Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 500 mg/kg was derived.

The parental No Observed Effect Level (NOEL) was established at 150 mg/kg based on non-adverse microscopic changes in the thyroids of males at 500 mg/kg.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 September 2016 to 30 December 2016
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)
Deviations:
yes
Remarks:
see "Any other information on materials and methods incl. tables"
Qualifier:
according to guideline
Guideline:
other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
yes
Remarks:
see "Any other information on materials and methods incl. tables"
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
"Any other information on materials and methods incl. tables"
Qualifier:
equivalent or similar to guideline
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
yes
Remarks:
see "Any other information on materials and methods incl. tables"
Qualifier:
equivalent or similar to guideline
Guideline:
other: Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
Deviations:
yes
Remarks:
"Any other information on materials and methods incl. tables"
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
Deviations:
yes
Remarks:
"Any other information on materials and methods incl. tables"
Qualifier:
equivalent or similar to guideline
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.
Deviations:
yes
Remarks:
see "Any other information on materials and methods incl. tables"
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
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 6,6’-di-tert-butyl-2,2’-thiodi-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:
Internal identification number 207570/A
Identification LOWINOX® TBP-6
Appearance White powder
Batch C034J0059
Purity/Composition 99.8 %
Test item storage At room temperature
Stable under storage conditions until 12 May 2017 (expiry date)
Species:
rat
Strain:
other: Crl:WI(Han) (outbred, SPF-Quality).
Details on species / strain selection:
This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and
reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.

Source F0:Charles River Deutschland, Sulzfeld, Germany.

Age at start pretest: Females - approximately 11 weeks.

Age at start F0-treatment: Males - approximately 10 weeks / Females: approximately 13 weeks.

Number of F0-animals: 48 females and 40 males.

At the end of the pretest phase, 40 females with at least two regular estrous cycles were selected at random and further used in the study. The remaining females were removed from the study.

Acclimatization F0: At least 5 days prior to start of pretest (females) or treatment (males).

Health inspection F0: At least upon receipt of the animals.

Randomization F0: Before initiation of pretest, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.

Identification F0: During pretest (females) and treatment (males and females): by earmark and tattoo. Reserve females were numbered R1 through R8 at random by indelible marker. Any reserve female replacing an allocated female prior to treatment received identification by earmark and tattoo.

Mating procedures
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
 
Parturition
The females were allowed to litter normally. Postnatal day (PND) 1 was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). Females that were littering were left undisturbed.
Number of pups: 424 pups.

Identification of pups
On PND 1, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

Culling
To reduce variability among the litters, on PND 4 eight pups from each litter of equal sex distribution (if possible) were selected. Blood samples were collected from two of the surplus pups (see section 0). Selective elimination of pups, e.g. based upon body weight, or anogenital distance was not done. Whenever the number of male or female pups prevented having four of each sex per litter, partial adjustment (for example, five males and three females) was acceptable.

Animal Husbandry
Room number: Room A0.07 (A0.23 for motor activity measurements).
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 room air changes/hour, and a 12-hour light/12-hour dark cycle: the photoperiod is between 07:00 and 19:00 hrs daily. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages (MIV type, height 18 cm).

Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).

Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).

Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).

Lactation
Females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30 40 minutes.

General
Sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.

Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water: Free access to tap-water.
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.
Route of administration:
oral: gavage
Mass median aerodynamic diameter (MMAD):
196.378 µm
Vehicle:
propylene glycol
Details on exposure:
Treatment - Parental Animals
Method
Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures. (Test Facility Study No. 513607 was used for DCS).
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Frequency: Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Treatment period: Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females that delivered were treated for 50-56 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 38-45 days.
Routinely, females that are littering are left undisturbed. In this study, female nos. 52, 55 and 59 (Group 2) and 73 (Group 4) were not dosed on one occasion as they were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was considered not to affect the toxicological evaluation.

Treatment - Pups
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.
Details on mating procedure:
Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating was confirmed, the males and females were separated. A maximum of 14 days was allowed for mating, after which females who had not shown evidence of mating were separated from their males.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verification of doses was conducted using a Liquid chromatography diode array detector (LC-DAD)

Procedural recovery samples
The mean recoveries of the procedural recovery samples fell within the criterion of 90-110%. It demonstrated that the analytical method was adequate for the determination of the test item in the test samples.

Test samples
In the Group 1 formulation, no test item was detected.
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 90% and 110%).
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Duration of treatment / exposure:
Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females that delivered were treated for 50-56 days, i.e. during 2 weeks prior to Repeated dose toxicity: oral - Charles River Laboratories Den Bosch B.V. - 513606 mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the daybefore scheduled necropsy. Females which failed to deliver healthy offspring were treated for 38-45 days.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Details on study schedule:
Pups were not dosed directly but could have potentially been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10 males and 10 females per dosing group
Control animals:
yes, concurrent vehicle
Details on study design:
Based on the results of a dose range finding study in which toxicity was noted at 500 mg/kg but not at 250 mg/kg (Test Facility Study No. 513610), the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 50, 150 and 500 mg/kg.
Positive control:
None.
Parental animals: Observations and examinations:
Mortality / Viability: At least twice daily.

Clinical signs: Clinical observations (detailed clinical signs and arena) were conducted from start of treatment onwards up to the day prior to necropsy at least at 1 hour (± 30 min)) after dosing based on the peak period of anticipated effects. Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for s
everity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Functional Observations
The following functional observations tests were performed on each individual animal of the selected 5 animals/sex/group:
• hearing ability (HEARING), pupillary reflex (PUPIL L/R), and static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a co mputerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or finer movements like grooming, weaving or movements of
the head. The selected males were tested during Week 4 of treatment and the selected females were tested once during the last week of lactation (e.g. PND 6-13). These tests were performed after observation for clinical signs (incl. arena observation, if applicable) and started at 1 hour (± 30 min) after dosing.

Body weights: Males and females were weighed on the first day of treatment (prior to first dosing) and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on PND 1, 4, 7 and 13.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

General reproduction data:
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded.

Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females were examined for evidence of abortion or premature delivery. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold,physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Oestrous cyclicity (parental animals):
Daily vaginal lavage was performed to determine the stage of estrous beginning 14 days prior to treatment (pretest), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage continued for those females with no evidence of copulation until termination of the mating period. During pretest, this was done for 48 females. On the day of scheduled necropsy, a vaginal lavage was taken to determine the stage of estrous.
Sperm parameters (parental animals):
Not specified
Litter observations:
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups were determined on PND 1 and daily thereafter. If possible, defects or cause of death were evaluated.

Clinical signs: At least once daily, detailed clinical observations were made for all animals. Only days on which clinical signs were present between first and last litter check are presented in the respective tables.

Body weights: Live pups were weighed on PND 1, 4, 7 and 13.

Sex: Sex was determined for all pups on PND 1 and 4. Sex ratio (% male pups / % female pups) was calculated per group.

Anogenital distance: Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight.

Areola/nipple retention: On PND 13, all males in each litter were examined for the number of areola/nipples.

Clinical Laboratory Investigations (F0-Generation only)
Blood samples were collected at the end of the treatment period on the day of scheduled necropsy from the selected 5 animals/sex/group under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m.
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and tubes treated with Li-heparin for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.

Haematology
The following haematology parameters were determined in blood prepared with K3-EDTA as an anti-coagulant, using the ADVIA® 2120i Hematology System (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands):
Parameter (Abbreviation) Unit
White blood cells (WBC) 109/L
Differential leucocyte count:
Neutrophils %WBC
Lymphocytes %WBC
Monocytes %WBC
Eosinophils %WBC
Basophils %WBC
Red blood cells 1012/L
Reticulocytes %RBC
Red blood cell distribution width (RDW) %
Haemoglobin mmol/L
Haematocrit L/L
Mean corpuscular volume (MCV) fL
Mean corpuscular haemoglobin (MCH) fmol
Mean corpuscular haemoglobin concentration (MCHC) mmol/L
Platelets 109/L

The following clotting parameters were determined in plasma prepared with citrate as anti-coagulant, using the STA Compact® (Diagnostica Stago S.A.S., Asnières, France):
Parameter (Abbreviation) Unit
Prothrombin Time (PT) s
Activated Partial Thromboplastin Time (APTT) s

Clinical Biochemistry
The following clinical biochemistry parameters were determined using the AU400 (Beckman Coulter Nederland B.V., Woerden, The Netherlands). All parameters were determined in plasma, except for bile acids which were determined in serum:
Parameter (Abbreviation) Unit
Alanine aminotransferase (ALAT) U/L
Aspartate aminotransferase (ASAT) U/L
Alkaline Phosphatase (ALP) U/L
Total protein g/L
Albumin g/L
Total Bilirubin μmol/L
Bile acids μmol/L
Urea mmol/L
Creatinine μmol/L
Glucose mmol/L
Cholesterol mmol/L
Sodium mmol/L
Potassium mmol/L
Chloride mmol/L
Calcium mmol/L
Inorganic Phosphate (Inorg. Phos) mmol/L

Blood Sampling for Thyroid Hormone Analysis
F1-generation, PND 4 pups:
From 2 surplus pups per litter at culling (if possible). Blood samples from the 2 pups per litter were collected into one serum tube. If only 1 surplus pup per litter was available at culling, as much as possible blood was collected from this single pup.
Blood samples were collected by decapitation, between 7.00 and 10.30 a.m.. Blood samples from the 2 pups per litter (0.4 mL in total) were collected into one serum tube (Greiner Bio-One GmbH, Kremsmünster, Austria) for possible future measurement of thyroxine (T4). After clotting and centrifugation, serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months were discarded.

