Registration Dossier

Administrative data

Description of key information

Two repeated dose toxicity studies are available. (1) In the OECD 422 study, a NOEL for systemic toxicity was not established for either sex. The NOAEL for females was considered to be 50 mg/kg bw/day. The microscopic changes evident in the heart of males at all doses, 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 considered to be 50 mg/kg bw/day.  The NOEL for reproductive toxicity was considered to be 150 mg/kg bw/day. (2) In the most recent OECD 408 study with a 6-week recovery period, The No Observed Adverse Effect Levels (NOAEL) for males (bile duct hyperplasia associated with inflammatory cell infiltration present in the livers of many animals treated with 120 mg/kg bw/day were considered adverse; no microscopic changes noted in the heart) and females were considered to be 30 and 120 mg/kg bw/day, respectively. The most sensitive NOAEL, 30 mg/kg bw/d, from the subchronic study, was chosen for DNEL calculations.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
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.
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
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.

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.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral 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.
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
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:
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.
Positive control:
None
Observations and examinations performed and frequency:
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.

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

Sacrifice and pathology:
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.
Other examinations:
Reproductive Performance
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

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.
Statistics:
Due to the nature and quantity of this data, please see section "Any other information on materials and methods incl. tables"
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "Details on Results" section below
Mortality:
mortality observed, treatment-related
Description (incidence):
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):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "Details on Results" section below
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Please refer to "Details on Results" section below
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
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
Histopathological findings: neoplastic:
no effects observed
Details on results:
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.

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
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.

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.
Dose descriptor:
other: No NOEL for systemic toxicity established for either sex
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 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.
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
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.
Critical effects observed:
not specified

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.

 

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 4post partumhowever was 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.

 

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 2post 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

Group mean values for total litter weights, offspring body weights and body weight changes, a summary incidence of clinical signs and surface righting reflex are given in Tables13,16and 17. Individual values and observations are given in Appendices12,15and16.

 

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 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.

 

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. 

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. According to the GHS classification and Labelling of Chemicals, this test item may warrant classification as Category 2 STOT-RE (cardiovascular/hematological: heart) pending confirmation from the proposed OECD 408 test results.

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) 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) 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 4post partumat 500 mg/kg bw/day.

According to the GHS classification and Labelling of Chemicals, and the EU CLP criteria, this test item may warrant classification as Category 2 STOT-RE (cardiovascular/hematological: heart) pending confirmation from the proposed OECD 408 test results. This tentative conclusion was based on the reported LOAEL of 50 mg/kg/d for males.

NOTE: The reported LOAEL was mainly based on the rare microscopic myocardial effects reported in the study. However, it is to be noted that the data owner is not aware of the latter type of microscopic findings for any other organic peroxide. No functional correlates (e.g., clinical signs, heart weight changes, death) accompanied these microscopic changes in the heart either. Additionally, it cannot be ruled out that prior exogenous or endogenous (subclinical) confounding factors could have predisposed the rats towards the observed microscopic changes ascribed to the test item. Oral gavage studies (28 -day and 90 -day) on CAS#24748 -23 -0, one of the major constituents of the test item did not reveal the above cardiac microscopic findings. A subchronic repeated dose study (90 -days) of the test substance with an appropriate study design may be necessary to clarify the above cardiac microscopic finding. 

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 03 October 2017 Experimental Completion Date: 11 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Identification (Chemical Name) : 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives
Batch Number : 1411519843
Trade name : Trigonox 501
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 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
Appearance : Clear Colorless liquid
Purity : 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives: 36.9% Spirdane D60: 57% (solvent)
Label : Trigonox 501-CS40, 1411519843
Date Received : 02 August 2017
Storage Conditions : Approximately 4 °C in the dark
Expiry Date : 01 March 2024
No correction for purity was made.
Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for eight days during which time their health status was assessed. A total of one hundred and twenty animals (sixty males and sixty females) were accepted into the study. At the start of treatment the males weighed 187 to 226g, the females weighed 130 to 169g, and were approximately six weeks old.

Animal Care and Husbandry
The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK) was used. 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). The diet, drinking water, bedding and environmental enrichment were 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 Envigo Research Limited, 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.

