Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 21st November 2014 Experimental completion date: 20th August 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Liquid
Details on test material:
Identification : PP-R-001 (CAS#1613243-54-1)
Physical State/Appearance :Clear, colorless liquid
Chemical Name :1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives
IUPAC Name : Reaction mass of 3,6,9-triethyl-3,6,9-trimethyl-1,2,4,5,7,8-hexoxonane and 3,6-diethyl-3,6,9-trimethyl-9-n-propyl-1 ,2,4,5,7,8-hexoxonane and 3-ethyl-3,6,9-trimethyl-6,9-di-n-propyl-1,2,4,5,7,8-hexoxonane and 3,6,9-trimethyl-3,6, 9-tri-n-propyl-1,2,4,5,7,8-hexoxonane
CAS Number : 1613243-54-1
Purity : not supplied
Batch Number : BYK004856-98
Label : PP-R-001 Batch: BYK004856-098 Exp: 03/04/2017 To maintain quality store in original container below 25 °C
Date Received : 24 September 2014
Storage Conditions :approximately 4 °C in the dark
Expiry Date : 03 April 2017

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nineteen days during which time their health status was assessed. A total of ninety six animals (forty eight males and forty eight females) were accepted into the study. At the start of treatment the males weighed 282 to 347g, the females weighed 189 to 220g, and were approximately twelve weeks old.

Initially, all animals were housed in groups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the pairing phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.

The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. A certificate of analysis of the batch of diet used is given in Appendix 30. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for paired animals and mated females during gestation and lactation. Mated females were also given softwood flakes, as bedding, throughout gestation and lactation. The diet, drinking water, bedding and environmental enrichment was considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from these targets.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least twenty days. Formulations were therefore prepared fortnightly and stored at approximately 4 °C in the dark.

Samples of the test item formulation were taken and analyzed for concentration of PP-R-001 (CAS#1613243-54-1) at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 26. The results indicate that the prepared formulations were within ± 5% of the nominal concentration.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test item concentration in the test samples was determined by gas chromatography (GC) using an external standard technique. The test item gave a chromatographic profile consisting of a profile of multiple peaks.

Preparation of standard solutions
Stock solutions of test item in methanol were prepared for external standard calibration. An aliquot, approximately 0.05 g of test item was accurately weighed into a 10 mL volumetric flask and brought to volume with methanol to yield a solution with a concentration of 0.5 mg/mL. Aliquots of this stock standard solution were used to prepare working standard solutions in methanol with a concentration of 0.1 mg/mL.

On each occasion standard solutions derived from two stock standard solutions were used for calculation.

Analysis of samples
The formulations received were extracted with methanol. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with methanol. this was then ultra-sonicated for 15 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further distilled with methanol to achieve the working concentration.

Preparation of accuracy samples
Samples of Arachis Oil BP were accurately fortified with known amounts of test item equivalent to the highest and lowest anticipated dose concentrations. These samples were then prepared for analysis.

Preparation of linearity standards
A range of standard solutions were prepared in methanol from a stock solution of 0.513 mg/mL by serial dilution covering the concentration range 0 to 0.2052 mg/mL.

Instrumental Setup
GC system: Agilent Technologies 6890, incorporating autosampler and workstation
Column: RXI-5 ms (15 m x 0.25 nm id x 1 µm film)
Oven temperature programme: Oven: 40°C for 1 minute with 20°C/minute to 300°C for 2 minutes
Injection temperature: Cool on column
Flame ionisation detector temperature: 300°C
Injection volume: 1 µL
Retention time: -2.5 to 7.5 mins

Study samples and storage
Representative samples were dispatched to the analytical laboratories internally (under ambient conditions) and stored at room temperature until analysis.
Duration of treatment / exposure:
Up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females)
Frequency of treatment:
Daily
Details on study schedule:
Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. Prior to the start of treatment and once weekly thereafter, all animals were observed for signs of functional/behavioral toxicity.
iii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iv. Following evidence of mating (designated as Day 0 post coitum) the males were returned to their original cages and females were transferred to individual cages.
v. On completion of the pairing phase (during Week 6), five selected males per dose group were evaluated for functional/sensory responses to various stimuli.
vi. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
vii. At Day 4 post partum, the first five selected females per dose group that maintained a litter were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from the first five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were euthanized and examined macroscopically on Day 43 or Day 44.
ix. Blood samples were taken from the first five selected females that maintained a litter from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and surviving offspring were euthanized and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
50 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
12 males and 12 females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before dosing, soon after dosing, and one hour after dosing during the working week, at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.