F1-generation, PND 13-15 pups:
From 2 pups per litter (if possible from one male and one female) at planned necropsy.
As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m.. Blood was collected into serum tubes.
After clotting and centrifugation, serum from each sample was divided into 2 aliquots: 150 µL serum for measurement of thyroxine (T4) and the remaining volume of serum for possible future measurement of thyroid-stimulating hormone (TSH).
Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months will be discarded.

F0-generation, males and females:
End of study from all animals at planned necropsy; this included females on Day 14-16 of lactation, all females that failed to deliver healthy pups and all males after at least 4 weeks of treatment (including all males that failed to sire).
Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30 a.m. The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available. Blood samples (0.9 mL) were drawn from the retro-orbital sinus and collected into serum tubes (Greiner Bio-One GmbH, Kremsmünster, Austria).
After clotting and centrifugation, serum was used as listed below.
Males: 1 aliquot of 150 µL serum was used for measurement of thyroxine (T4), and the remaining volume of serum was stored for possible future measurement of thyroid-stimulating hormone (TSH).
Females: The serum was stored for possible future measurement of thyroxine (T4) and/or thyroid-stimulating hormone (TSH).
Serum samples were stored at ≤-75°C. Under these storage conditions, the samples are stable for 6 months. Any samples remaining after 6 months will be discarded.
In general:
Total T4 was measured in serum using the Immulite 1000® (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands):
Parameter (Abbreviation) Unit
Thyroxine (T4) µg/dL

 

Postmortem examinations (parental animals):
F0-generation - Pathology
F0-generation - Termination
The animals were deprived of food overnight (with a maximum of 24 hours) before blood sampling, but water was available.
All animals surviving to the end of the observation period were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.
Necropsy was conducted on the following days:
Condition Day of necropsy
Males Following completion of the mating period (a minimum of 28 days of dose administration).
Females which delivered PND 14-16.
Females which failed to deliver (nos. 77, 69) Post-coitum Day 25 (female no. 77 with evidence of mating) or 18 days after the last day of the mating period (female no. 69 without evidence of mating).
Females with total litter loss (nos. 51, 79) Within 24 hours of total litter loss

F0-generation – Macroscopic Examination
After sacrifice, all animals were subjected to a full post mortem necropsy, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The number of former implantation sites were recorded for all paired females.
Samples of the following tissues and organs were collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
Selected 5 animals/sex/group (see Allocation):
Identification marks: not processed
(Nasopharynx)
Adrenal glands
(Esophagus)
(Aorta)
Ovaries
Brain - cerebellum, mid-brain, cortex (7-levels)
(Pancreas)
Caecum
Peyer's patches [jejunum, ileum] if detectable
Cervix
Pituitary gland
Clitoral gland
Preputial gland
Colon
Prostate gland
Coagulation gland
Rectum
(Cowper’s gland)
(Salivary glands - mandibular, sublingual)
Duodenum
Sciatic nerve
Epididymides
Seminal vesicles
Eyes (with optic nerve (if detectable) and Harderian gland)
Skeletal muscle
(Skin)
Mammary gland area (males and females)
Spinal cord -cervical, midthoracic, lumbar
Femur including joint
Spleen
(Glans penis)
Sternum with bone marrow
(Levator ani plus bulbocavernosus muscle complex (LABC))
Stomach
Testes
Heart
Thymus
Ileum
Thyroid including parathyroid if detectable
Jejunum
(Tongue)
Kidneys
Trachea
(Lacrimal gland, exorbital)
Urinary bladder
(Larynx)
Uterus
Liver
Vagina
Lung, infused with formalin
All gross lesions
Lymph nodes - mandibular, mesenteric

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
All remaining animals:
Cervix
Preputial gland
Clitoral gland
Prostate gland
Coagulation gland
Seminal vesicles
Cowper’s glands
Testes
Epididymides
Thyroid including parathyroid if detectable
Glans penis
Uterus
Levator ani plus bulbocavernosus muscle complex (LABC)
Vagina
All gross lesions
Mammary gland area (males and females)
Identification marks: not processed
Ovaries

F0-generation – Organ Weights
Terminal body weights were recorded from all rats at scheduled necropsy.
The following organ weights were recorded from the following animals on the scheduled day of necropsy:
Selected 5 animals/sex/group (see Allocation):
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Prostate
Seminal vesicles including coagulating glands
Spleen
Testes
Thymus
Thyroid, including parathyroid if detectable
Uterus, including cervix
All remaining animals:
Epididymides
Prostate
Seminal vesicles including coagulating glands
Testes
Thyroid (including parathyroid if detectable)
Absolute organ weights and organ to body weight ratios are reported.

F0-generation - Histotechnology
All organ and tissue samples, as defined under Histopathology (following section), were processed, embedded and cut at a thickness of 2-4 micrometers. These slides were stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands). The additional slides of the testes (to examine staging of spermatogenesis) were stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).
F0-generation – Histopathology
The following slides were examined by a pathologist:
• The preserved organs and tissues of the selected 5 animals/sex of Groups 1 and 4 (see Allocation).
• Additional slides of the testes of the selected 5 males of Groups 1 and 4 (see Allocation) and all males that failed to sire (see table below).
• The mammary gland of the females with total litter loss.
• All gross lesions of all animals (all dose groups).
• Thyroid gland of the selected 5 males of Groups 2 and 3, based on (possible) treatment-related changes in this organ in Group 4.
• The reproductive organs of all males that failed to sire and all females that failed to deliver healthy pups:

Group Female/Male nos. Reason
1 --- ---
2 51/11 Total litter loss
3 69/29 Not mated
4 77/37 Female was not pregnant
79/39 Total litter loss

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
A peer review on the histopathology data was performed by a second pathologist.
 
Postmortem examinations (offspring):
F1-generation Pups - Pathology
F1-generation Pups - Termination
Pups, younger than 7 days were euthanized by decapitation.
All remaining pups (PND 7-15), except those used for blood sampling on PND 13-15, were sacrificed using Euthasol® 20% (AST Farma B.V., Oudewater, The Netherlands) by intraperitoneal (ip) injection.
Pups 13 and 14 (litter 48), 3 and 4 (litter 61) and 8 (litter 72) found dead during the weekend were fixed in an identified container containing 70% ethanol (Klinipath, Duiven, The Netherlands) as they were not necropsied on the same day.
On PND 4 (at culling), from 2 pups per litter, blood samples (0.4 mL in total) were collected by decapitation between 7.00 and 10.30 a.m. Blood samples from the 2 pups per litter were collected into one serum tube.
On PND 13-15, from 2 pups per litter (if possible from one male and one female) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands), between 7.00 and 10.30 a.m., followed by exsanguination. Blood was collected into serum tubes.
Necropsy was conducted on the following days:
Condition Day of necropsy
Culling PND 4.
Terminal sacrifice PND 13-15.
Spontaneous deaths As soon as possible after death and always within 24 hours.

F1-generation pups - Macroscopic Examination
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded.
At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling (see also section 5.10 and 5.12.1), was preserved in 10% buffered formalin.
The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Statistics:
Statistical Analyses
The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
Live birth index
Percentage live males at First Litter Check
Percentage live females at First Litter Check
Viability index
Lactation index
Offspring viability indices:
Post-implantation survival index
Mating index
Precoital time
Fertility index
Conception index
Gestation index
Duration of gestation
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs of toxicity were noted during daily clinical observations or during weekly arena observations.
Slight salivation was noted after dosing, starting after about two weeks of treatment, at all dose levels, most frequently at 150 and 500 mg/kg. This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e.after dosing).

One female at 500 mg/kg (no. 76) showed lethargy, shallow respiration and piloerection towards the end of the gestation period (on one or two days). Based on the incidental occurrence of these signs of distress and the absence of abnormalities in her body weight development and food consumption, these findings were considered not to be toxicologically relevant.

Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain were not affected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes in food consumption before or after allowance for body weight were noted.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in haematology parameters of treated rats.
Isolated statistically significant variations noted in haematology parameters in males were unrelated to treatment or not toxicologically relevant due to the lack of a dose-related response or a slightly high concurrent control value.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No toxicologically relevant changes occurred in clinical biochemistry parameters of treated rats.
The statistically significant variations noted in clinical biochemistry parameters were unrelated to treatment or not toxicologically relevant due to the lack of a dose-related response, slight magnitude of the difference from controls (values in treated rats remained within normal limits), and/or a slightly low concurrent control value.

Thyroid hormone analyses:
Serum levels of T4 in F0 males were not affected by treatment.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals.
Grip strength was similar across the groups, except for a statistically significantly lower hind limb grip strength in males at 500 mg/kg. This finding was not accompanied by a decrease in fore limb grip strength and the values at 500 mg/kg were within the normal range for male rats of this strain and age. Therefore, the lower hind limb grip strength in males at 500 mg/kg was considered not to be toxicologically relevant.

The variation in motor activity did not indicate a relation with treatment. All groups showed a similar ha bituation profile with a decreasing trend in activity over the duration of the test period.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In the thyroid gland of males, an increased incidence and severity (up to slight) of follicular cell hypertrophy was present starting at 150 mg/kg/day, and colloid alteration was present at 500 mg/kg (up to slight).

The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations. This included the recorded findings of control animals which erroneously received the test item.
Histopathological findings: neoplastic:
not specified
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Mating index
Mating index was not affected by treatment. Except for one female at 150 mg/kg (no. 69), all females showed evidence of mating.

Fertility and Conception index
Fertility and conception index were not affected by treatment.
Except for one female at 500 mg/kg (no. 77), all mated females were pregnant. This incidental non-pregnancy without related histopathology changes in reproductive organs was considered to be unrelated to treatment.