The animals were randomised and allocated to dose groups on arrival. Subsequent group mean body weights were checked on Day -2 and animals reallocated where necessary to ensure similarity between the dose 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.
Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe.
Vehicle:
arachis oil
Details on oral exposure:
The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP. Recovery group animals were maintained for a further forty-two days following termination of treatment.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of test item formulations were taken and analyzed on four occasions for concentration of 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicated that the prepared formulations were within 92 to 100 % of the nominal concentration.

Preparation of Calibration Standards
Stock solutions of test item in dilution solvent were prepared for external standard calibration. An aliquot, approximately 0.05 g of test item was exactly weighed into a 100 mL volumetric flask and brought to volume with dilution solvent 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 dilution solvent with a concentration of 0.1 mg/mL.

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

Calibration solutions were injected onto the instrument, at the beginning and end of each sample analysis sequence as a minimum.

To assess the calibration range of the method, a range of standard solutions were prepared in dilution solvent from a stock standard solution of 0.495 mg/mL by serial dilution covering the conecentration range 0.0495 mg/mL to 0.165 mg/mL.

Preparation of Test Samples
The formulations received were extracted with extraction solvent. An aliquot of test item formulation was accurately weighed into a volumetric flask and brought to volume with extraction solvent. This was then ultra-sonicated for 15 minutes and centrifuged at 4500 rpm for 10 minutes. Where necessary, sample solutions were further diluted with dilution solvent to achieve the working concentration.

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

The 2.5 mg/mL level recoveries were prepared at each analysis occasion and used to correct the concentration of procedural recovery.

The concentration of Test Item in the final solution was quantified by Gas Chromatography using Flame Ionisation detection as detailed in the instrument parameters section below.

Instrumentation Parameters
GC system: Agilent Technologies 6890, incorporating autosampler and workstation
Column: Rxi-5MS (15 m x 0.25 mm 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
FID temperature: 300°C
Injection volume: 1 µL
Retention time: ~2.5-7.5 mins

Data Evaluation and Calculations
The peak area response for Test Item in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression of calibration standard response versus calibration standard concentration. The area response at the peak observed at the characteristic retention time for Test Item in sample and procedural recovery chromatograms were measured.
Duration of treatment / exposure:
90 days treatment, followed by a 42-day reovery period for the highest dose group, plus a group dosed with vehicle alone
Frequency of treatment:
Daily
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
120 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Ten male, ten females per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
The dose levels were chosen in collaboration with the Sponsor and were based on the results of previous toxicity work (Envigo Research Limited Study Number 41402890; Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the rat (OECD 422)). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.
Positive control:
None
Observations and examinations performed and frequency:
Serial Observations

General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. During the treatment-free period, animals were observed daily. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performance tests were also performed on all non-recovery animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each non-recovery 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
Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Non-recovery animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour 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 grip strength meter was used. Each non-recovery 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. 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 scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

The following parameters were observed:
Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

Ophthalmoscopic Examination
The eyes of all animals were examined pre-treatment and for non-recovery control and non-recovery high dose animals before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using an ophthalmoscope was performed.

Estrous Cycle Assessment
Vaginal smears were taken daily for non-recovery females during the final three weeks of treatment. The stage of the estrous cycle was recorded for each day.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all non-recovery animals from each test and control group at the end of the study (Day 90) and on all recovery group animals at the end of the treatment-free period (Day 132). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 91 and 133.
Animals were not fasted prior to sampling.

Urinalysis investigations were performed on all non-recovery test and control group animals during Week 12 and on all recovery group animals during the final week of the recovery period. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.

Hematology
The following parameters were measured on plasma from 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)
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
Aspartate aminotransferase (ASAT)
Glucose
Alanine aminotransferase (ALAT)
Total protein (Tot.Prot.)
Alkaline phosphatase (AP)
Albumin
Creatinine (Creat)
Albumin/Globulin (A/G) ratio (by calculation)
Triglycerides (Tri)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)
Gamma-glutamyl transferase (γGT)
Inorganic phosphorus (P)

Urinalysis
The following parameters were measured on collected urine:
Volume
Ketones
Specific Gravity
Bilirubin
pH
Urobilinogen
Protein
Blood
Glucose
Appearance
Sacrifice and pathology:
Terminal Investigations

Necropsy
On completion of the dosing period or in the case of recovery group animals, at the end of the treatment-free period all animals were killed by intravenous overdose of a suitable barbiturate followed by exsanguination.

