Registration Dossier

Administrative data

Endpoint:
repeated dose toxicity: oral, other
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 January 2017 and 07 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 January 2017 and 07 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
Please refer to the materials and methods section for more information.
GLP compliance:
yes (incl. certificate)
Limit test:
no
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:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven 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 330 to 371g, the females weighed 194 to 221g, 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, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 5. 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 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 reco
rds. 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 random ization 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 exposure:

The test item was administered daily 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. 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. For the purpose of this study the test item was prepared at the appropriate concentrations as a susp
ension in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least eleven days. Formulations were therefore prepared weekly and stored at approximately 4 ºC in the dark.
Samples of each test item formulation were taken on six occasions and analyzed for concentration of LA-63P at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 20% of the nominal concentration.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of estrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test item formulation were taken on six occasions and analyzed for concentration of LA-63P at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 20% of the nominal concentration. The test item concentration in the test samples was determined by HPLC-MS. The test itm gave a chromatographic profile consisting of a single peak.
The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision. The homogeneity was confirmed for Test item in Arachis Oil BP formulations at nominal concentrations of 2.5mg/ml and 25mg/ml. The stability was confirmed for test item in Arachis Oil BP formulations at nominal concentrations of 2.5mg/ml and 25mg/ml when stored refrigerated for 11 and 14 days.
Frequency of treatment:
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.
Details on study schedule:
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.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 animals, per sex, per dose level.
Control animals:
yes, concurrent no treatment
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, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43 or 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

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)
Parental animals: Observations and examinations:
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 (except for females during parturition where applicable). All observations were recorded.

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 during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

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 Assessment
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

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

Sacrifice and pathology
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or 44. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 postpartum. Surviving offspring were terminated via intra cardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy were killed 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.

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
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 (with coagulating
Epididymides
gland)
Esophagus
Skin (hind limb)
Eyes *
Spinal cord (cervical, mid thoracic
Gross lesions
and lumbar)
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Testes
Kidneys
Thyroid/Parathyroid
Liver
Trachea
Lungs (with bronchi)
Thymus
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary gland
Uterus & Cervix
Muscle (skeletal)
Vagina