Precoital time
Precoital time was not affected by treatment.

Number of implantation sites
Number of implantation sites was considered not to be adversely affected by treatment.
The mean number of implantation sites at 500 mg/kg was slightly lower than that in the control group. The lower mean value at 500 mg/kg was largely due to the low number of implantation sites in two females (nos. 73 and 79 with five and two implantation sites, respectively). Such low numbers of implantation sites occasionally occur in untreated controls. Moreover, the difference from the control group was not statistically significant. Therefore, this finding was considered not to be toxicologically relevant.
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
Length and regularity of the estrous cycle were not affected by treatment.
Most females had regular cycles of 4 days. Control female no. 46 had an irregular cycle and extended di-estrus during paring was noted for female no. 69 at 150 mg/kg. These findings were considered to be unrelated to treatment because their incidence was within normal limits and showed no dose-related trend.
Reproductive function: sperm measures:
not specified
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
There were 1/10 couples treated at 50 mg/kg (female/male no. 51/11, total litter loss), 1/10 couples treated at 150 mg/kg (female/male no. 69/29, not mated) and 2/10 couples treated at 500 mg/kg (female/male no. 77/37, not pregnant; no. 79/39, total litter loss) that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain the reproductive failure or total litter loss.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and spermatogenic staging profiles were normal for all males examined.
No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg).
No treatment-related or toxicologically relevant changes were noted in any of reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable
Key result
Critical effects observed:
no
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment.
The nature and incidence of the clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.
Note: Only days on which clinical signs were present between first and last litter check are presented in the table.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No mortality occurred during the study period.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights of pups were considered not to be affected by treatment.
Compared to controls, mean body weights of male and female pups at 500 mg/kg were higher from birth until PND 13. The relative differences gradually decreased from about 20% at PND 1 (statistically significant) to about 10% at PND 13 (no longer statistically significant). The higher means at 500 mg/kg were particularly due to the higher (exceeding normal limits) weights and weight gain of the pups of one small litter (no. 73, two male and two female pups). The higher pup weights in this small litter are consistent with the generally recognized inverse relation between litter size and pup body weight. Pups of the other litters at 500 mg/kg had normal body weights at birth and thereafter (except for those of litter no. 71 which had slightly lower weights at PND 13). For these reasons, the higher mean pup weights at 500 mg/kg were considered to reflect normal biological variation rather than an effect of the test item.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 levels in male and female PND 13-15 pups were not affected by treatment.
Urinalysis findings:
not specified
Sexual maturation:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
effects observed, non-treatment-related
Histopathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment.
The nature and incidence of the few findings noted (mostly in pups found dead) remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Gestation index and duration
Gestation index and duration of gestation were not affected by treatment.

Parturition/maternal care
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Post-implantation survival index
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was not affected by treatment. The survival indices across the groups were 90-96%.

Live birth index
Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices across the groups were 97 or 99%.
At first litter check, four pups of the control group (litter nos. 46-48), three pups at 150 mg/kg (litter nos. 61 and 64) and one pup at 500 mg/kg (litter no. 72) were found dead. One female at 50 mg/kg (no. 51) had only one dead pup at first litter check (total litter loss). This pup mortality was considered to be unrelated to treatment because the mortality incidence was within normal limits and showed no dose-related trend.
The mean number of living pups at first litter check was slightly lower at 500 mg/kg. The difference from the concurrent control group was not statistically significant and could be explained by the small size of two litters in the 500 mg/kg group (litter nos. 73 (four pups) and 79 (two pups)) which correlated with low numbers of implantation sites in the dams. Such small litter sizes occasionally occur in untreated controls. All other litters at 500 mg/kg had normal sizes (between 8 and 14 pups). For these reasons, the lower mean litter size at 500 mg/kg was considered not to be toxicologically relevant.

Viability index
Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices across the groups were 94-98%.
Female no. 79 at 500 mg/kg had total litter loss (one of her two pups went missing on PND 2, the other on PND 4). In addition, six pups of the control group (of four litters), two pups at 50 mg/kg (of two litters), six pups at 150 mg/kg (of four litters), and one pup at 500 mg/kg went missing (presumably cannibalized) or were found dead between PND 2-4 (mostly at PND 2). This post-natal loss was considered to be unrelated to treatment as the incidence showed no dose-related trend and remained within the range considered normal for pups of this age.

Lactation index
Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment. The lactation index was 99% at 150 mg/kg (one pup of litter no. 70 went missing on PND 7) and 100% in the other groups.

Sex ratio
Sex ratio was not affected by treatment.

Anogenital distance
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.

Areola/nipple retention
Treatment up to 500 mg/kg had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13
Behaviour (functional findings):
not specified
Developmental immunotoxicity:
not specified
No developmental toxicity was observed up to the highest dose level tested (500 mg/kg).
No treatment-related or toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopy).
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
150 mg/kg bw/day (nominal)
Treatment related:
yes

                    

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG

 

GROUP 3

150 MG/KG

 

GROUP 4

500 MG/KG

 

AT WEEK 4

 

 

 

 

 

HEARING

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

PUPIL L

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

PUPIL R

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

STATIC R

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

GRIP FORE

MEAN

1288

1162

1439

1373

GRAM

ST.DEV

176

120

178

187

 

N

5

5

5

5

GRIP HIND

MEAN

612

557

581

497 *

GRAM

ST.DEV

65

57

44

93

 

N

5

5

5

5

 

 

FEMALES

 

 

 

 

GROUP 1

 

GROUP 2

 

GROUP 3

 

GROUP 4

 

 

CONTROL

50 MG/KG

150 MG/KG

500 MG/KG

 

LACTATION

 

 

 

 

 

HEARING

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

PUPIL L

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

PUPIL R

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

STATIC R

MEDIAN

0

0

0

0

SCORE 0/1

N

5

5

5

5

GRIP FORE

MEAN

840

1025

870

1009

GRAM

ST.DEV

122

206

228

245

 

N

5

5

5

5

GRIP HIND

MEAN

613

690

621

680

GRAM

ST.DEV

88

67

66

81

 

N

5

5

5

5


 

MOTOR ACTIVITY MALES

 

AT WEEK 4

TEST SUMMARY

 

 

 

 

 

 

 

GROUP 1

 

GROUP 2

 

GROUP 3

 

GROUP 4

 

 

CONTROL

50 MG/KG

150 MG/KG

500 MG/KG

 

Total Movements

 

MEAN1

 

3794

 

2571

 

2772

 

3065

 

ST.DEV

2378

182

1016

669

 

N

5

5

5

5

Ambulations

 

MEAN1

852

587

625

842

 

ST.DEV

594

72

334

280

 

N

5

5

5

5

 

 

*/** Wilcoxon test significant at 5% (*) or 1% (**) level

1Group mean of all intervals

2combined


 

 

 

MOTOR ACTIVITY TEST SUMMARY

FEMALES

AT LACTATION

 

 

GROUP 1

 

GROUP 2

 

GROUP 3

 

GROUP 4

 

CONTROL

50 MG/KG

150 MG/KG

500 MG/KG

 

TotalMovements                MEAN1

 

3492

 

3270

 

2229

 

3235

ST.DEV

1449

1151

413

818

N

5

5

5

5

Ambulations                        MEAN1

848

680

545

773

ST.DEV

355

345

140

284

N

5

5

5

5

 

 

*/** Wilcoxon test significant at 5% (*) or 1% (**) level

1Group mean of all intervals combined


 

BODY WEIGHTS (GRAM) SUMMARY MALES

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG

 

GROUP 3

150 MG/KG

 

GROUP 4

500 MG/KG

 

PRE MATING

DAY 1

 

MEAN

 

298

 

297

 

294

 

300

WEEK 1

ST.DEV

7.4

11.1

13.0

9.4

 

N

10

10

10

10

DAY 8

MEAN

319

320

315

321

WEEK 2

ST.DEV

8.5

16.4

17.5

12.6

 

N

10

10

10

10

 

MATING PERIOD

DAY 1

 

MEAN

 

339

 

341

 

335

 

343

WEEK 1

ST.DEV

9.5

20.6

21.5

16.7

 

N

10

10

10

10

DAY 8

MEAN

351

357

347

357

WEEK 2

ST.DEV

10.4

20.4

19.3

17.7

 

N

10

10

10

10

DAY 15

MEAN

368

375

365

374

WEEK 3

ST.DEV

12.2

22.7

21.3

20.4

 

N

10

10

10

10

 

 

FEMALES

 

 

 

 

GROUP 1

 

GROUP 2

 

GROUP 3

 

GROUP 4

 

 

CONTROL

50 MG/KG

150 MG/KG

500 MG/KG

 

PRE MATING

DAY 1

 

MEAN

 

221

 

225

 

222

 

226

WEEK 1

ST.DEV

8.2

11.5

7.6

14.3

 

N

10

10

10

10

DAY 8

MEAN

222

226

224

228

WEEK 2

ST.DEV

8.9

12.5

9.5

15.8

 

N

10

10

10

10

 

MATING PERIOD

DAY 1

 

MEAN

 

229

 

232

 

231

 

235

WEEK 1

ST.DEV

9.2

13.9

8.7

17.3

 

N

10

10

10

10

DAY 8

MEAN

 

 

263

 

WEEK 2

ST.DEV N

 

 

--- 1

 

DAY 15

WEEK 3

MEAN ST.DEV

 

 

268

---

 

 

N

 

 

1

 

DAY 22

MEAN

 

 

257

 

WEEK 4

ST.DEV N

 

 

--- 1

 

DAY 29

MEAN

 

 

260

 

WEEK 5

ST.DEV N

 

 

--- 1

 

 

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 

F0-GENERATION                                                                                                                                                                                       

 

BODY WEIGHTS (GRAM) SUMMARY FEMALES

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

POST COITUM

DAY 0

 

MEAN

 

231

 

235

 

232

 

238

 

ST.DEV.