All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Sperm Analysis
At necropsy, the left testis and epididymis were removed from all males, dissected from connective tissue and weighed separately.

For the epididymis, the distal region was incised and a sample of the luminal fluid was collected and transferred to a buffer solution for analysis of sperm motility. The semen sample was assessed using an automated semen analyzer to determine the numbers of motile, progressively motile and non-motile sperm.

For the testis, the tunica albuginea was removed and the testicular tissue was stored frozen at approximately -20°C.

The cauda epididymis was separated from the body of the epididymis and weighed. The cauda epididymis was frozen at approximately -20°C.

Morphological assessment was performed on a sample of a minimum of 200 sperm, where possible, to determine the number with apparent structural anomalies.

Assessment of morphology was only performed for non-recovery control and non-recovery high dose males. As there were no treatment-related findings, these evaluations were not extended to males from other dose groups.

Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:
Adrenals
Ovaries
Brain
Spleen
Epididymides
Testes
Heart
Thymus
Kidneys
Uterus
Liver

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals
Ovaries
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Pituitary
Bone & bone marrow (sternum)
Prostate
Brain (including cerebrum, cerebellum and pons)
Rectum
Caecum
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)
Testes
Jejunum
Thymus
Kidneys
Thyroid/Parathyroid
Liver
Tongue
Lungs (with bronchi)
Trachea
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary gland
Uterus (with cervix)
Muscle (skeletal)
Vagina

All tissues were dispatched to the Test Site (Propath UK Ltd.,Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing. All tissues from non-recovery control and 120 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.

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

Pathology
Microscopic examination was conducted by the Study Pathologist. A peer review of the findings observed was conducted by V Mowat at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS.
Statistics:
Please refer to the section "Any other information on materials and methods incl. tables"
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
PLEASE REFER TO TABLE 1 - ATTACHED

Animals of either sex treated with 120 mg/kg bw/day showed incidences of increased salivation from Day 13 (males) and Day 14 (females) onwards. Four males treated with 30 mg/kg bw/day also showed incidences of increased salivation between Days 26 and 86 and one male treated with 10 mg/kg bw/day showed isolated instances of increased salivation on nine occasions. Isolated instances of noisy respiration were evident in two males treated with 120 mg/kg bw/day, two females treated with 30 mg/kg bw/day and one female treated with 10 mg/kg bw/day. Observations such as increased salivation and noisy respiration are commonly observed following the oral administration of an unpalatable or irritant test item formulation and represent difficulties in dosing particular animals rather than evidence of true systemic toxicity.

Generalized fur loss was evident in a number of control and treated animals throughout the study period. Observations of this nature are commonly observed in group housed animals and are considered to be incidental. Red/brown stained fur/snout/eyes/head were also evident in a number of control and treated animals throughout the study period and an isolated incident of noisy respiration was evident in one control female. These observations were considered to be incidental.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
PLEASE REFER TO TABLE 7 - ATTACHED

Males treated with 120 mg/kg bw/day showed a reduction in body weight gain during the first three weeks of treatment. Statistical significance (p<0.05) was achieved during Week 2 and Week 3. Body weight gains were 10% lower during Week 1 and 16% lower during Weeks 2 and 3. Recovery was evident thereafter, with body weight gain for these males being comparable to controls during the remainder of the study, and as such the effect was considered non-adverse.

No such effects were evident in females treated with 120 mg/kg bw/day or in animals of either sex treated with 30 or 10 mg/kg bw/day.

Females treated with 10 mg/kg bw/day showed a statistically significant reduction (p<0.05) in body weight gain during Week 12. In isolation and in the absence of a similar effect at higher dosages, the intergroup difference was considered not to be of toxicological significance.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in food consumption or food conversion efficiencies.
Food efficiency:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in food consumption or food conversion efficiencies.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no treatment-related changes in water consumption.