Tissues were dispatched to the Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: M Dow). The tissues from five selected control and 50 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 50 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 50 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.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A review of the histopathology results for the study was conducted by an appointed Responsible Scientist (V Mowat)
working at a designated Test Site.
Litter observations:
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)
Reproductive indices:
Pre-coital Interval
Fertility Indices
Gestation and Parturition Data
Parturition Index
Offspring viability indices:
Implantation Losses (%)
Live Birth and Viability Indices
Sex Ratio (% males)
Clinical signs:
no effects observed
Description (incidence and severity):
There were no toxicologically significant findings in animals treated at 10, 25 or 50 mg/kg bw/day. All males and eight females at 50 mg/kg bw/day showed increased post dose salivation between Days 13 and 43. One male treated with 25 mg/kg bw/day also showed increased salivation between Days 29 and 32. One male each from the 10 and 50 mg/kg bw/day dose groups showed noisy respiration on Days 27 and 36, respectively. Observations of this nature are often encountered observed following the pral administration of an unpalatable test item formulation rather than true systemic toxicity. Incidental findings included one male treated with 50 mg/kg bw/day showing staining around the eyes on Days 27 and 28 and a control male showed staining red staining of the urine between Days 9 to 11.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 50mg/kg bw/day showed a reduction in body weight gain throughout the treatment period. Statistically significance (p<0.05-0.01) was achieved during the first five weeks of treatment and overall body weight gain was 71% lover when compared to controls. Males treated with 25mg/kg bw/day showed a statistically significant reduction (p<0.05) in body weight gain during the final week of treatment and a 13% reduction in overall body weight gain. No such
effects were detected in males treated with 10mg/kg bw/day. Females from all treatment groups showed a statistically significant reduction (p<0.05) in body weight gain during the first week of treatment. Recovery was evident thereafter and body weight gain during gestation and lactation was comparable to controls
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males treated at 50 mg/kg bw/day showed lower dietary intake throughout the treatment period, whereas the food consumption of males exposed to 25 or 10 mg/kg bw/day remained similar to the controls throughout the study. Males treated with 50 mg/kg bw/day showed lower food conversion efficiency throughout the treatment period. During the pre-pairing phase all female dose groups showed lower food consumptions when compared to controls. However, a true dose related response was not evident during Week 2. Females from all dose groups also showed lower food conversion efficiency during the first week of treatment. These reductions are associated with the lower body weight gains and reduced food
consumption evident in these females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no indications of an adverse effect of treatment on water intake in test animals in comparison with controls throughout the study.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters examined. All male dose groups showed a statistically significant increase (p<0.05) in total leukocyte count and platelet count. Although a true dose related response was evident for total leukocyte count and as the majority of individual values were above and the background control range, no dose related response was evident for platelet count and all individual values were within the background control range. Therefore, in the absence of any histopathological correlates, these findings were considered to be of no toxicological significance. At 50 mg/kg bw/day males showed a statistically significant increase (p<0.05) in neutrophils and a
statistically significant reduction (p<0.05) in mean corpuscular hemoglobin concentration. The majority of the individual values for neutrophils were above the background control range. However, individual values for mean corpuscular haemoglobin concentration were within historical control range. In the absence of any histopathological correlates these findings were considered to be of no toxicological significance.
Females from all dose groups showed a statistically significant reduction (p<0.05) in eosinophils. All individual values were within the background control range and in the absence of any histopathological correlates, this finding was considered to be of no toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes identified in the blood chemistry parameters examined. All dose groups for either sex showed a statistically significant increase in aspartate aminotransferase, a dose related increase was noted for the males only. The majority of individual values from 50 or 25 mg/kg bw/day were above the background control ranges. All male dose groups showed a statistically significant reduction in total proteins, in a dose related manner. All individual values were within the background control ranges. In the absence of any histopathological correlates, these findings were considered to be of no toxicological importance. At 50 mg/kg bw/day males showed a statistically significant reduction in albumin and statistically significant increase in albumin/globulin ratio, with no dose related response. All individual values for both parameters were within the background control ranges. In the absence of any histopathological correlates, these findings were considered to be of no toxicological importance. At 50 or 25 mg/kg bw/day males showed a statistically significant reduction in cholesterol in a dose related manner. All individual values were within the background control ranges. In the absence of any histopathological correlates, these findings were considered to be of no toxicological importance.
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
One female treated with 25 mg/kg bw/day showed an isolated instance of vocalisation during assessment in Week 4 of treatment. In the absence of a similar effect at 50mg/kg bw/day this was considered to be incidental. One control female showed pilo-erection, tip-toe gait and hunched posture during assessment of the final week of treatment. In the absence of treatment this was considered incidental.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At 50 mg/kg bw/day males showed a statistically significant reduction in pituitary, liver and the prostate gland weights both absolute and relative to terminal body weights. The majority of individual values for these tissues were within the background control ranges. The relative weights for liver was statistically significantly highere (P<0.05) compared to controls. All the individual values were within background control ranges and in the absence of histopathological correlates these findings were
considered to be of no toxicological significance. Males receiving 50 or 25 mg/kg bw/day showed a statistically significant reduction in seminal vesicle weights both absolute and relative (pituitary only) to terminal body weights.
The majority of individual values were within the background control ranges and with no histopathological correlates this finding was considered to be no toxicological significance. At 25 mg/kg bw/day males showed a statistically significant increase (p<0.05) in brain weight both absolute and relative to terminal body weight. Individual values were within the background control ranges. At 25 or 10 mg/kg bw/day males showed a statistically significant increase in heart weights
both absolute and relative to terminal body weights. Individual values were within the background
control ranges. In the absence of histopathological correlates these findings were considered to be of no toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no macroscopic findings considered to be related to administration of the test item. At 25 mg/kg bw/day one male (No. 58) showed a small left epididymis and a further one male (No.52) showed a mass on the liver. One control male (No.5) was observed to have small epididymides and testes, with increased pelvic space in both kidneys, and red discolouration of the lungs. One additional male (No.3) also had increased pelvic space in both kidneys. At 10 and 25 mg/kg bw/day
two and one females respectively exhibited red discolouration of the lungs. In the absence of any histopathological correlates or similar observation at 50mg/kg bw/day, the intergroup findings were considered to be incidental.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Description (incidence and severity)
Mesenteric Lymph Node
Four males and one female receiving 50 mg/kg bw/day were shown to have foamy macrophages of minimal or mild severity in the mesenteric lymph nodes.
Spleen
All males and three females receiving 50 mg/kg bw/day were shown to have foamy macrophages of minimal or mild severity in the spleen.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating:
No treatment-related effects were detected in mating performance. The majority of females showed evidence of mating within four days of pairing, with the exception of one control female that mated eight days after pairing.

