9

 

Thyroid (relative to brain), %

1.170

1.203

1.267

1.394

NS

(difference %)

2.8

8.2

19.2

 

Notes: Data (group mean values, n=12-13) were rounded to one to four decimal places. Paired organs were weighed together or total weight of the pair were analysed. Thyroid and parathyroid weights were measured together.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s Multiple Range Test, NS: Statistically not significant compared to control

 

Organ weight data (females)

Organ weight

Dose groups

 

Control

Low dose

Mid dose

High dose

 

Terminal body weight, g

337.5

359.2

360.7

355.4

NS

(difference %)

6.4

6.9

5.3

 

Liver (absolute), g

12.965

15.461

16.988**

16.775**

DU

(difference %)

19.3

31.0

29.4

 

Liver (relative to body), %

3.832

4.307*

4.707**

4.728**

DN

(difference %)

12.4

22.8

23.4

 

Liver (relative to brain), %

646.02

770.14**

846.45**

834.76**

DN

(difference %)

19.2

31.0

29.2

 

Thyroid (absolute), g

0.0217

0.0224

0.0246

0.0256*

DU

(difference %)

3.1

13.4

18.0

 

Thyroid (relative to body), %

0.0065

0.0063

0.0068

0.0072

NS

(difference %)

-3.2

5.5

11.3

 

Thyroid (relative to brain), %

1.087

1.116

1.229

1.272*

DU

(difference %)

2.7

13.1

17.0

 

Notes: Data (group mean values, n=10-11) were rounded to one or three decimal places. Paired organs were weighed together or total weight of the pair were analysed. Thyroid and parathyroid weights were measured together.

Statistical significance compared to control: * = p<0.05, ** = p<0.01

DN: Dunnett’s Multiple Range Test, DU: Dunn test, NS: Statistically not significant compared to control

Applicant's summary and conclusion

Conclusions:
In summary, daily administration of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] test item by oral gavage to Wistar rats at dose levels of 100, 300 or 1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) during the treatment period of this study did not result in test item related mortality, clinical signs, or significant changes in body weight / body weight gain, food consumption, haematology, coagulation, clinical chemistry or urinalysis parameters.

No test item-related macroscopic findings were recorded in any of the dose groups at necropsy. Increased liver weights were observed in all treated groups, associated with test item-related microscopic findings (centrilobular/periportal hepatocellular hypertrophy) in the Mid and High dose groups (males and females), the intensity ranged from minimal to slight. Hypertrophy was considered to be an adaptive, non-adverse change. A slight microvesicular vacuolation in the liver seen at a low incidence in High dose males was regarded as a non-adverse finding. There were no other organ weight or histopathological findings related to treatment.
No endocrine disruptor effect of test item was observed in the study.

In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was considered to be 1000 mg/kg bw/day for the female and male parental (adult) generation, and also for the F1 generation (pups).
Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test in the Rats was to obtain information on the toxicity of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] test item following repeated daily administration by oral gavage to Wistar rats. The study included a reproductive / developmental toxicity-screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and development of the F1 offspring from conception to post-natal day (PND) 13.

 

Male and female Wistar rats were treated for 2 weeks pre-mating and then during the mating / post-mating periods. This was 28 days in total for males. Females were treated throughout gestation and up to and including postpartum/lactation day (PPD) 13. The Experimental design was as follows:

 

Gr. No.

Group designation

Dose level
(mg/kg bw/day)

Concentration (mg/mL)

Dose volume

(mL/kg bw)

Animal numbers

Male

Female

1

Control

0

0

5

1001-1012

1501-1512

2

Low dose

100

20

2001-2012

2501-2512

3

Mid dose

300

60

3001-3012, 3109

3501-3512, 3609

4

High dose

1000

200

4001-4012

4501-4512

 

Parameters measured during the study included signs of morbidity and mortality twice daily, daily or weekly detailed observation of clinical signs, neurological assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. Neurological assessment of functional observation battery (including the measurements of the landing foot splay and grip strength) and measurement of motor activity was performed during the last week of the treatment. In addition, the reproductive performance, pregnancy, parturition and postpartum/lactation period were monitored in the adult animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND 13.

At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues / organs were sampled and preserved in appropriate fixatives from the adult animals and offspring. The thyroxine (T4) levels in the adult males andPND 13 pups were also assessed. For the adult animals, a detailed histological examination was performed on the selected list of retained organs of 5 Control and High dose animals per sex as well as reproduction organs of all animals in the Low and Mid dose groups which failed to sire / conceive. Additionally, livers were examined histologically on all High, Mid dose and Low dose animals.

All test item formulations were within the range of 95-109% of nominal concentration and were found to be homogenous. No test item was detected in the vehicle control samples. Based on these results, test item formulations were considered suitable for the study purposes.

 

In summary, daily administration of 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] test item by oral gavage to Wistar rats at dose levels of 100, 300 or
1000 mg/kg bw/day (Low, Mid and High dose groups, respectively) during the treatment period of this study did not result in test item related mortality, clinical signs, or significant changes in body weight / body weight gain, food consumption, haematology, coagulation, clinical chemistry or urinalysis parameters.

 

No test item related effect was detected during neurotoxicity assessment.

 

No test item effect on oestrus cycle of parental females were noted.

 

No test item related changes were noted in the reproductive parameters, gestation, parturition and lactation.

 

There were no adverse effects on the F1 offspring viability, clinical signs, physical or sexual development. No test item related macroscopic finding were recorded for F1 pups at necropsy.

No test item-related macroscopic findings were recorded in any of the dose groups at necropsy. Increased liver weights were observed in all treated groups, associated with test item-related microscopic findings (centrilobular/periportal hepatocellular hypertrophy) in the Mid and High dose groups (males and females), the intensity ranged from minimal to slight. Hypertrophy was considered to be an adaptive, non-adverse change. A slight microvesicular vacuolation in the liver seen at a low incidence in High dose males was regarded as a non-adverse finding. There were no other organ weight or histopathological findings related to treatment.

 

No endocrine disruptor effect of test item was observed in the study.

 

In conclusion, under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for 2,2’-methylenebis[6-(1-methylcyclohexyl)-p-cresol] was considered to be 1000 mg/kg bw/day for the female and male parental (adult) generation, and also for the F1 generation (pups).