Visual inspection of water bottles did not reveal any inter-group differences.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes observed during ophthalmoscopic examination of animals of both sexes from the control group and high dose group during Week 12 of the treatment period.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
PLEASE

There were no toxicologically significant changes in the hematological parameters examined.

Animals of either sex from all treatment groups showed a statistically significant increase (p<0.05-0.01) in neutrophil count. Although the majority of individual values were above historical control range, a true dose related response was not evident in either sex and four control male and eight control female values were also above the historical control range. In the absence of any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Females treated with 120 mg/kg bw/day showed a statistically significant increase (p<0.05) in total leukocyte count and statistically significant reductions (p<0.05) in mean corpuscular hemoglobin concentration and platelet count. The majority of individual values for total leukocyte count and platelet count and all of the individual values for mean corpuscular hemoglobin concentration were within historical control ranges and in the absence of any associated histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Following the treatment-free period, females that were previously given 120 mg/kg bw/day showed a statistically significant increase (p<0.05) in activated partial thromboplastin time. The majority of individual values were within the historical control range and in the absence of a similar effect in 120 mg/kg bw/day females at the end of the treatment period or any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters examined.

Females treated with 120 mg/kg bw/day showed a statistically significant reduction (p<0.01) in bilirubin. With the exception of two values which were below the historical control range, all remaining individual values were within the historical control range. Three control values were also below the historical control range. In the absence of any associated histopathological correlates the intergroup difference was considered not to be of toxicological importance. Females from all treatment groups showed a statistically significant reduction (p<0.05-0.01) in triglyceride levels. In the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological importance.

Males treated with 30 mg/kg bw/day showed a statistically significant increase (p<0.05) in phosphorus whilst males treated with 10 mg/kg bw/day showed a statistically significant increase (p<0.05) in albumin/globulin ratio. The majority of the individual values were within historical control ranges and in the absence of a similar effect at 120 mg/kg bw/day, the intergroup differences were considered not to be of toxicological importance.

Following the treatment-free period, females that were previously given 120 mg/kg bw/day showed a statistically significant reduction (p<0.05) in glucose. All of the individual values were within the historical control range and in the absence of a similar effect in 120 mg/kg bw/day females at the end of the treatment period or any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment-related effects detected in the urinalysis parameters measured.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
One female treated with 30 mg/kg bw/day had hunched posture during Week 9 assessments. This was not observed during the routine daily clinical observations nor in animals of either sex treated with 120 mg/kg bw/day. Therefore, in isolation, this was considered incidental and unrelated to treatment.

Functional Performance Tests
There were no treatment-related changes in functional performance.

Females treated with 30 mg/kg bw/day showed a statistically significant increase (p<0.05) in overall activity. In the absence of a true dose related response or any clinical signs of neurotoxicity, the intergroup difference was considered not to be toxicologically significant.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
PLEASE REFER TO TABLE 17 - ATTACHED

Animals of either sex treated with 120 mg/kg bw/day showed a statistically significant increase (p<0.01) in liver weight both absolute (5%) and relative (13%) to terminal body weight. Males from this treatment group also showed a statistically significant increase (p<0.01) in absolute (11%) and relative (18%) kidney weight. Following the treatment-free period animals of either sex previously treated with 120 mg/kg bw/day continued to show a statistically significant increase (p<0.05) in absolute and relative liver weight and males also continued to show a statistically significant increase (p<0.05) in absolute and relative kidney weights. Although the majority of the individual values for both organs were within the normal expected ranges, together with the associated histopathological changes evident in the kidneys and liver, a relationship to treatment cannot be excluded.

No toxicologically significant effects were detected in animals of either sex treated with 30 or 10 mg/kg bw/day.

Males treated with 30 and 10 mg/kg bw/day showed a statistically significant increase (p<0.05) in kidney weights both absolute and relative to terminal body weight. A true dose related response was not evident and with the exception of one relative value at 10 mg/kg bw/day, all the remaining individual values were within the historical control ranges. In the absence of any associated histopathological correlates at these dosages, the intergroup differences were considered not to be of toxicological significance.

Females treated with 120 mg/kg bw/day showed a statistically significant increase (p<0.05) in kidney weights both absolute and relative to terminal body weight. Although the majority of individual relative values were above the historical control range, the majority of individual absolute values were within the historical control range. In the absence of any associated histopathological correlates at this dosage, in this sex, the intergroup difference was considered not to be of toxicological significance.