10.3

14.5

10.0

15.7

 

N

10

10

9

9

DAY 4

MEAN

245

248

248

256

 

ST.DEV.

10.9

16.1

11.2

16.0

 

N

10

10

9

9

DAY 7

MEAN

253

256

255

264

 

ST.DEV.

10.1

16.7

12.0

18.5

 

N

10

10

9

9

DAY 11

MEAN

269

271

271

277

 

ST.DEV.

9.9

17.6

14.3

20.6

 

N

10

10

9

9

DAY 14

MEAN

279

284

283

286

 

ST.DEV.

11.3

19.7

15.5

20.9

 

N

10

10

9

9

DAY 17

MEAN

299

304

305

309

 

ST.DEV.

11.6

25.2

16.9

22.8

 

N

10

10

9

9

DAY 20

MEAN

336

341

346

341

 

ST.DEV.

16.3

34.2

18.6

28.4

 

N

10

10

9

9

 

LACTATION

DAY 1

 

MEAN

 

260

 

263

 

264

 

272

 

ST.DEV.

9.3

20.4

16.3

21.9

 

N

10

10

9

9

DAY 4

MEAN

273

276

276

281

 

ST.DEV.

15.5

19.8

15.8

24.0

 

N

10

9

9

9

DAY 7

MEAN

284

287

289

292

 

ST.DEV.

15.6

18.7

16.5

23.2

 

N

10

9

9

8

DAY 13

MEAN

302

299

297

306

 

ST.DEV.

16.0

21.5

17.5

21.9

 

N

10

9

9

8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level Explanations for excluded data are listed in the tables of the individual values


 

BODY WEIGHT GAIN (%) SUMMARY MALES

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG

 

GROUP 3

150 MG/KG

 

GROUP 4

500 MG/KG

 

PRE MATING

DAY 1

 

MEAN

 

0

 

0

 

0

 

0

WEEK 1

ST.DEV

0.0

0.0

0.0

0.0

 

N

10

10

10

10

DAY 8

MEAN

7

8

7

7

WEEK 2

ST.DEV

2.0

2.2

2.1

1.6

 

N

10

10

10

10

 

MATING PERIOD

DAY 1

 

MEAN

 

14

 

15

 

14

 

14

WEEK 1

ST.DEV

2.9

3.5

3.0

2.6

 

N

10

10

10

10

DAY 8

MEAN

18

20

18

19

WEEK 2

ST.DEV

3.5

3.7

3.1

2.9

 

N

10

10

10

10

DAY 15

MEAN

24

26

24

24

WEEK 3

ST.DEV

4.2

4.2

3.4

3.5

 

N

10

10

10

10

 

 

FEMALES

 

 

 

 

 

 

 

 

GROUP 1

 

GROUP 2

 

GROUP 3

 

GROUP 4

 

 

CONTROL

50 MG/KG

150 MG/KG

500 MG/KG

 

PRE MATING

DAY 1

 

MEAN

 

0

 

0

 

0

 

0

WEEK 1

ST.DEV

0.0

0.0

0.0

0.0

 

N

10

10

10

10

DAY 8

MEAN

0

0

1

1

WEEK 2

ST.DEV

2.5

1.4

1.7

1.8

 

N

10

10

10

10

 

MATING PERIOD

DAY 1

 

MEAN

 

3

 

3

 

4

 

4

WEEK 1

ST.DEV

2.6

2.5

2.9

3.5

 

N

10

10

10

10

DAY 8

MEAN

 

 

15

 

WEEK 2

ST.DEV N

 

 

--- 1

 

DAY 15

WEEK 3

MEAN ST.DEV

 

 

17

---

 

 

N

 

 

1

 

DAY 22

MEAN

 

 

12

 

WEEK 4

ST.DEV N

 

 

--- 1

 

DAY 29

MEAN

 

 

14

 

WEEK 5

ST.DEV N

 

 

--- 1

 

 

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 

F0-GENERATION                                                                                                                                                                                       

 

BODY WEIGHT GAIN (%) SUMMARY FEMALES

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

POST COITUM

DAY 0

 

MEAN

 

0

 

0

 

0

 

0

 

ST.DEV.

0.0

0.0

0.0

0.0

 

N

10

10

9

9

DAY 4

MEAN

6

6

7

8

 

ST.DEV.

1.8

1.8

1.8

2.8

 

N

10

10

9

9

DAY 7

MEAN

9

9

10

11

 

ST.DEV.

1.7

1.9

2.2

3.3

 

N

10

10

9

9

DAY 11

MEAN

16

15

17

16

 

ST.DEV.

2.5

2.1

3.1

3.4

 

N

10

10

9

9

DAY 14

MEAN

20

20

22

20

 

ST.DEV.

2.1

2.5

3.4

2.8

 

N

10

10

9

9

DAY 17

MEAN

30

29

32

30

 

ST.DEV.

3.0

4.5

3.5

4.7

 

N

10

10

9

9

DAY 20

MEAN

45

45

49

43

 

ST.DEV.

3.0

8.3

3.6

7.6

 

N

10

10

9

9

 

LACTATION

DAY 1

 

MEAN

 

0

 

0

 

0

 

0

 

ST.DEV.

0.0

0.0

0.0

0.0

 

N

10

10

9

9

DAY 4

MEAN

5

5

5

3

 

ST.DEV.

3.1

2.9

3.0

3.4

 

N

10

9

9

9

DAY 7

MEAN

10

9

10

8

 

ST.DEV.

3.1

3.4

4.1

2.4

 

N

10

9

9

8

DAY 13

MEAN

16

14

13

13

 

ST.DEV.

3.6

4.4

6.8

2.6

 

N

10

9

9

8

 

 

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level Explanations for excluded data are listed in the tables of the individual values

 

 


 

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY MALES

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG

 

GROUP 3

150 MG/KG

 

GROUP 4

500 MG/KG

 

PRE MATING

DAYS 1-8

 

MEAN

 

22

 

22

 

22

 

23

WEEKS 1-2

ST.DEV

0.9

0.1

0.1

0.7

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

21

22

22

23

WEEKS 2-3

ST.DEV

0.2

0.1

0.5

1.3

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER PRE MATI...

 

MEAN

 

22

 

22

 

22

 

23

MATING PERIOD

 

 

 

 

 

DAYS 1-8

MEAN

23

23

23

23

WEEKS 1-2

ST.DEV

0.7

0.2

0.4

1.0

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

22

22

22

23

WEEKS 2-3

ST.DEV

0.5

0.1

0.8

1.0

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER MATING P...

 

MEAN

 

22

 

22

 

22

 

23

 

FEMALES

 

 

 

 

 

 

 

 

GROUP 1

 

GROUP 2

 

GROUP 3

 

GROUP 4

 

 

CONTROL

50 MG/KG

150 MG/KG

500 MG/KG

 

PRE MATING

DAYS 1-8

 

MEAN

 

15

 

15

 

15

 

15

WEEKS 1-2

ST.DEV

0.6

0.7

0.7

0.7

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

14

14

15

16

WEEKS 2-3

ST.DEV

0.6

0.4

0.4

0.6

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER PRE MATI...

 

MEAN

 

15

 

15

 

15

 

15


 

F0-GENERATION                                                                                                                                                                                       

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY FEMALES

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

POST COITUM

DAYS 0-4

 

MEAN

 

16

 

16

 

16

 

19

 

ST.DEV.

2.8

2.8

3.0

3.8

 

N

10

10

9

9

DAYS 4-7

MEAN

18

18

18

20

 

ST.DEV.

1.6

2.2

1.7

3.4

 

N

10

10

9

9

DAYS 7-11

MEAN

19

18

19

20

 

ST.DEV.

1.3

2.3

2.4

3.2

 

N

10

10

9

9

DAYS 11-14

MEAN

21

21

22

22

 

ST.DEV.

1.1

2.2

3.0

2.4

 

N

10

10

9

9

DAYS 14-17

MEAN

20

20

20

22

 

ST.DEV.

0.6

2.4

2.2

2.9

 

N

10

10

9

9

DAYS 17-20

MEAN

23

22

24

24

 

ST.DEV.

2.2

2.5

2.5

2.8

 

N

10

10

9

9

 

MEAN OF MEANS

 

 

19

 

19

 

20

 

21

LACTATION

DAYS 1-4

 

MEAN

 

25

 

27

 

25

 

26

 

ST.DEV.

4.1

3.0

2.8

5.3

 

N

6

6

8

9

DAYS 4-7

MEAN

37

38

37

37

 

ST.DEV.

4.3

3.5

3.6

5.2

 

N

10

9

9

8

DAYS 7-13

MEAN

49

46

46

47

 

ST.DEV.

3.3

3.8

4.4

5.4

 

N

10

9

9

8

 

MEAN OF MEANS

 

 

37

 

37

 

36

 

36

 

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level Explanations for excluded data are listed in the tables of the individual values

 

 


 

 

 

 

GROUP 1 CONTROL

 

GROUP 2

50 MG/KG

 

GROUP 3

150 MG/KG

 

GROUP 4

500 MG/KG

 

PRE MATING

DAYS 1-8

 

MEAN

 

68

 

70

 

69

 

71

WEEKS 1-2

ST.DEV

1.9

0.2

1.1

2.3

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

67

69

69

70

WEEKS 2-3

ST.DEV

0.0

0.4

2.3

4.1

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER PRE MATI...