Fertility:
There were no treatment-related effects on fertility.
One control female, five females treated with 10 mg/kg bw/day, one female treat with 25 mg/kg bw/day and one female treat with 50 mg/kg bw/day were found to be non-pregnant following positive evidence of mating. Histopathological examination revealed in the corresponding control male, marked tubular atrophy, a condition considered likely to have caused infertility. There were no other significant findings in the remaining males or females appeared to be cycling normally. In the absence of a dose related response these findings were considered to be of no toxicological significance as there was no dose related response.

Gestation Length:
There were no treatment-related differences in gestation lengths. The distribution for treated females was comparable to controls as gestation lengths were between 22 and 23 ½ days.
Key result
Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
reproductive performance
Key result
Critical effects observed:
no
Dermal irritation (if dermal study):
not examined
There was no effect of treatment with the test item at any dose level on the mean number of implantations or pre and pose-implantation losses. Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum from all treated dose groups was comparable with controls indicating lack of any effect on offspring viability.
There were no treatment-related intergroup differences in Sex Ratio (percentage male offspring) for litters from test items treated groups when compared with controls. Statistical analysis of the data did not reveal any significant intergroup differences.
One female treated with 25mg/kg bw/day was pregnant but failed to give birth to any live offspring. In the absence of the similar effect of 50mg/kg bw/day, the intergroup differences was consider to be incidental.
The group mean number of corpora lutea for 50 mg/kg bw/day females was statistically significantly lower than controls (p<0.05). Individual values were within the background control ranges and in the absence of any associated changes, this finding was considered to be of no toxicological significance.

There was no indication of an effect of treatment with the test item on offspring body weights and body weight development up to Day 4 post partum. Litter weights, and surface righting reflex for all treated groups were also comparable with controls.
Clinical signs detected in pups from control and treated dose groups included small, no milk in stomach, missing and bruising. Such findings are often observed in this type of study and were considered not to be related to treatment with the test item.

There were no clinical signs of toxicity observed and no macroscopic abnormalities detected in offspring at terminal kill.
Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no
Treatment related:
no

The oral administration of LA-63P to rats for a period of up to seven weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 10, 25 and 50 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 50 mg/kg bw/day.

Males treated with 50 mg/kg bw/day were affected with reductions in body weight development throughout the treatment period resulting in lower overall body weight gains compared to controls. The food consumptions and food conversion efficiency was also lower throughout the treatment period which accompanied the lower body weight development. At 25 mg/kg bw/day males exhibited slight fluctuations in body weight gains during treatment resulting in lower overall body weight compared to controls with no accompanying effects to food intake. The overall effect on body weight development at 25 mg/kg bw/day was considered not to be adverse in nature. Females from all treatment groups showed a reduction in body weight gain and food conversion efficiency during the first week of treatment, however, recovery was evident thereafter and comparable to controls during gestation and lactation.

There were no effects detected for water consumption and there were no clinical signs of systemic toxicity. However, a number of animals treated with 50 mg/kg bw/day exhibited increase salivation and/or noisy respiration during the treatment period. Observations of this nature are often associated with gavage administration and are normally considered to reflect an unpalatable test formulation rather than test item toxicity.