Behavioral Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:

Gait
Hyper/Hypothermia
Tremors
Skin color
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behavior
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Purpose-built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time on each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).

The following parameters were observed:

Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until pairing. During pairing phase females were weighed daily until mating was confirmed. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum. Body weights were also recorded at terminal kill.

Food Consumption
During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated for females during gestation and lactation.

Water Consumption
Water intake was measured daily during the pre-pairing phase of the study.

Reproductive Performance
Normal range data for reproductive parameters and offspring are presented in Appendix 32 and Appendix 33.

Mating
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages (unless required for additional pairing). Mated females were housed individually during the period of gestation and lactation.

Pregnancy and Parturition
Each pregnant female was observed at least three times a day (early morning, mid-day and as late as possible during the normal working day) around the period of expected parturition. Observations were carried out at approximately 0830 and as late as possible at weekends and public holidays. The following was recorded for each female:

i. Date of pairing
ii. Date of mating
iii. Date and time of observed start of parturition
iv. Date and time of observed completion of parturition

Laboratory Investigations
Hematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

Hematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:

Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:

Urea
Calcium (Ca++)
Glucose
Inorganic phosphorus (P)
Total protein (Tot.Prot.)
Aspartate aminotransferase (ASAT)
Albumin
Alanine aminotransferase (ALAT)
Albumin/Globulin (A/G) ratio (by calculation)
Alkaline phosphatase (AP)
Sodium (Na+)
Creatinine (Creat)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Bile acids
Oestrous cyclicity (parental animals):
A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded.
Sperm parameters (parental animals):
Semen was not assessed until post-mortem
Litter observations:
Litter Data
On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.

For each litter the following was recorded:

i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
Pathology
Necropsy
Adult males were euthanized by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or Day 44. Adult females were euthanized by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were euthanized via intracardiac overdose of suitable barbiturate agent. Any females which failed to achieve pregnancy were euthanized on Day 26 post coitum.

For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each horn was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.

All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were dissected free from fat and weighed before fixation from the first five selected males and the first five selected females that maintained a litter to Day 4 post partum from each dose group. Tissues shown in bold were weighed from all remaining animals:
Adrenals
Prostate
Brain
Seminal vesicles
Epididymides
Spleen
Heart
Testes
Kidneys
Thymus
Liver
Thyroid (weighed post-fixation with Parathyroid)
Ovaries
Uterus (weighed with Cervix)
Pituitary (post fixation)

Histopathology
Samples of the following tissues were removed from five selected males and five selected females from each dose group and preserved in buffered 10% formalin, except where stated. Tissues shown in bold were preserved from all remaining animals:

Adrenals
Muscle (skeletal)
Aorta (thoracic)
Ovaries
Bone & bone marrow (femur including stifle joint)
Pancreas
Bone & bone marrow (sternum)
Pituitary
Brain (including cerebrum, cerebellum and pons)
Prostate
Caecum
Rectum
Coagulating gland
Salivary glands (submaxillary)
Colon
Sciatic nerve
Duodenum
Seminal vesicles
Epididymides•
Skin
Esophagus
Spinal cord (cervical, mid-thoracic and lumbar)
Eyes*
Gross lesions
Spleen
Heart
Stomach
Ileum (including peyer’s patches)
Thyroid/parathyroid
Jejunum
Trachea
Kidneys
Testes•
Liver
Thymus
Lungs (with bronchi) #
Urinary bladder
Lymph nodes (mandibular and mesenteric)
Uterus/Cervix
Mammary gland
Vagina

Tissues were dispatched to the Test Site (Huntingdon Life Sciences Ltd., Eye Research Centre, Eye, Suffolk, IP23 7PX) for processing. The tissues from five selected control and 500 mg/kg bw/day dose group animals, and any animals which did not achieve a pregnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with hematoxylin and eosin for subsequent microscopic examination. The tissues shown in bold from the remaining control and 500 mg/kg bw/day animals and animals which did not achieve a pregnancy were also processed. In addition, sections of testes from all control and 500 mg/kg bw/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.