 

MEAN

 

68

 

69

 

69

 

71

MATING PERIOD

 

 

 

 

 

DAYS 1-8

MEAN

64

64

65

65

WEEKS 1-2

ST.DEV

1.3

1.0

0.8

2.7

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

58

58

60

61

WEEKS 2-3

ST.DEV

0.8

0.4

2.0

2.3

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER MATING P...

 

MEAN

 

61

 

61

 

63

 

63

 

FEMALES

 

 

 

 

 

 

 

 

GROUP 1

 

GROUP 2

 

GROUP 3

 

GROUP 4

 

 

CONTROL

50 MG/KG

150 MG/KG

500 MG/KG

 

PRE MATING

DAYS 1-8

 

MEAN

 

67

 

66

 

68

 

67

WEEKS 1-2

ST.DEV

1.0

1.5

2.0

1.3

 

N (CAGE)

2

2

2

2

DAYS 8-15

MEAN

65

63

67

68

WEEKS 2-3

ST.DEV

0.9

0.3

0.7

1.1

 

N (CAGE)

2

2

2

2

MEAN OF MEANS OVER PRE MATI...

 

MEAN

 

66

 

64

 

67

 

68


 

F0-GENERATION                                                                                                                                                                                       

RELATIVE FOOD CONSUMPTION (G/KG BODY WEIGHT/DAY) SUMMARY FEMALES

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

POST COITUM

DAYS 0-4

 

MEAN

 

66

 

65

 

65

 

73

 

ST.DEV.

10.6

9.3

12.7

14.4

 

N

10

10

9

9

DAYS 4-7

MEAN

70

69

72

76

 

ST.DEV.

6.2

4.7

5.3

9.4

 

N

10

10

9

9

DAYS 7-11

MEAN

70

67

71

73

 

ST.DEV.

4.4

5.2

6.5

8.5

 

N

10

10

9

9

DAYS 11-14

MEAN

74

72

77

76

 

ST.DEV.

4.3

4.5

8.7

5.4

 

N

10

10

9

9

DAYS 14-17

MEAN

68

66

66

70

 

ST.DEV.

2.2

4.5

5.8

6.1

 

N

10

10

9

9

DAYS 17-20

MEAN

68

66

69

71

 

ST.DEV.

7.0

6.4

7.2

6.8

 

N

10

10

9

9

 

MEAN OF MEANS

 

 

69

 

67

 

70

 

73

LACTATION

DAYS 1-4

 

MEAN

 

93

 

98

 

91

 

92

 

ST.DEV.

12.8

8.5

7.8

18.1

 

N

6

6

8

9

DAYS 4-7

MEAN

130

133

126

125

 

ST.DEV.

12.1

14.5

11.1

13.4

 

N

10

9

9

8

DAYS 7-13

MEAN

161

154

155

154

 

ST.DEV.

9.9

10.3

12.8

16.0

 

N

10

9

9

8

 

MEAN OF MEANS

 

 

128

 

128

 

124

 

124

 

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level Explanations for excluded data are listed in the tables of the individual values

 

 


 

HAEMATOLOGY SUMMARY MALES

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

WBC

 

MEAN

 

6.9

 

7.5

 

5.7

 

6.8

10E9/L

ST.DEV

2.0

1.4

0.9

1.3

 

N

5

5

5

5

Neutrophils

MEAN

14.3

14.6

13.6

12.1

%WBC

ST.DEV

3.1

3.9

3.4

1.7

 

N

5

5

5

4

Lymphocytes

MEAN

82.9

82.7

83.6

85.1

%WBC

ST.DEV

2.7

4.6

3.3

1.9

 

N

5

5

5

4

Monocytes

MEAN

1.7

1.8

1.8

1.9

%WBC

ST.DEV

0.3

0.5

0.6

0.3

 

N

5

5

5

4

Eosinophils

MEAN

1.0

0.9

1.0

0.9

%WBC

ST.DEV

0.5

0.5

0.2

0.2

 

N

5

5

5

4

Basophils

MEAN

0.1

0.1

0.1

0.1

%WBC

ST.DEV

0.1

0.0

0.1

0.0

 

N

5

5

5

4

Red blood cells

MEAN

8.88

8.71

8.78

8.62

10E12/L

ST.DEV

0.21

0.29

0.32

0.34

 

N

5

5

5

5

Reticulocytes

MEAN

2.2

2.5

2.7

2.7

%RBC

ST.DEV

0.3

0.4

0.3

0.6

 

N

5

5

5

5

RDW

MEAN

11.8

12.9

12.1

12.1

%

ST.DEV

0.6

2.1

0.7

0.3

 

N

5

5

5

5

Haemoglobin

MEAN

10.0

9.8

10.0

9.7

mmol/L

ST.DEV

0.2

0.2

0.3

0.2

 

N

5

5

5

5

Haematocrit

MEAN

0.451

0.447

0.463

0.449

L/L

ST.DEV

0.013

0.007

0.018

0.013

 

N

5

5

5

5

MCV

MEAN

50.7

51.4

52.8

52.2

fL

ST.DEV

1.5

1.7

2.1

1.7

 

N

5

5

5

5

MCH

MEAN

1.12

1.13

1.14

1.13

fmol

ST.DEV

0.03

0.04

0.04

0.03

 

N

5

5

5

5

MCHC

MEAN

22.19

22.05

21.58 *

21.67

mmol/L

ST.DEV

0.33

0.52

0.30

0.22

 

N

5

5

5

5

Platelets

MEAN

837

757

755

683 *

10E9/L

ST.DEV

136

20

48

50

 

N

5

5

5

5

 

+/++ Steel-test significant at 5% (+) or 1% (++) level

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

 

 

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

PT

 

MEAN

 

17.9

 

18.6

 

18.1

 

18.4

s

ST.DEV

0.6

0.3

0.5

0.4

 

N

5

5

5

5

APTT

MEAN

15.0

17.1

15.4

16.4

s

ST.DEV

1.3

2.3

1.4

2.4

 

N

5

5

5

5


 

HAEMATOLOGY SUMMARY FEMALES

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

WBC

 

MEAN

 

7.3

 

7.2

 

7.6

 

8.1

10E9/L

ST.DEV

1.0

1.3

1.3

2.1

 

N

5

5

5

5

Neutrophils

MEAN

52.3

45.6

43.5

45.6

%WBC

ST.DEV

9.1

8.4

4.4

6.4

 

N

5

5

5

5

Lymphocytes

MEAN

41.5

48.7

50.9

48.8

%WBC

ST.DEV

7.8

8.2

4.2

6.0

 

N

5

5

5

5

Monocytes

MEAN

4.2

4.5

4.3

4.9

%WBC

ST.DEV

1.4

1.3

0.7

1.2

 

N

5

5

5

5

Eosinophils

MEAN

2.0

1.1

1.1

0.6

%WBC

ST.DEV

1.5

0.5

0.3

0.4

 

N

5

5

5

5

Basophils

MEAN

0.1

0.1

0.1

0.1

%WBC

ST.DEV

0.0

0.0

0.0

0.0

 

N

5

5

5

5

Red blood cells

MEAN

7.79

7.89

7.70

7.57

10E12/L

ST.DEV

0.50

0.70

0.46

0.58

 

N

5

5

5

5

Reticulocytes

MEAN

3.4

3.2

2.9

3.5

%RBC

ST.DEV

0.7

0.5

0.5

0.5

 

N

5

5

5

5

RDW

MEAN

13.1

13.7

14.7

13.4

%

ST.DEV

1.1

1.1

1.3

0.5

 

N

5

5

5

5

Haemoglobin

MEAN

9.3

9.4

9.2

9.2

mmol/L

ST.DEV

0.9

1.0

0.6

0.7

 

N

5

5

5

5

Haematocrit

MEAN

0.433

0.434

0.431

0.442

L/L

ST.DEV

0.036

0.047

0.038

0.047

 

N

5

5

5

5

MCV

MEAN

55.6

55.0

56.0

58.3

fL

ST.DEV

1.7

2.2

2.0

1.8

 

N

5

5

5

5

MCH

MEAN

1.19

1.19

1.19

1.21

fmol

ST.DEV

0.05

0.04

0.02

0.02

 

N

5

5

5

5

MCHC

MEAN

21.43

21.66

21.28

20.77

mmol/L

ST.DEV

0.32

0.40

0.52

0.60

 

N

5

5

5

5

Platelets

MEAN

782

793

803

801

10E9/L

ST.DEV

78

36

126

184

 

N

5

5

5

5


 

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

PT

 

MEAN

 

17.3

 

17.4

 

17.7

 

17.3

s

ST.DEV

0.5

1.4

0.8

0.6

 

N

5

5

5

5

APTT

MEAN

13.1

13.4

12.7

14.2

s

ST.DEV

1.7

2.5

0.7

2.0

 

N

5

5

5

5


 

CLINICAL BIOCHEMISTRY SUMMARY MALES

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

ALAT

 

MEAN

 

49.8

 

47.7

 

43.1

 

44.6

U/L

ST.DEV

20.5

10.4

9.3

6.8

 

N

5

5

5

5

ASAT

MEAN

86.4

82.8

85.2

85.8

U/L

ST.DEV

4.0

4.7

10.0

13.3

 

N

5

5

5

5

ALP

MEAN

179

162

151

169

U/L

ST.DEV

62

57

59

39

 

N

5

5

5

5

Total protein

MEAN

65.0

64.3

63.0

63.0

g/L

ST.DEV

2.1

1.6

1.2

2.1

 

N

5

5

5

5

Albumin

MEAN

34.7

34.1

33.7

33.2 *

g/L

ST.DEV

1.2

0.7

0.6

1.1

 