The histopathological examination revealed foamy macrophages present in the mesenteric lymph node and the spleen these were considered likely to be related to the absorption or clearance of the test item or its metabolites. There were no other associated pathological changes and thus this is considered likely to be a non-adverse change within the confines of this study.

Mating performance and fertility was unaffected by treatment and there were no treatment related effects detected in the offspring parameters observed.

As no specific target organ toxicity was observed and all adverse effects were considered to be a consequence of local irritant type effects, under CLP Regulation (Classification, Labelling and Packaging of substances and mixtures) this study should not be classified for this end point. LA-63P is therefore considered not classified as STOT RE (Specific target organ toxicity - repeat exposure) as the test item did not cause any specific target organ

toxicity. However, reduced body weight for males at 50 mg/kg bw/day was considered an adverse effect therefore the data can be used to calculate DNEL (Derived No-Effect Level) to address potential adverse effects.

Conclusions:
In conclusion, based on the findings of this study, due to the irritant nature of the test item, the prolonged treatment period at 50 mg/kg bw/day (maximum dose) showed an adverse reduction in body weight development throughout the treatment period in males resulting in 71% lower overall body weight gain compared to controls, which accompanied reduction in food consumption. These effects were initially observed in all female treatment groups during Week 1 however recovery was evident thereafter. At 25 mg/kg bw/day males exhibited slight fluctuations in body weight gains during treatment resulting in lower overall body weight (13%) compared to controls. These findings were considered to be associated with the irritant/sensitising properties of the test item as identified in the Fourteen Day Range Finding Study (Envigo Study number: QB15NM), Acute Eye Irritation (Envigo Study number: WB71VG) performed to OECD TG405 and Local Lymph Node Assay (Envigo
Study number: 1824100) performed to OECD TG429. Although there were adverse effects at 50 mg/kg bw/day (maximum dose), there was no evidence of adverse target organ effects,
hence, this may not be indicative of true systemic toxicity. Taking this into account the No Observed Adverse Effect Level (NOAEL) for toxicity could therefore be considered to be at
least 50 mg/kg bw/day (maximum dose) for animals of either sex. The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 50 mg/kg bw/day (maximum dose).
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 10, 25 and 50 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 4 post 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 44, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on Day 26 post 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 during the study.

Clinical Observations

There were no clinical signs of systemic toxicity at 10, 25 or 50 mg/kg bw/day.

Behavioral Assessment

There were no treatment-related effects detected.

Functional Performance Tests

There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

There was adverse effects on body weight gain for males treated with 50 mg/kg bw/day. Statistically significantly lower body weight gains were evident throughout the treatment period. Males treated with 25mg/kg bw/day also showed a reduction in overall body weight gain. No such effects were evident in males treated with 10mg/kg bw/day.

Females from all dose groups initially showed lower body weight gains during the first week of treatment. Recovery thereafter was evident.

Food Consumption

At 50 mg/kg bw/day males showed lower dietary intake throughout the treatment period.

During the pre-pairing phase, all female dose groups showed lower food consumptions when compared to controls. Food consumption during gestation and lactation for treated females was comparable to controls. .

Reductions in food conversion efficiency was observed in both males and females and generally followed the fluctuations seen in body weight gains and food consumptions.

Water Consumption

There were no effects on water consumption during the study.

Reproductive Performance

Mating

There were no treatment-related effects detected in mating performance.

Fertility

There were no treatment-related effects on fertility.

Gestation Lengths

There were no differences in gestation lengths. The distribution for treated females was comparable to controls as gestation lengths were between 22 and 23 ½ days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no detrimental effect of treatment with the test item on the mean number of corpora lutea, implantations, pre and post-implantation losses, litter size, sex ratio and subsequent offspring survival to Day 5 of age.

Offspring Growth and Development

There was no detrimental effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights or clinical signs up to Day 5 of age.

Laboratory Investigations

Hematology

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

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemistry parameters examined.

Pathology

Necropsy

There were no macroscopic findings considered to be related to the administration of the test item.