Detailed qualitative examination of the testes was undertaken, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell-or stage-specificity of testicular findings was noted.

Since there were indications of treatment-related liver (both sexes), spleen (both sexes), kidneys (males only) and heart (males only) changes, examination was subsequently extended to include similarly prepared sections of the liver (both sexes), spleen (both sexes), kidneys (males only) and heart (males only) from selected animals in the low and intermediate groups.

Microscopic examination was conducted by the Study Pathologist (Roger Alison Ltd, Caerfyrddin Fach, Cilcennin, Lampeter, SA48 8RN, United Kingdom). A peer review was performed randomly on the slides for independent evaluation by the Test Facility (W Henderson). A complete histopathology phase report is presented in Appendix 25 and represents the consensus view of both pathologists.
Postmortem examinations (offspring):
All offspring were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Statistics:
Due to the nature and quantity of this dataplease see section "Any other information on materials and methods inc. tables"
Reproductive indices:
Reproductive Indices
Mating Performance and Fertility

The following parameters were calculated from the individual data during the mating period of the parental generation:

i. Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii. Fertility Indices
For each group the following were calculated:

Mating Index (%) = (Number of animals mated / Number of animlas paired) x 100

Pregnancy Index (%) = (Number of pregnant females / Number of animals mated) x 100

Gestation and Parturition Data
The following parameters were calculated from individual data during the gestation and parturition period of the parental generation:

i. Gestation Length
Calculated as the number of days of gestation including the day for observation of mating and the start of parturition.

ii. Parturition Index
The following was calculated for each group:

Parturition Index (%) = (Number of females delivering live offspring / Number of pregnant females) x 100
Offspring viability indices:
Litter Responses
The standard unit of assessment was considered to be the litter, therefore values were first calculated for each litter and the group mean was calculated using their individual litter values. Group mean values included all litters reared to termination (Day 5 of age).

i. Implantation Losses (%)
Mean percentage pre-implantation and post-implantation loss were calculated for each female/litter as follows:

Pre–implantation loss (%) = ((Number of corpora lutea - Number of implantati on sites) / Number of corpora lutea) x100

Post–implantation loss (%) = ((Number of implantati on sites - Total number of offspring born) / Number of implantati on sites) x100

Live Birth and Viability Indices
The following indices were calculated for each litter as follows:

Live Birth Index (%) = (Number of offspring alive on Day 1 / Number of offspring born) x 100

Viability Index (%) = (Number of offspring alive on Day 4 / Number of offspring alive on Day 1) x 100

Sex Ratio (% males)
Sex ratio was calculated for each litter value on Days 1 and 4 post partum, using the following formula:

(Number of male offspring / Total number of offspring) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "Details on results" section below
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "Details on results" section below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Please refer to "Details on results" section below
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "Details on results" section below
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "Details on results" section below
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "Details on results" section below
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Adult Reponses
Mortality
There were no unscheduled deaths.

Clinical Observations
Animals of either sex treated with 500 mg/kg bw/day showed incidences of increased salivation from Day 7 (females) and Day 8 (males) until termination. Eight males and four females treated with 150 mg/kg bw/day also showed incidences of increased salivation from Day 28 to Day 33 (females) and Day 34 (males).

No such effects were evident in animals of either sex treated with 50 mg/kg bw/day.

Functional Observations
Behavioral Assessments
There were no treatment-related changes in the behavioural parameters at 50, 150 or 500 mg/kg bw/day.

Functional Performance Tests
There were no toxicologically significant changes in functional performance considered to be related to treatment at 50, 150 or 500 mg/kg bw/day.