N

5

5

5

5

Total bilirubin

MEAN

2.5

2.0 *

2.1

2.1

umol/L

ST.DEV

0.3

0.2

0.3

0.3

 

N

5

5

5

5

Urea

MEAN

8.1

8.0

7.9

7.7

mmol/L

ST.DEV

2.5

1.1

1.5

0.2

 

N

5

5

5

5

Creatinine

MEAN

41.1

41.1

41.1

40.3

umol/L

ST.DEV

2.9

1.2

1.5

1.3

 

N

5

5

5

5

Glucose

MEAN

7.94

8.26

8.55

8.19

mmol/L

ST.DEV

1.07

1.27

1.19

0.82

 

N

5

5

5

5

Cholesterol

MEAN

1.92

1.89

1.85

2.03

mmol/L

ST.DEV

0.16

0.25

0.36

0.40

 

N

5

5

5

5

Bile Acids

MEAN

28.7

13.6 *

16.1 *

16.1 *

umol/L

ST.DEV

11.8

2.6

6.5

6.7

 

N

5

5

5

5

Sodium

MEAN

141.0

140.6

141.5

141.2

mmol/L

ST.DEV

0.2

0.3

1.5

0.4

 

N

5

5

5

5

Potassium

MEAN

3.59

4.02 **

3.75

3.82 *

mmol/L

ST.DEV

0.11

0.13

0.16

0.13

 

N

5

5

5

5

Chloride

MEAN

102

103

102

103

mmol/L

ST.DEV

1

1

1

1

 

N

5

5

5

5

Calcium

MEAN

2.60

2.58

2.56

2.60

mmol/L

ST.DEV

0.08

0.05

0.03

0.03

 

N

5

5

5

5

 

 

 

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 
  - Page 19 -

- Page 19 -


 

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

Inorg.Phos

 

MEAN

 

1.99

 

2.05

 

1.94

 

1.99

mmol/L

ST.DEV

0.20

0.20

0.18

0.21

 

N

5

5

5

5

Total T4

MEAN

4.93

4.00

4.06

4.34

ug/dL

ST.DEV

1.58

0.76

0.81

0.92

 

N

10

10

10

10

 

 

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

                                              

                                              


 

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

ALAT

 

MEAN

 

75.1

 

59.9

 

79.7

 

76.5

U/L

ST.DEV

17.8

11.6

29.2

13.9

 

N

5

5

5

5

ASAT

MEAN

118.1

100.0

112.1

112.1

U/L

ST.DEV

24.7

15.0

29.2

22.4

 

N

5

5

5

5

ALP

MEAN

108

125

162

129

U/L

ST.DEV

29

38

67

62

 

N

5

5

5

5

Total protein

MEAN

59.0

61.5

62.9

60.8

g/L

ST.DEV

9.7

2.1

1.3

2.3

 

N

5

5

5

5

Albumin

MEAN

31.7

32.8

33.0

31.8

g/L

ST.DEV

4.3

0.1

0.6

1.5

 

N

5

5

5

5

Total bilirubin

MEAN

2.7

2.3

2.3

2.2

umol/L

ST.DEV

0.3

0.6

0.4

0.4

 

N

5

5

5

5

Urea

MEAN

14.2

10.9

11.2

10.2

mmol/L

ST.DEV

4.9

0.6

0.9

2.1

 

N

5

5

5

5

Creatinine

MEAN

47.5

45.8

44.4

43.2

umol/L

ST.DEV

3.0

2.5

3.0

3.1

 

N

5

5

5

5

Glucose

MEAN

7.03

7.98

7.92

7.96

mmol/L

ST.DEV

1.00

1.07

0.98

0.70

 

N

5

5

5

5

Cholesterol

MEAN

1.80

2.15

2.30

2.18

mmol/L

ST.DEV

0.17

0.35

0.39

0.56

 

N

5

5

5

5

Bile Acids

MEAN

60.7

58.0

39.3

32.6

umol/L

ST.DEV

60.7

72.3

26.1

7.8

 

N

5

5

5

5

Sodium

MEAN

134.6

137.8 **

136.8 *

137.2 **

mmol/L

ST.DEV

0.9

1.7

1.2

0.9

 

N

5

5

5

5

Potassium

MEAN

3.62

3.53

3.98

3.79

mmol/L

ST.DEV

0.29

0.46

0.58

0.50

 

N

5

5

5

5

Chloride

MEAN

94

96

97

97

mmol/L

ST.DEV

2

3

1

2

 

N

5

5

5

5

Calcium

MEAN

2.53

2.63

2.70

2.61

mmol/L

ST.DEV

0.23

0.12

0.10

0.04

 

N

5

5

5

5


 

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

Inorg.Phos

 

MEAN

 

3.08

 

2.91

 

2.83

 

2.68

mmol/L

ST.DEV

0.38

0.55

0.53

0.34

 

N

5

5

5

5

Total T4

MEAN

---

---

---

---

ug/dL

ST.DEV

---

---

---

---

 

N

0

0

0

0


 

MACROSCOPIC FINDINGS SUMMARY MALES

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

Animals examined

 

10

 

10

 

10

 

10

Animals without findings

7

7

9

10

Animals affected

3

3

1

0

Epididymides

 

 

 

 

Nodule(s) Cowper's gland

1

1

0

0

Agenesis

0

1

0

0

Thymus Focus/foci

 

1

 

1

 

1

 

0

Mandibular lymph n

 

 

 

 

Discolouration

1

0

0

0

 

FEMALES

 

 

 

 

 

 

GROUP 1

 

GROUP 2

 

GROUP 3

 

GROUP 4

 

CONTROL

50 MG/KG

150 MG/KG

500 MG/KG

 

INTERCURRENT DEATH

 

 

 

 

Animals examined

 

1

 

1

Animals affected

 

1

 

1

General observations

 

 

 

 

Total litter loss

 

1

 

1

END OF TREATMENT

Animals examined

 

10

 

9

 

10

 

9

Animals without findings

7

4

5

5

Animals affected

3

5

5

4

Stomach Focus/foci

 

3

 

1

 

1

 

1

Uterus Contains fluid

 

0

 

0

 

1

 

1

Clitoral glands

 

 

 

 

Focus/foci

0

2

3

1

Thyroid gland

Reduced in size

 

0

 

1

 

0

 

0

Thymus Focus/foci

 

0

 

1

 

0

 

0

Reduced in size

0

1

0

0

Discolouration

0

1

1

0

Skin

Alopecia

 

0

 

1

 

1

 

1

 

 

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

 

 

 

 

 

                                   

                                   


 

ORGAN WEIGHTS (GRAM) SUMMARY MALES

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

BODY W.

 

MEAN

 

349

 

356

 

345

 

354

(GRAM)

ST.DEV

12

24

19

19

 

N

10

10

10

10

BRAIN

MEAN

2.07

2.05

2.01

2.04

(GRAM)

ST.DEV

0.09

0.07

0.05

0.04

 

N

5

5

5

5

HEART

MEAN

0.928

1.011

0.895

1.003

(GRAM)

ST.DEV

0.069

0.069

0.023

0.092

 

N

5

5

5

5

LIVER

MEAN

9.22

9.22

9.05

9.34

(GRAM)

ST.DEV

0.93

0.93

0.73

1.19

 

N

5

5

5

5

THYROIDS

MEAN

0.015

0.015

0.018 *

0.016

(GRAM)

ST.DEV

0.002

0.002

0.003

0.003

 

N

10

10

10

10

THYMUS

MEAN

0.334

0.409

0.353

0.365

(GRAM)

ST.DEV

0.071

0.052

0.042

0.125

 

N

5

5

5

5

KIDNEYS

MEAN

2.40

2.46

2.39

2.43

(GRAM)

ST.DEV

0.06

0.12

0.19

0.15

 

N

5

5

5

5

ADRENALS

MEAN

0.060

0.064

0.072

0.058

(GRAM)

ST.DEV

0.008

0.008

0.006

0.007

 

N

5

5

5

5

SPLEEN

MEAN

0.586

0.672

0.640

0.639

(GRAM)

ST.DEV

0.055

0.091

0.110

0.089

 

N

5

5

5

5

TESTES

MEAN

3.46

3.57

3.59

3.47

(GRAM)

ST.DEV

0.28

0.32

0.27

0.19

 

N

10

10

10

10

PROSTATE GLAND

MEAN

0.893

0.942

1.043

0.916

(GRAM)

ST.DEV

0.140

0.183

0.111

0.104

 

N

10

10

10

10

EPIDIDYMIDES

MEAN

1.134

1.096

1.091

1.077

(GRAM)

ST.DEV

0.085

0.118

0.065

0.028

 

N

10

10

10

10

SEMINAL VESICLES

MEAN

1.638

1.415 *

1.424 *

1.514

(GRAM)

ST.DEV

0.155

0.164

0.215

0.210

 

N

10

10

10

10

 

 

 

 

 


 

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

BODY W.