Organ Weights

There were no toxicologically significant effects detected on the organ weights measured.

Histopathology

There were foamy macrophages present from minimal or mild severity in the mesenteric lymph node and spleen which was considered likely to be a non-adverse change within the confines of this study with no other associated pathological changes.

Conclusion

In conclusion, based on the findings of this study, due to the irritant nature of the test item, the prolonged treatment period at 50 mg/kg bw/day (maximum dose) showed an adverse reduction in body weight development throughout the treatment period in males resulting in 71% lower overall body weight gain compared to controls, with accompanied reduction in food consumption. These effects were initially observed in all female treatment groups during Week 1 however recovery was evident thereafter. At 25 mg/kg bw/day males exhibited slight fluctuations in body weight gains during treatment resulting in lower overall body weight

(13%) compared to controls. These findings were considered to be associated with the irritant/sensitising properties of the test item as identified in the Fourteen Day Range Finding Study (Envigo Study number: QB15NM), Acute Eye Irritation (Envigo Study number: WB71VG) performed to OECD TG405 and Local Lymph Node Assay (Envigo Study number: 1824100) performed to OECD TG429. Although there were adverse effects at 50 mg/kg bw/day (maximum dose), there was no evidence of adverse target organ effects, hence, this may not be indicative of true systemic toxicity. Taking this into account the No Observed Adverse Effect Level (NOAEL) for toxicity could therefore be considered to be at least 50 mg/kg bw/day (maximum dose) for animals of either sex.

The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 50 mg/kg bw/day (maximum dose).

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

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:
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Blackthorn, Bicester, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for seven 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 330 to 371g, the females weighed 194 to 221g, 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, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 5. 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 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 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.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily 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.
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.
Vehicle:
arachis oil
Details on oral exposure:
For the purpose of this study the test item was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results show the formulations to be stable for at least eleven days. Formulations were therefore prepared weekly and stored at approximately 4 ºC in the dark.
Samples of each test item formulation were taken on six occasions and analyzed for concentration of LA-63P at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 20% of the nominal concentration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test item formulation were taken on six occasions and analyzed for concentration of LA-63P at Envigo Research Limited, Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 20% of the nominal concentration. The test item concentration in the test samples was determined by HPLC-MS. The test itm gave a chromatographic profile consisting of a single peak.

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, method accuracy and precision.
The homogeneity was confirmed for Test item in Arachis Oil BP formulations at nominal concentrations of 2.5mg/ml and 25mg/ml. The stability was confirmed for test item in Arachis Oil BP formulations at nominal concentrations of 2.5mg/ml and 25mg/ml when stored refrigerated for 11 and 14 days.
Frequency of treatment:
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.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
25 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12 animals, per sex, per dose level.
Control animals:
yes, concurrent no treatment
Details on study design:
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, five selected females per dose group were evaluated for functional/sensory responses to various stimuli.
viii. Blood samples were taken from five males from each dose group for hematological and blood chemical assessments on Day 42. The male dose groups were killed and examined macroscopically on Day 43 or 44.
ix. Blood samples were taken from five randomly selected females from each dose group for hematological and blood chemical assessment on Day 4 post partum. At Day 5 post partum, all females and offspring were killed and examined macroscopically. Any female which did not produce a pregnancy was also killed and examined macroscopically.

Examinations

Observations and examinations performed and frequency:
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 (except for females during parturition where applicable). All observations were recorded.

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 during gestation and lactation.

Water Consumption
Water intake was observed daily by visual inspection of water bottles for any overt changes.

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 Assessment
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

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


Sacrifice and pathology:
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or 44. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of a suitable barbiturate agent. Any females which failed to achieve pregnancy were killed 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.