Males treated with 500 and 150 mg/kg bw/day showed a statistically significant increase in the first hind limb grip strength. Males treated with 150 mg/kg bw/day also showed a statistically significant increase in overall mobility. In the absence of a true dose related response or any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance. Females treated with 500 mg/kg bw/day showed a statistically significant reduction in overall mobility. In the absence of any supporting clinical observations to suggest an effect of neurotoxicity, the intergroup differences were considered to be of no toxicological significance.

Sensory Reactivity Assessments
There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 50, 150 or 500 mg/kg bw/day.

Body Weight
Males treated with 500 mg/kg bw/day showed a reduction in body weight gain during the first four weeks of treatment. Statistical significance was evident during Weeks 1, 2 and 4 and actual body weight losses were evident in four males on Day 15. Slight recovery in body weight gain was evident in these males during the final two weeks of treatment, however overall body weight gain was reduced.

No adverse effects were detected in body weight gain in treated females during maturation or lactation however body weight gain for females treated with 500 mg/kg bw/day during the final week of gestation was statistically significantly lower than controls.

No such effects were detected in animals of either sex treated with 150 or 50 mg/kg bw/day.

Food Consumption
No adverse effects were detected in food consumption for treated males throughout the treatment period or in treated females during maturation, gestation or lactation.

Fluctuations in food conversion efficiency were evident in males treated with 500 mg/kg bw/day however these generally followed the reductions that were evident in body weight gains.

Water Consumption
Animals of either sex treated with 500 mg/kg bw/day and females treated with 150 mg/kg bw/day showed an increase in overall water consumption during maturation.

No such effects were detected in males treated with 150 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day.

Reproductive Performance
Mating
No treatment-related effects were detected in mating performance.

Fertility
No treatment-related effects were detected in fertility.

One control female and two females treated with 500 mg/kg bw/day were not pregnant following positive evidence of mating. No histopathological abnormalities were observed in either the males or females to explain the failure of the animals to breed successfully.

Gestation Length
Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.

Laboratory Investigations
Hematology
Males treated with 500 mg/kg bw/day showed statistically significant reductions in hemoglobin, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. The effect on mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration also extended to males treated with 150 mg/kg bw/day. Females treated with 500 mg/kg bw/day showed statistically significant reductions in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration and statistically significant increases in erythrocyte count, lymphocytes and total leukocyte count. Although some of the individual values were within the normal expected ranges, together with the associated histopathological changes in the spleen, a relationship to treatment cannot be excluded.

No toxicologically significant effects were detected in females treated with 150 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day.

Males from all treatment groups showed a statistically significant increase in neutrophil count. In the absence of a true dose related response the intergroup differences were considered of no toxicological significance.

Blood Chemistry
Animals of either sex treated with 500 mg/kg bw/day showed statistically significant increases in total protein and alanine aminotransferase. Females from this treatment group also showed statistically significant increases in bile acid, bilirubin, cholesterol and aspartate aminotransferase and a statistically significant reduction in albumin/globulin ratio. Males from this treatment group also showed a statistically significant increase in sodium concentration. The effect on albumin/globulin ratio extended to females treated with 150 mg/kg bw/day. The majority of individual values for total protein, alanine aminotransferase, bile acid, bilirubin, cholesterol and aspartate aminotransferase were outside of the normal expected ranges for these parameters. Although the majority of the individual values for all of the remaining intergroup differences were within the normal expected ranges, together with the associated histopathological changes, a relationship to treatment cannot be excluded.

No toxicologically significant effects were detected in males treated with 150 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day.

Males treated with 500 and 150 mg/kg bw/day showed a statistically significant increase in creatinine and urea. Males treated with 50 mg/kg bw/day also showed a statistically significant increase in creatinine and a statistically significant reduction in bilirubin. The majority of the individual values were within the normal expected ranges and in the absence of true dose related responses, the intergroup differences were considered to be of no toxicological significance.


Pathology
Necropsy

Adults
At necropsy, seven males treated with 500 mg/kg bw/day had an enlarged liver; two of these males also had a dark liver. A further three males had a dark liver. Six males from this treatment group had mottled kidneys and four of these males also had enlarged kidneys. Three females treated with 500 mg/kg bw/day had a dark and enlarged liver and a further female had an enlarged liver.