 

MEAN

 

349

 

356

 

345

 

354

(GRAM)

ST.DEV

12

24

19

19

 

N

10

10

10

10

BRAIN

MEAN

0.59

0.58

0.58

0.58

(%)

ST.DEV

0.04

0.04

0.03

0.03

 

N

5

5

5

5

HEART

MEAN

0.264

0.285

0.258

0.282

(%)

ST.DEV

0.025

0.021

0.011

0.007

 

N

5

5

5

5

LIVER

MEAN

2.62

2.59

2.61

2.62

(%)

ST.DEV

0.17

0.12

0.12

0.15

 

N

5

5

5

5

THYROIDS

MEAN

0.004

0.004

0.005 *

0.004

(%)

ST.DEV

0.001

0.001

0.001

0.001

 

N

10

10

10

10

THYMUS

MEAN

0.094

0.116

0.102

0.101

(%)

ST.DEV

0.017

0.016

0.008

0.028

 

N

5

5

5

5

KIDNEYS

MEAN

0.68

0.69

0.69

0.68

(%)

ST.DEV

0.03

0.03

0.04

0.02

 

N

5

5

5

5

ADRENALS

MEAN

0.017

0.018

0.021 *

0.016

(%)

ST.DEV

0.002

0.002

0.003

0.002

 

N

5

5

5

5

SPLEEN

MEAN

0.167

0.189

0.184

0.180

(%)

ST.DEV

0.020

0.020

0.025

0.023

 

N

5

5

5

5

TESTES

MEAN

0.99

1.01

1.04

0.98

(%)

ST.DEV

0.07

0.09

0.10

0.06

 

N

10

10

10

10

PROSTATE GLAND

MEAN

0.257

0.264

0.302 *

0.259

(%)

ST.DEV

0.045

0.046

0.033

0.026

 

N

10

10

10

10

EPIDIDYMIDES

MEAN

0.325

0.309

0.317

0.305

(%)

ST.DEV

0.026

0.037

0.027

0.018

 

N

10

10

10

10

SEMINAL VESICLES

MEAN

0.471

0.399 *

0.412 *

0.427

(%)

ST.DEV

0.051

0.052

0.051

0.053

 

N

10

10

10

10


 

ORGAN WEIGHTS (GRAM) SUMMARY FEMALES

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

BODY W.

 

MEAN

 

266

 

266

 

263

 

267

(GRAM)

ST.DEV

12

17

15

28

 

N

10

9

10

10

BRAIN

MEAN

1.91

1.91

1.96

1.94

(GRAM)

ST.DEV

0.02

0.05

0.10

0.07

 

N

5

5

5

5

HEART

MEAN

0.805

0.841

0.805

0.874

(GRAM)

ST.DEV

0.076

0.072

0.051

0.097

 

N

5

5

5

5

LIVER

MEAN

8.79

9.22

9.20

9.99

(GRAM)

ST.DEV

0.34

0.82

0.78

1.02

 

N

5

5

5

5

THYROIDS

MEAN

0.014

0.013

0.015

0.015

(GRAM)

ST.DEV

0.002

0.003

0.003

0.002

 

N

10

9

10

10

THYMUS

MEAN

0.214

0.182

0.199

0.236

(GRAM)

ST.DEV

0.038

0.052

0.049

0.042

 

N

5

5

5

5

KIDNEYS

MEAN

1.80

1.98

1.98

2.03

(GRAM)

ST.DEV

0.10

0.14

0.28

0.23

 

N

5

5

5

5

ADRENALS

MEAN

0.075

0.078

0.069

0.076

(GRAM)

ST.DEV

0.009

0.005

0.015

0.004

 

N

5

5

5

5

SPLEEN

MEAN

0.518

0.527

0.521

0.570

(GRAM)

ST.DEV

0.089

0.056

0.088

0.078

 

N

5

5

5

5

OVARIES

MEAN

0.111

0.121

0.111

0.119

(GRAM)

ST.DEV

0.010

0.024

0.012

0.010

 

N

5

5

5

5

UTERUS

MEAN

0.386

0.503

0.464

0.559

(GRAM)

ST.DEV

0.052

0.111

0.084

0.211

 

N

5

5

5

5


 

ORGAN/BODY WEIGHT RATIOS (%) SUMMARY FEMALES

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

END OF TREATMENT

BODY W.

 

MEAN

 

266

 

266

 

263

 

267

(GRAM)

ST.DEV

12

17

15

28

 

N

10

9

10

10

BRAIN

MEAN

0.72

0.72

0.76

0.69

(%)

ST.DEV

0.03

0.04

0.02

0.04

 

N

5

5

5

5

HEART

MEAN

0.305

0.317

0.310

0.308

(%)

ST.DEV

0.025

0.015

0.009

0.024

 

N

5

5

5

5

LIVER

MEAN

3.33

3.47

3.55

3.52

(%)

ST.DEV

0.12

0.25

0.25

0.27

 

N

5

5

5

5

THYROIDS

MEAN

0.005

0.005

0.006

0.006

(%)

ST.DEV

0.001

0.001

0.001

0.001

 

N

10

9

10

10

THYMUS

MEAN

0.080

0.068

0.077

0.083

(%)

ST.DEV

0.011

0.018

0.021

0.010

 

N

5

5

5

5

KIDNEYS

MEAN

0.68

0.74

0.76

0.71

(%)

ST.DEV

0.03

0.04

0.08

0.03

 

N

5

5

5

5

ADRENALS

MEAN

0.028

0.029

0.027

0.027

(%)

ST.DEV

0.004

0.000

0.005

0.002

 

N

5

5

5

5

SPLEEN

MEAN

0.197

0.199

0.201

0.201

(%)

ST.DEV

0.038

0.019

0.031

0.025

 

N

5

5

5

5

OVARIES

MEAN

0.042

0.045

0.043

0.042

(%)

ST.DEV

0.005

0.006

0.005

0.002

 

N

5

5

5

5

UTERUS

MEAN

0.146

0.188

0.178

0.195

(%)

ST.DEV

0.016

0.031

0.030

0.066

 

N

5

5

5

5


 

 

REPRODUCTIONDATASUMMARY

 

GROUP 1CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

Females paired

 

10

 

10

 

10

 

10

Females mated

10

10

9

10

Pregnant females

10

10

9

9

Females with total litter loss on Day 1

0

1

0

0

Females with living pups on Day 1

10

9

9

9

 

Mating index (%)

 

100

 

100

 

90

 

100

(Females mated / Females paired) * 100

 

 

 

 

Fertility index (%)

100

100

90

90

(Pregnant females / Females paired) * 100

 

 

 

 

Conception index (%)

100

100

100

90

(Pregnant females / Females mated) * 100

 

 

 

 

Gestation index (%)

100

90

100

100

(Females with living pups on Day 1 / Pregnant females) * 100

 

 

 

 



 

F0-GENERATION - POSTCOITUM                                                                                                                                                         

 

DAY OF THE PAIRING PERIOD

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

NUMBER OF FEMALES MATED

1

 

2

 

1

 

2

 

4

2

3

1

3

2

3

3

4

3

1

4

2

4

1

3

MEDIAN PRECOITAL TIME

3

3

2

2

MEAN PRECOITAL TIME

2.5

3.1

2.3

2.3

N

10

10

9

10


 

IMPLANTATION SITES FEMALES

SUMMARY

 

 

 

 

GROUP 1

 

GROUP 2

 

GROUP 3

 

GROUP 4

 

 

CONTROL

50 MG/KG

150 MG/KG

500 MG/KG

 

NECROPSY

Implantations

 

MEAN

 

12.5

 

12.1

 

13.4

 

10.0

 

ST.DEV

2.4

4.4

1.6

4.2

 

N

10

10

9

9


 

F0-GENERATION - LACTATION

GROUP 1 CONTROL

GROUP 2

GROUP 3

GROUP 4

50 MG/KG

150 MG/KG

500 MG/KG

LITTERS TOTAL

10

10

9

9

DURATION OF GESTATION MEAN (+)

21.2

21.6

21.1

21.7

ST.DEV.

0.4

0.5

0.3

0.7

  N

10

10

9

9

DEAD PUPS AT FIRST LITTER CHECK

LITTERS AFFECTED (#)

3

1

2

1

TOTAL

4

1

3

1

MEAN (+)

0.4

0.1

0.3

0.1

ST.DEV.

0.7

0.3

0.7

0.3

N

10

10

9

9

LIVING PUPS AT FIRST LITTER CHECK

% OF MALES / FEMALES (#)

51 / 49

52 / 48

45 / 55

53 / 47

TOTAL

113

108

109

85

MEAN (+)

11.3

10.8

12.1

9.4

ST.DEV.

2

4.6

2.3

4.2

N

10

10

9

9

POSTNATAL LOSS

% OF LIVING PUPS

5.3

1.9

5.5

3.5

LITTERS AFFECTED (#)

4

2

4

2

TOTAL (#)

6

2

6

3

MEAN (+)

0.6

0.2

0.7

0.3

ST.DEV.

1

0.4

0.9

0.7

N

10

10

9

9

CULLED PUPS

TOTAL

28

35

32

22

LIVING PUPS DAY 4 P.P.

TOTAL

79

71

71

60

MEAN (+)

7.9

7.1

7.9

6.7

ST.DEV.

0.3

2.5

0.3

2.8

N

10

10

9

9

BREEDING LOSS DAYS 5 - 13 P.P.

% OF LIVING PUPS AT DAY 4 P.P.

0

0

1.4

0

LITTERS AFFECTED (#)

0

0

1

0

TOTAL (#)

0

0

1

0

MEAN (+)

0

0

0.1

0

ST.DEV.

0

0

0.3

0

N

10

10

9

9

LIVING PUPS DAY 13 P.P.

% OF MALES / FEMALES (#)

48 / 52

51 / 49

49 / 51

53 / 47

TOTAL

79

71

70

60

MEAN (+)

7.9

7.1

7.8

6.7

ST.DEV.