Females treated with 30 and 10 mg/kg bw/day showed a statistically significant increase (p<0.01) in liver weight both absolute and relative to terminal body weight. With the exception of two absolute values and one relative value at 30 mg/kg bw/day, all the remaining individual values were within the historical control ranges. In the absence of any associated histopathological correlates at these dosages, the intergroup differences were considered not to be of toxicological significance.

Following the treatment-free period, males that were previously given 120 mg/kg bw/day showed a statistically significant reduction (p<0.05) in right testis weight both absolute and relative to terminal body weight. In the absence of a similar effect in 120 mg/kg bw/day males at the end of the treatment period or any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
One male treated with 120 mg/kg bw/day had a dark liver at necropsy.
No toxicologically significant effects were detected in females treated with 120 mg/kg bw/day or in animals of either sex treated with 30 or 10 mg/kg bw/day.

The following macroscopic abnormalities were detected, however, they were either present in the control group or were not associated with any treatment-related findings in the treated groups and therefore were considered to be incidental. Small and dark (left only) adrenals in one control male, reddened lungs in one control male and one recovery control female, dark lungs in one recovery control female, a mass (approximately 25 mm x 10 mm) in the adipose tissue in the abdominal cavity in one recovery control female, a mass on the left lobe of the liver in a control male, enlarged spleen in one male treated with 30 mg/kg bw/day and a dark liver and mass (approximately 8 mm x 4 mm) in the adipose tissue near the right epididymis in one male treated with 120 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Kidneys: Hyaline droplets were increased in five males treated with 120 mg/kg bw/day. There was no notable associated pathology. Although tubular basophilia was present only in treated non-recovery males it was also present in two recovery control males and was therefore considered to be incidental.

Complete recovery was evident following the treatment-free period, with no difference between control and previously treated males apparent.

Liver: Bile duct hyperplasia was present in five males treated with 120 mg/kg bw/day with associated inflammatory cell infiltration evident in three of these males. Pigment deposits were also present in the bile ducts of seven males treated with 120 mg/kg bw/day.

Following the treatment-free period, bile duct hyperplasia persisted, albeit at a lower incidence and at minimal severity in two males previously treated with 120 mg/kg bw/day and pigment deposits in the bile ducts was evident in five males previously treated with 120 mg/kg bw/day. Pigment was apparent in the bile ducts of one female previously treated with 120 mg/kg bw/day.

Centrilobular hypertrophy was evident in five males and seven females treated with 120 mg/kg bw/day. Recovery was complete following the treatment-free period.

No other changes were noted which could be attributed to treatment with the test item at terminal kill.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Estrous Cycle
Assessment of estrous cycles during the final three weeks of treatment for non-recovery females did not indicate any obvious effect of treatment at 10, 30 or 120 mg/kg bw/day, with the majority of non-recovery females showing regular cycling.

Sperm Analysis
No adverse effects on sperm concentration, motility, progressive motility or morphology were observed in treated males when compared to controls.

Statistical analysis of the sperm concentration, motility and progressive motility data did not reveal any significant intergroup differences.
Key result
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
30 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
30 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
The oral (gavage) administration of 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives (CAS No. 1613243-54-1) for ninety consecutive days, to Wistar rats of both sexes, at dose levels of 10, 30 or 120 mg/kg bw/day resulted in adverse liver changes in males treated with 120 mg/kg bw/day, non-adverse kidney changes in males treated with 120 mg/kg bw/day and adaptive liver changes in females treated with 120 mg/kg bw/day. No treatment-related effects were evident in animals of either sex treated with 30 or 10 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 30 mg/kg bw/day for both sexes. The No Observed Adverse Effect Level (NOAEL) for males was also considered to be 30 mg/kg bw/day.

Hypertrophy in the liver of females treated with 120 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 120 mg/kg bw/day.