No such effects were detected in animals of either sex treated with 150 or 50 mg/kg bw/day.

One female treated with 150 mg/kg bw/day had pale adrenals and increased pelvic space in the kidneys at necropsy. In the absence of a similar effect at 500 mg/kg bw/day or any associated histopathological correlates in this sex the intergroup difference was considered to be incidental and of no toxicological significance.

Organ Weights
Animals of either sex treated with 500 mg/kg bw/day and males treated with 150 mg/kg bw/day showed a statistically significant increase in liver and spleen weight both absolute and relative to terminal body weight. Males from these treatment groups also showed a statistically significant increase in absolute and relative kidney weight.

No toxicologically significant effects were detected in females treated with 150 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day.

Females treated with 500 mg/kg bw/day showed a statistically significant increase in absolute and relative kidney weight. Although all of the individual values for relative weights were outside of normal ranges, in the absence of any associated histopathological correlates the intergroup difference was considered to be of limited toxicological importance. Females from all treatment groups showed a statistically significant reduction in pituitary weight, both absolute and relative to terminal body weight. Females treated with 50 mg/kg bw/day also showed a statistically significant reduction in thyroid weight. The majority of the individual values were within normal ranges for rats of the strain and age used and in the absence of any associated histopathological correlates or a true dose related response, the intergroup differences were considered not to be of toxicological significance. Males treated with 500 mg/kg bw/day showed a statistically significant reduction in absolute and relative thymus weight. The majority of the individual values were within normal ranges for rats of the strain and age used and in the absence of any associated histopathological correlates, the intergroup difference was considered not to be of toxicological significance.

Histopathology
The following microscopic abnormalities were detected:

Heart: myocarditis was evident in males from all treatment groups. The changes were grade 1 or 2 (minimal or slight severity) and showed a clear dose response in male animals in incidence and severity. This is a focal or multi focal change most often within the ventricle walls.

Liver: hypertrophy, bile duct hyperplasia, brown pigment in the bile ducts, peribiliary inflammation, single cell necrosis and increased glycogen was evident in animals of either sex treated with 500 mg/kg bw/day. Hypertrophy, bile duct hyperplasia, brown pigment in the bile ducts and peribiliary inflammation was also evident in animals of either sex treated with 150 mg/kg bw/day and in males treated with 50 mg/kg bw/day. Hypertrophy was also evident in females treated with 50 mg/kg bw/day.

Kidneys: hyaline droplets were evident in males from all treatment groups. Tubular basophilia and granular casts were also evident in males treated with 500 mg/kg bw/day and tubular basophilia was also evident in males treated with 150 mg/kg bw/day.

Spleen: A small increase in severity of hematopoiesis in the spleen was evident in animals of either sex treated with 500 mg/kg bw/day. Subsequent examination of the 50 and 150 mg/kg bw/day dose groups did not reveal a relationship to dose therefore these findings were considered to be incidental.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: Hypertrophy in the liver of females treated with 50 mg/kg bw/day was considered to represent an adaptive response to treatment therefore a No Observed Adverse Effect Level for females was considered to be 50 mg/kg bw/day.
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Remarks:
Reproductive toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 150 mg/kg bw/day based on reduced offspring viability, offspring body weight gain, litter size and litter weights on Days 1 and 4 post partum at 500 mg/kg bw/day.
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on findings at high dose level, which are considered to be adverse and the lack of significant findings at 150 mg/kg bw/day, the NOAEL for the reproductive toxicity is considered to be 150 mg/kg bw/day

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "Details on results" section below
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "Details on results" section below
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

Litter Responses
In total eleven females from the control and 50 mg/kg bw/day dose group, twelve females from the 150 mg/kg bw/day dose group and nine females from the 500 mg/kg bw/day dose group gave birth to a live litter and successfully reared young to Day 5 of age. The following assessment of litter response is based on all litters reared to termination on Day 5 of lactation/age.