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
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 (with coagulating
Epididymides
gland)
Esophagus
Skin (hind limb)
Eyes *
Spinal cord (cervical, mid thoracic
Gross lesions
and lumbar)
Heart
Spleen
Ileum (including peyer’s patches)
Stomach
Jejunum
Testes
Kidneys
Thyroid/Parathyroid
Liver
Trachea
Lungs (with bronchi)
Thymus
Lymph nodes (mandibular and mesenteric)
Urinary bladder
Mammary gland
Uterus & Cervix
Muscle (skeletal)
Vagina

Tissues were dispatched to the Test Site (Propath UK Ltd., Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: M Dow). The tissues from five selected control and 50 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 50 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 50 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.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A review of the histopathology results for the study was conducted by an appointed Responsible Scientist (V Mowat) working at a designated Test Site. A complete histopathology phase report is presented in Annex 1 and represents the consensus view of both pathologists.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no toxicologically significant findings in animals treated at 10, 25 or 50 mg/kg bw/day.
All males and eight females at 50 mg/kg bw/day showed increased post dose salivation between Days 13 and 43. One male treated with 25 mg/kg bw/day also showed increased salivation between Days 29 and 32. One male each from the 10 and 50 mg/kg bw/day dose groups showed noisy respiration on Days 27 and 36, respectively. Observations of this nature are often encountered observed following the pral administration of an unpalatable test item formulation rather than true systemic toxicity.
Incidental findings included one male treated with 50 mg/kg bw/day showing staining around the eyes on Days 27 and 28 and a control male showed staining red staining of the urine between Days 9 to 11.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 50mg/kg bw/day showed a reduction in body weight gain throughout the treatment period. Statistically significance (p<0.05-0.01) was achieved during the first five weeks of treatment and overall body weight gain was 71% lover when compared to controls.
Males treated with 25mg/kg bw/day showed a statistically significant reduction (p<0.05) in body weight gain during the final week of treatment and a 13% reduction in overall body weight gain. No such effects were detected in males treated with 10mg/kg bw/day.
Females from all treatment groups showed a statistically significant reduction (p<0.05) in body weight gain during the first week of treatment. Recovery was evident thereafter and body weight gain during gestation and lactation was comparable to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumptions are given in Table 9 and are presented graphically in Figure 3 and Figure 4. Individual values for females during gestation and lactation are presented in Appendix 9. Food efficiency for males and for females during the pre-mating phase is given in Table 10.
Males treated at 50 mg/kg bw/day showed lower dietary intake throughout the treatment period, whereas the food consumption of males exposed to 25 or 10 mg/kg bw/day remained similar to the controls throughout the study. Males treated with 50 mg/kg bw/day showed lower food conversion efficiency throughout the treatment period.
During the pre-pairing phase all female dose groups showed lower food consumptions when compared to controls. However, a true dose related response was not evident during Week 2.
Females from all dose groups also showed lower food conversion efficiency during the first week of treatment. These reductions are associated with the lower body weight gains and reduced food consumption evident in these females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were no indications of an adverse effect of treatment on water intake in test animals in comparison with controls throughout the study.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters examined.
All male dose groups showed a statistically significant increase (p<0.05) in total leukocyte count and platelet count. Although a true dose related response was evident for total leukocyte count and as the majority of individual values were above and the background control range, no dose related response was evident for platelet count and all individual values were within the background control range. Therefore, in the absence of any histopathological correlates, these findings were considered to be of no toxicological significance.
At 50 mg/kg bw/day males showed a statistically significant increase (p<0.05) in neutrophils and a statistically significant reduction (p<0.05) in mean corpuscular hemoglobin concentration. The majority of the individual values for neutrophils were above the background control range. However, individual values for mean corpuscular haemoglobin concentration were within historical control range. In the absence of any histopathological correlates these findings were considered to be of no toxicological significance.
Females from all dose groups showed a statistically significant reduction (p<0.05) in eosinophils. All individual values were within the background control range and in the absence of any histopathological correlates, this finding was considered to be of no toxicological significance.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no toxicologically significant changes identified in the blood chemistry parameters examined.