Following the treatment-free period, complete recovery in the kidney changes and adaptive liver changes (centrilobular hypertrophy) were evident in recovery animals and the adverse liver changes (bile duct hyperplasia and pigment deposits) were evident at lower incidence and severity.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives (CAS No. 1613243-54-1) and is compatible with the following regulatory guideline:

·        The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

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 ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 10, 30 and 120 mg/kg bw/day (animals designated as non-recovery). A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of ten males and ten females, were treated with the high dose (120 mg/kg bw/day) or the vehicle alone for ninety consecutive days and then maintained without treatment for a further forty-two days (animals designated as recovery).

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. Vaginal smears were performed for all non-recovery females during the final three weeks of treatment. Ophthalmoscopic examination was also performed on all animals prior to the start of treatment and on non-recovery control group and non-recovery high dose animals during Week 12.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results

Mortality

There were no unscheduled deaths.

Clinical Observations

Incidences of increased salivation was evident in animals of either sex treated with 120 mg/kg bw/day throughout the treatment period and in males treated with 30 and 10 mg/kg bw/day albeit to a lesser extent. Isolated instances of noisy respiration were evident in a small number of males treated with 120 mg/kg bw/day and females treated with 30 and 10 mg/kg bw/day. Observations of this nature are considered not to be related to systemic toxicity, and are due to the oral administration of an unpalatable or irritant test item formulation.

Behavioral Assessment

There were no treatment-related changes in the behavioral parameters measured.

Functional Performance Tests

There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

Males treated with 120 mg/kg bw/day showed a reduction in body weight gain during the first three weeks of treatment. Body weight gains were 10% to 16% lower than controls during this period. Improvement was evident thereafter.  No such effects were evident in females treated with 120 mg/kg bw/day or in animals of either sex treated with 30 or 10 mg/kg bw/day.

Food Consumption

No effect on food consumption or food conversion efficiency was evident in treated animals when compared to controls.

Water Consumption

Visual inspection of water bottles did not reveal any inter-group differences.

Ophthalmoscopy

Ophthalmoscopic examination of animals of both sexes from the non-recovery control and non-recovery 120 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.

Estrous Cycle

Assessment of estrous cycles for non-recovery females during the final three weeks of treatment did not indicate any obvious effect of treatment at 10, 30 or 120 mg/kg bw/day.

Hematology

There were no toxicologically significant effects detected in the hematological parameters measured.


Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters measured.

Urinalysis

There were no treatment-related effects detected in the urinalysis parameters measured.

Necropsy

One male treated with 120 mg/kg bw/day had a dark liver at necropsy.

No such effects were detected in females treated with 120 mg/kg bw/day or in animals of either sex treated with 30 or 10 mg/kg bw/day.

Sperm Analysis

No adverse effects on sperm concentration, motility, progressive motility or morphology were observed in treated males when compared to controls.

Organ Weights

Animals of either sex treated with 120 mg/kg bw/day showed an increase in liver weight both absolute (5%) and relative (13%) to terminal body weight. Males from this treatment group also showed an increase in absolute (11%) and relative (18%) kidney weight. Following the treatment-free period animals of either sex previously treated with 120 mg/kg bw/day continued to show an increase in liver weights and males continued to show an increase in kidney weights. No toxicologically significant effects were detected in animals of either sex treated with 30 or 10 mg/kg bw/day.

Histopathology

The following treatment-related microscopic abnormalities were detected:

Kidneys:Hyaline droplets were increased in five males treated with 120 mg/kg bw/day. There was no notable associated pathology. Although tubular basophilia was present only in treated non-recovery males it was also present in two recovery control males and was therefore considered to be incidental.

Complete recovery was evident following the treatment-free period, with no difference between control and previously treated males apparent.

Liver:Bile duct hyperplasia was present in five males treated with 120 mg/kg bw/day with associated inflammatory cell infiltration evident in three of these males. Pigment deposits were also present in the bile ducts of seven males treated with 120 mg/kg bw/day.

Following the treatment-free period, bile duct hyperplasia persisted, albeit at a lower incidence and at minimal severity in two males previously treated with 120 mg/kg bw/day and pigment deposits in the bile ducts was evident in five males previously treated with 120 mg/kg bw/day. Pigment was apparent in the bile ducts of one female previously treated with 120 mg/kg bw/day.

Centrilobular hypertrophy was evident in five males and seven females treated with 120 mg/kg bw/day. Recovery was complete following the treatment-free period.