Offspring Litter Size, Sex Ratio and Viability
No significant differences were detected for corpora lutea or implantation counts for treated animals when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

Of the litters born, litter size at birth was comparable to controls. Litter size on Days 1 and 4 post partum however was reduced in litters from females treated with 500 mg/kg bw/day although statistical significance was not achieved. Offspring viability on Day 4 post partum was reduced in these litters however statistical significance was not achieved. A total litter loss was also observed for one female treated with 500 mg/kg bw/day by Day 3 post partum. No such effects were detected in litters from females treated with 150 or 50 mg/kg bw/day.

An increase in pre and post implantation losses were evident in 500 mg/kg bw/day litters. Although statistical significance was not achieved and litter size at birth was unaffected, an effect of treatment cannot be excluded, in view of the effect on overall offspring survival.

There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

One female treated with 50 mg/kg bw/day had a total litter loss by Day 2 post partum. In the absence of a true dose related response or any other effects on the remaining litter parameters measured at this level, the intergroup difference was considered to be incidental and unrelated to treatment.

Offspring Growth and Development
As a consequence of the reduced litter size, females treated with 500 mg/kg bw/day showed a statistically significant reduction in litter weight on Days 1 and 4 post partum. Offspring body weight gain was also slightly reduced and male offspring body weight on Days 1 and 4 of lactation was statistically significantly reduced. Surface righting reflex was statistically significantly reduced in litters from females treated with 500 mg/kg bw/day and an increase in the number of offspring recorded missing or found dead was also increased in these litters.

No such effects on litter weight or offspring body weight gains were detected in females treated with 150 or 50 mg/kg bw/day.

Surface righting reflex was also statistically significantly reduced in litters from females treated with 150 mg/kg bw/day. The majority of individual litter values were within normal ranges and in the absence of any other effects on the remaining litter parameters measured at this level, the intergroup difference was considered to be incidental and unrelated to treatment.

Pathology
Necropsy

Offspring
No treatment-related macroscopic abnormalities were detected for offspring. The incidental findings observed were those occasionally observed in reproductive studies of this type and were considered to be unrelated to toxicity of the test item.

Effect levels (F1)

open allclose all
Dose descriptor:
NOEL
Remarks:
reproductive toxicity
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 150 mg/kg bw/day based on reduced offspring viability, offspring body weight gain, litter size and litter weights on Days 1 and 4 post partum at 500 mg/kg bw/day.
Dose descriptor:
NOAEL
Remarks:
Reproductive toxicity
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The ‘No Observed Effect Level’ (NOEL) for reproductive toxicity was considered to be 150 mg/kg bw/day based on reduced offspring viability, offspring body weight gain, litter size and litter weights on Days 1 and 4 post partum at 500 mg/kg bw/day.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

No NOEL for systemic toxicity established for either sex. The oral administration of PP-R-001 (CAS#1613243-54-1) to rats by gavage, at dose levels of 50, 150 and 500 mg/kg bw/day, resulted in treatment related effects in animals of either sex from all treatment groups.

Applicant's summary and conclusion

Conclusions:
The oral administration of PP-R-001 (CAS#1613243-54-1) to rats by gavage, at dose levels of 50, 150 and 500 mg/kg bw/day, resulted in treatment related effects in animals of either sex from all treatment groups. A ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore not established for either sex.

Hypertrophy in the liver of females treated with 50 mg/kg bw/day was considered to represent an adaptive response to treatment therefore a No Observed Adverse Effect Level (NOAEL) for females was considered to be 50 mg/kg bw/day. The microscopic change evident in the heart of males from all treatment groups and the brown pigment in bile ducts, bile duct hyperplasia and peribilary inflammation in the liver of 50 mg/kg bw/day males were considered to represent an adverse effect of treatment. The Lowest Observed Adverse Effect Level (LOAEL) for males was therefore considered to be 50 mg/kg bw/day.

The ‘No Observed Effect Level’ (NOEL) and NOAEL for reproductive toxicity was considered to be 150 mg/kg bw/day based on reduced offspring viability, offspring body weight gain, litter size and litter weights on Days 1 and 4 post partum at 500 mg/kg bw/day.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring development) and is designed to be compatible with the requirements of the OECD Guidelines for Testing of Chemicals No. 422 “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).