0.3

2.5

0.4

2.8

N

10

10

9

9

 

 

Steel-test significant at 5% (+) or 1% (++) level


DEVELOPMENTAL DATA

 

 

 

GROUP 1

 

GROUP 2

 

GROUP 3

 

GROUP 4

 

CONTROL

50 MG/KG

150 MG/KG

500 MG/KG

 

Total number of offspring born

 

117

 

109

 

112

 

86

Total number of uterine implantation sites

125

121

121

90

Number of live offspring on Day 1 after littering

113

108

109

85

Number of live offspring on Day 4 (before culling)

107

106

103

82

Number of live offspring on Day 4 (after culling)

79

71

71

60

Number of live offspring on Day 13 after littering

79

71

70

60

 

Post-implantation survival index (%)

 

94

 

90

 

93

 

96

(Total number of offspring born/Total number of uterine implantation sites) * 100

 

 

 

 

Live birth index (%)

97

99

97

99

(Number of live offspring on Day 1 after littering/Total number of offspring born) * 100

 

 

 

 

Viability index (%)

95

98

94

96

(Number of live offspring on Day 4 (before culling)/Number of live offspring on Day 1 after littering)*100

 

 

 

 

Lactation index (%)

100

100

99

100

(Number of live offspring on Day 13 after littering/Number of live offspring on Day 4 (after culling)) * 100

 

 

 

 

BODY WEIGHTS OF PUPS(GRAM)

 

 

F0-GENERATION -LACTATION                                                                                                                                                              

 

DAY

SEX

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

1

M

MEAN

6.0

6.5

6.0

7.1 *

 

 

ST.DEV.

0.7

0.6

0.9

1.3

 

 

N

10

9

9

9

 

F

MEAN

5.7

6.2

5.6

6.8 *

 

 

ST.DEV.

0.8

0.7

0.9

1.4

 

 

N

10

9

9

9

 

M+F

MEAN

5.9

6.4

5.8

7.0 *

 

 

ST.DEV.

0.7

0.6

0.9

1.4

 

 

N

10

9

9

9

4

M

MEAN

8.7

9.4

8.7

10.5

 

 

ST.DEV.

1.3

1.0

1.3

2.4

 

 

N

10

9

9

8

 

F

MEAN

8.4

8.9

8.1

10.2 *

 

 

ST.DEV.

1.3

1.2

1.3

2.3

 

 

N

10

9

9

8

 

M+F

MEAN

8.5

9.2

8.4

10.4 *

 

 

ST.DEV.

1.2

1.0

1.3

2.4

 

 

N

10

9

9

8

7

M

MEAN

14.4

15.1

14.3

16.6 *

 

 

ST.DEV.

1.5

1.1

1.6

3.1

 

 

N

10

9

9

8

 

F

MEAN

14.0

14.5

13.3

16.2

 

 

ST.DEV.

1.3

1.2

1.7

3.3

 

 

N

10

9

9

8

 

M+F

MEAN

14.2

14.8

13.8

16.4

 

 

ST.DEV.

1.3

1.1

1.7

3.2

 

 

N

10

9

9

8

13

M

MEAN

28.5

28.5

27.7

30.9

 

 

ST.DEV.

1.6

1.2

2.1

5.1

 

 

N

10

9

9

8

 

F

MEAN

27.7

27.7

26.3

29.8

 

 

ST.DEV.

1.6

1.4

2.4

5.2

 

 

N

10

9

9

8

 

M+F

MEAN

28.1

28.1

26.9

30.4

 

 

ST.DEV.

1.5

1.3

2.2

5.1

 

 

N

10

9

9

8

 

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level


 

F0-GENERATION -LACTATION                                                                                                                                                              

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

anogenital dist M mm

MEAN

2.48

2.59

2.60

2.70

 

ST. DEV.

0.14

0.09

0.13

0.27

 

N

10

9

9

9

anogenital dist F mm

MEAN

1.05

0.97

0.94

1.11

 

ST.DEV.

0.17

0.02

0.18

0.21

 

N

10

9

9

9

Number of nipples

MEAN

0.00

0.00

0.00

0.00

 

MEDIAN (+)

0.00

0.00

0.00

0.00

 

N

10

9

9

8

 

  

 

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level


 

 

CORRECTED ANOGENITAL DISTANCE SUMMARY FEMALES

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

PND 1

norm anog dist M

 

MEAN

 

1.37

 

1.39

 

1.43

 

1.41

mm

ST.DEV

0.07

0.03

0.10

0.06

 

N

10

9

9

9

norm anog dist F

MEAN

0.59

0.53

0.53

0.58

mm

ST.DEV

0.10

0.02

0.10

0.09

 

N

10

9

9

9


 

CLINICAL BIOCHEMISTRY SUMMARY MALES

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

PUPS (PND 13-15)

Total T4

 

MEAN

 

6.60

 

7.36

 

5.76

 

6.65

ug/dL

ST.DEV

1.36

2.01

1.13

0.83

 

N

10

9

9

8


 

CLINICAL BIOCHEMISTRY SUMMARY FEMALES

 

 

GROUP 1 CONTROL

GROUP 2

50 MG/KG

GROUP 3

150 MG/KG

GROUP 4

500 MG/KG

 

PUPS (PND 13-15)

Total T4

 

MEAN

 

6.09

 

6.32

 

5.62

 

5.88

ug/dL

ST.DEV

1.31

1.03

1.05

1.01

 

N

10

9

9

8

 

Conclusions:
No reproduction toxicity was observed up to the highest dose level tested (500 mg/kg).
No treatment-related or toxicologically relevant changes were noted in any of reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

No developmental toxicity was observed up to the highest dose level tested (500 mg/kg).
No treatment-related or toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopy).

In conclusion, based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 500 mg/kg was derived.
The parental No Observed Effect Level (NOEL) was established at 150 mg/kg based on non-adverse microscopic changes in the thyroids of males at 500 mg/kg.
Executive summary:

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of LOWINOX® TBP-6 in rats by oral gavage.

The study was based on the following guidelines:

• OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2016.

• OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

• OECD 421, Reproduction/Developmental Toxicity Screening Test, July 2016.

• OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

• EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

• OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

• OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

Based on the results of a dose range finding study in which toxicity was noted at 500 mg/kg but not at 250 mg/kg (Test Facility Study No. 513610 ), the dose levels for this combined 28 -day oral gavage study with reproduction/developmental toxicity screening test were selected to be 50, 150 and 500 mg/kg.

The test item, LOWINOX® TBP-6, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated for 50-56 days, i.e. during 2 weeks prior to mating, during

mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were treated for 38-45 days.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of

treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment, PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopy).

Formulations were analyzed once during the study to assess accuracy and homogeneity.

There were no toxicologically relevant changes in the in-life parameters up to 500 mg/kg.

Slight salivation was noted after dosing at all dose levels, most frequently at 150 and 500 mg/kg. This was considered to be a physiological response rather than a sign of systemic toxicity. Treatment-related microscopic changes were present in the thyroid of male rats, characterized by an increased incidence and severity of follicular cell hypertrophy starting at 150 mg/kg, and alteration of the colloid at 500 mg/kg. These findings were regarded as non-adverse based on their low degree (up to slight) and the absence of other treatment-related findings.

No treatment-related or toxicologically relevant changes were noted in clinical pathology parameters, organ weights or findings at macroscopic examination.

In conclusion:

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 500 mg/kg was derived.

The parental No Observed Effect Level (NOEL) was established at 150 mg/kg based on non-adverse microscopic changes in the thyroids of males at 500 mg/kg.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
K1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test of LOWINOX® TBP-6 in rats by oral gavage.

The study was based on the following guidelines:

• OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2016.

• OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

• OECD 421, Reproduction/Developmental Toxicity Screening Test, July 2016.

• OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

• EC No 440/2008 B.7: "Repeated Dose (28 days) Toxicity (oral)", May 2008.

• OECD 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.

• OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

Based on the results of a dose range finding study in which toxicity was noted at 500 mg/kg but not at 250 mg/kg (Test Facility Study No. 513610 ), the dose levels for this combined 28 -day oral gavage study with reproduction/developmental toxicity screening test were selected to be 50, 150 and 500 mg/kg.

The test item, LOWINOX® TBP-6, formulated in propylene glycol, was administered daily by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females. Males were treated for 31 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were treated for 50-56 days, i.e. during 2 weeks prior to mating, during

mating, during post-coitum, and during 13-15 days of lactation. Females which failed to deliver healthy offspring were treated for 38-45 days.

The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of

treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment, PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues. In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopy).

Formulations were analyzed once during the study to assess accuracy and homogeneity.

There were no toxicologically relevant changes in the in-life parameters up to 500 mg/kg.

Slight salivation was noted after dosing at all dose levels, most frequently at 150 and 500 mg/kg. This was considered to be a physiological response rather than a sign of systemic toxicity. Treatment-related microscopic changes were present in the thyroid of male rats, characterized by an increased incidence and severity of follicular cell hypertrophy starting at 150 mg/kg, and alteration of the colloid at 500 mg/kg. These findings were regarded as non-adverse based on their low degree (up to slight) and the absence of other treatment-related findings.

No treatment-related or toxicologically relevant changes were noted in clinical pathology parameters, organ weights or findings at macroscopic examination.

In conclusion:

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 500 mg/kg was derived.

The parental No Observed Effect Level (NOEL) was established at 150 mg/kg based on non-adverse microscopic changes in the thyroids of males at 500 mg/kg.

Effects on developmental toxicity

Description of key information

No developmental toxicity was observed up to the highest dose level tested (500 mg/kg).

No treatment-related or toxicologically relevant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and early postnatal pup

development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 thyroid hormone levels and macroscopy).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
K1
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

Due to the lack of effects noted on the sexual function, fertility and development parameters, 6,6’-di-tert-butyl-2,2’-thiodi-p-cresol is not classified for reproductive toxicity in accordance with Regulation (EC) 1272/2008.

Additional information