Conclusion

The oral (gavage) administration of 1,2,4,5,7,8-Hexoxonane, 3,6,9-trimethyl-, 3,6,9-tris(Ethyl and Propyl) derivatives (CAS No. 1613243-54-1) for ninety consecutive days, to Wistar rats of both sexes, at dose levels of 10, 30 or 120 mg/kg bw/day resulted in adverse liver changes in males treated with 120 mg/kg bw/day, non-adverse kidney changes in males treated with 120 mg/kg bw/day and adaptive liver changes in females treated with 120 mg/kg bw/day. No treatment-related effects were evident in animals of either sex treated with 30 or 10 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was therefore considered to be 30 mg/kg bw/day for both sexes. The No Observed Adverse Effect Level (NOAEL) for males was also considered to be 30 mg/kg bw/day.

Hypertrophy in the liver of females treated with 120 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 120 mg/kg bw/day.

Following the treatment-free period, complete recovery in the kidney changes and adaptive liver changes (centrilobular hypertrophy) were evident in recovery animals and the adverse liver changes (bile duct hyperplasia and pigment deposits) were evident at lower incidence and severity.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
30 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
A well-conducted GLP study.
Organ:
liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The oral (gavage) administration of 1,2,4,5,7,8 -Hexoxonane, 3,6,9 -trimethyl-, 3,6,9 -tris(Ethyl and Propyl) derivatives (CAS No. 1613243 -54 -1) for ninety consecutive days (OECD 408), to Wistar rats of both sexes, at dose levels of 10, 30 or 120 mg/kg bw/day resulted in adverse liver changes in males treated with 120 mg/kg bw/day, non-adverse kidney changes in males treated with 120 mg/kg bw/day and adaptive liver changes in females treated with 120 mg/kg bw/day. No treatment-related effects were evident in animals of either sex treated with 30 or 10 mg/kg bw/day. The ‘No Observed Effect Level’ (NOEL) for systemic toxicity was considered to be 30 mg/kg bw/day for both sexes. Following the treatment-free period, complete recovery in the kidney changes and adaptive liver changes (centrilobular hypertrophy) were evident in recovery animals and the adverse liver changes (bile duct hyperplasia and pigment deposits) were evident at lower incidence and severity. The No Observed Adverse Effect Level (NOAEL) for males was also considered to be 30 mg/kg bw/day and the NOAEL for females was considered to be 120 mg/kg bw/day.

In a previous GLP, OECD 422 study “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996), oral gavage administration of CAS#1613243 -54 -1 to rats at 50, 150 and 500 mg/kg bw/day resulted in treatment related effects in animals of either sex from all treatment groups. A NOEL for systemic toxicity was not established for either sex. Hypertrophy (liver of females dosed with 50 mg/kg bw/day) was considered to represent an adaptive response to treatment, therefore a NOAEL for females was considered to be 50 mg/kg bw/day. The microscopic change evident in the heart of males (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. According to the GHS classification and Labelling of Chemicals, this test item may warrant classification as Category 2 STOT-RE (cardiovascular/hematological: heart). However, it is to be noted that the data owner is not aware of the latter type of cardiac microscopic findings for any other organic peroxide. No functional correlates (e.g., clinical signs, heart weight or death) accompanied these microscopic changes in the heart. Additionally, it cannot be ruled out that prior exogenous or endogenous (subclinical) confounding factors could have predisposed the rats towards the observed cardiac microscopic changes ascribed to the test item. It should also noted that the NOAEL for CAS#24748 -23 -0, one of the major constituents of the test item, was determined to be 50mg/kg/day in a 90 -day oral gavage study, and had somewhat similar histopathologic effects in the liver but had no myocardial effects. Subsequent subchronic (90 -days) study (results provided above) clarified the situation, resulting no STOT RE 2 classification, a clear NOAEL of 30 mg/kg bw/d and nor evidence of microscopic effects in the cardiac tissue at any dose.

Conclusion: the most sensitive NOAEL was considered to be 30 mg/kg bw/d (males) based on the standard subchronic GLP study results.

Justification for classification or non-classification

The data are conclusive but not sufficient for classification.