 

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

Methods…….

The test item was administered by gavage to three groups, each of twelve male and twelve female Wistar Han™:RccHan™:WIST strain rats, for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 150 and 500 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone (Arachis oil BP).

 

Clinical signs, behavioral assessments, body weight change and food and water consumption were monitored during the study. 

 

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation.

 

During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

 

Extensive functional observations were performed on five selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4post partum. Hematology and blood chemistry were evaluated prior to termination on five selected males and females from each dose group. 

 

Adult males were terminated on Day 43 or Day 44, followed by the termination of all females and offspring on Day 5post partum. Any female which did not produce a pregnancy was terminated on Day 26post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results…….

Adult Responses

Mortality: There were no unscheduled deaths.

 

Clinical Observations: Animals of either sex treated with 500 and 150 mg/kg bw/day showed incidences of increased salivation throughout the treatment period. No such effects were evident in animals of either sex treated with 50 mg/kg bw/day.

 

Behavioral Assessment: There were no treatment-related changes in the behavioural parameters at 50, 150 or 500 mg/kg bw/day.

 

Functional Performance Tests: There were no toxicologically significant changes in functional performance at 50, 150 or 500 mg/kg bw/day.

 

Sensory Reactivity Assessments: There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 50, 150 or 500 mg/kg bw/day.

 

Body Weight: Males treated with 500 mg/kg bw/day showed a reduction in body weight gain during the first four weeks of treatment and actual body weight losses were evident in four males on Day 15. Slight recovery was evident in these males during the final two weeks of treatment, however overall body weight gain was reduced. No adverse effects were detected in body weight gain in treated females during maturation or lactation however body weight gain for females treated with 500 mg/kg bw/day during the final week of gestation was reduced. No such effects were detected in animals of either sex treated with 150 or 50 mg/kg bw/day.

 

Food Consumption: No adverse effects were detected in food consumption for treated males throughout the treatment period or in treated females during maturation, gestation or lactation. Fluctuations in food conversion efficiency were evident in males treated with 500 mg/kg bw/day however these generally followed the reductions that were evident in body weight gains.

 

Water Consumption: Animals of either sex treated with 500 mg/kg bw/day and females treated with 150 mg/kg bw/day showed an increase in overall water consumption during maturation. No such effects were detected in males treated with 150 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day.

 

Reproductive Performance

Mating: No treatment-related effects were detected in mating performance.

 

Fertility: No treatment-related effects were detected in fertility.

 

Gestation Lengths: Gestation lengths were between 22 and 23½ days and the distribution of gestation lengths for treated females was essentially similar to control.

 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability: No significant differences were detected for corpora lutea or implantation counts for treated animals when compared to controls. Litter size on Days 1 and 4post partumwas slightly reduced in litters from females treated with 500 mg/kg bw/day although statistical significance was not achieved. Offspring viability on Day 4post partumwas reduced in these litters however statistical significance was not achieved. A total litter loss was also observed for one female treated with 500 mg/kg bw/day by Day 3post partum. No such effects were detected in litters from females treated with 150 or 50 mg/kg bw/day. There were no intergroup differences in sex ratio (percentage male offspring) for litters from treated groups compared to controls.

 

Offspring Growth and Development: Females treated with 500 mg/kg bw/day showed a statistically significant reduction in litter weight on Days 1 and 4post partum. Offspring body weight gain was also slightly reduced and male offspring body weight on Days 1 and 4 of lactation was statistically significantly reduced. Surface righting reflex was statistically significantly reduced in litters from females treated with 500 mg/kg bw/day and an increase in the number of offspring recorded missing or found dead was also increased in these litters. No such effects on litter weight or offspring body weight gains were detected in females treated with 150 or 50 mg/kg bw/day.

 

Laboratory Investigations

Hematology: Males treated with 500 mg/kg bw/day showed reductions in hemoglobin, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. The effect on mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration also extended to males treated with 150 mg/kg bw/day. Females treated with 500 mg/kg bw/day showed reductions in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration and increases in erythrocyte count, lymphocytes and total leukocyte count. No toxicologically significant effects were detected in females treated with 150 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day.

 

Blood Chemistry: Animals of either sex treated with 500 mg/kg bw/day showed increases in total protein and alanine aminotransferase. Females from this treatment group also showed increases in bile acid, bilirubin, cholesterol and aspartate aminotransferase and a reduction in albumin/globulin ratio. Males from this treatment group also showed an increase in sodium concentration. The effect on albumin/globulin ratio extended to females treated with 150 mg/kg bw/day. No toxicologically significant effects were detected in males treated with 150 mg/kg bw/day or animals of either sex treated with 50 mg/kg bw/day.

 

Pathology

Necropsy: At necropsy, seven males treated with 500 mg/kg bw/day had an enlarged liver; two of these males also had a dark liver. A further three males had a dark liver. Six males from this treatment group had mottled kidneys and four of these males also had enlarged kidneys. Three females treated with 500 mg/kg bw/day had a dark and enlarged liver and a further female had an enlarged liver. No such effects were detected in animals of either sex treated with 150 or 50 mg/kg bw/day.

 

Organ Weights: Animals of either sex treated with 500 mg/kg bw/day and males treated with 150 mg/kg bw/day showed a statistically significant increase in liver and spleen weight both absolute and relative to terminal body weight. Males from these treatment groups also showed a statistically significant increase in absolute and body weight relative kidney weight.

 

No toxicologically significant effects were detected in females treated with 150 mg/kg bw/day or in animals of either sex treated with 50 mg/kg bw/day.

Histopathology: The following microscopic abnormalities were detected:

 

Heart:myocarditis was evident in males from all treatment groups. The changes were grade 1 or 2 (minimal or slight severity) and showed a clear dose response in male animals in incidence and severity. This is a focal or multi focal change most often within the ventricle walls.

 

Liver:hypertrophy, bile duct hyperplasia, brown pigment in the bile ducts, peribiliary inflammation, single cell necrosis and increased glycogen was evident in animals of either sex treated with 500 mg/kg bw/day. Hypertrophy, bile duct hyperplasia, brown pigment in the bile ducts and peribiliary inflammation was also evident in animals of either sex treated with 150 mg/kg bw/day and in males treated with 50 mg/kg bw/day. Hypertrophy was also evident in females treated with 50 mg/kg bw/day.

 

Kidneys:hyaline droplets were evident in males from all treatment groups. Tubular basophilia and granular casts were also evident in males treated with 500 mg/kg bw/day and tubular basophilia was also evident in males treated with 150 mg/kg bw/day.

 

Spleen:A small increase in severity of hematopoiesis in the spleen was evident in animals of either sex treated with 500 mg/kg bw/day. Subsequent examination of the 50 and 150 mg/kg bw/day dose groups did not reveal a relationship to dose therefore these findings were considered to be incidental.

 

Conclusion

The oral administration ofPP-R-001 (CAS#1613243-54-1)to rats by gavage, at dose levels of 50, 150 and 500 mg/kg bw/day, resulted in treatment related effects in animals of either sex from all treatment groups. A ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore not established for either sex.

 

Hypertrophy in the liver of females treated with 50 mg/kg bw/day was considered to represent an adaptive response to treatment therefore a No Observed Adverse Effect Level for females was considered to be 50 mg/kg bw/day. The microscopic change evident in the heart of males from all treatment groups and the brown pigment in bile ducts, bile duct hyperplasia and peribilary inflammation in the liver of 50 mg/kg bw/day males were considered to represent an adverse effect of treatment. The Lowest Observed Adverse Effect Level (LOAEL) for males was therefore considered to be 50 mg/kg bw/day. 

 

The ‘No Observed Effect Level’ (NOEL) and 'No Observed Effect Level' NOAEL for reproductive toxicity were considered to be 150 mg/kg bw/day based on reduced offspring viability, offspring body weight gain, litter size and litter weights on Days 1 and 4 post partum at 500 mg/kg bw/day.