All dose groups for either sex showed a statistically significant increase in aspartate aminotransferase, a dose related increase was noted for the males only. The majority of individual values from 50 or 25 mg/kg bw/day were above the background control ranges. All male dose groups showed a statistically significant reduction in total proteins, in a dose related manner. All individual values were within the background control ranges. In the absence of any histopathological correlates, these findings were considered to be of no toxicological importance.
At 50 mg/kg bw/day males showed a statistically significant reduction in albumin and statistically significant increase in albumin/globulin ratio, with no dose related response.
All individual values for both parameters were within the background control ranges. In the absence of any histopathological correlates, these findings were considered to be of no toxicological importance.
At 50 or 25 mg/kg bw/day males showed a statistically significant reduction in cholesterol in a dose related manner. All individual values were within the background control ranges. In the absence of any histopathological correlates, these findings were considered to be of no toxicological importance..
Urinalysis findings:
not specified
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
One female treated with 25 mg/kg bw/day showed an isolated instance of vocalisation during assessment in Week 4 of treatment. In the absence of a similar effect at 50mg/kg bw/day this was considered to be incidental. One control female showed pilo-erection, tip-toe gait and hunched posture during assessment of the final week of treatment. In the absence of treatment this was considered incidental.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no treatment-related effects detected on the organ weights measured.
At 50 mg/kg bw/day males showed a statistically significant reduction in pituitary, liver and the prostate gland weights both absolute and relative to terminal body weights. The majority of individual values for these tissues were within the background control ranges. The relative weights for liver was statistically significantly highere (P<0.05) compared to controls. All the individual values were within background control ranges and in the absence of histopathological correlates these findings were considered to be of no toxicological significance.
Males receiving 50 or 25 mg/kg bw/day showed a statistically significant reduction in seminal vesicle weights both absolute and relative (pituitary only) to terminal body weights.
The majority of individual values were within the background control ranges and with no histopathological correlates this finding was considered to be no toxicological significance.
At 25 mg/kg bw/day males showed a statistically significant increase (p<0.05) in brain weight both absolute and relative to terminal body weight. Individual values were within the background control ranges. At 25 or 10 mg/kg bw/day males showed a statistically significant increase in heart weights both absolute and relative to terminal body weights. Individual values were within the background control ranges. In the absence of histopathological correlates these findings were considered to be of no toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no macroscopic findings considered to be related to administration of the test item.
At 25 mg/kg bw/day one male (No. 58) showed a small left epididymis and a further one male (No. 52) showed a mass on the liver. One control male (No.5) was observed to have small epididymides and testes, with increased pelvic space in both kidneys, and red discolouration of the lungs. One additional male (No.3) also had increased pelvic space in both kidneys. At 10 and 25 mg/kg bw/day two and one females respectively exhibited red discolouration of the lungs. In the absence of any histopathological correlates or similar observation at 50mg/kg bw/day, the intergroup findings were considered to be incidental.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Mesenteric Lymph Node
Four males and one female receiving 50 mg/kg bw/day were shown to have foamy macrophages of minimal or mild severity in the mesenteric lymph nodes.

Spleen
All males and three females receiving 50 mg/kg bw/day were shown to have foamy macrophages of minimal or mild severity in the spleen.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
50 mg/kg bw/day (nominal)
System:
other: This included reduced body weight gains throughout the treatment period in males at 50 mg/kg bw/day, with accompanied reduced food consumption.
Organ:
other: Body weight fluctuations.
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Any other information on results incl. tables

The oral administration of LA-63P to rats for a period of up to seven weeks (including two weeks pre-pairing, gestation and early lactation for females) at dose levels 10, 25 and 50 mg/kg bw/day resulted in treatment related effects in animals of either sex treated with 50 mg/kg bw/day.

Males treated with 50 mg/kg bw/day were affected with reductions in body weight development throughout the treatment period resulting in lower overall body weight gains compared to controls. The food consumptions and food conversion efficiency was also lower throughout the treatment period which accompanied the lower body weight development. At 25 mg/kg bw/day males exhibited slight fluctuations in body weight gains during treatment resulting in lower overall body weight compared to controls with no accompanying effects to food intake. The overall effect on body weight development at 25 mg/kg bw/day was considered not to be adverse in nature. Females from all treatment groups showed a reduction in body weight gain and food conversion efficiency during the first week of treatment, however, recovery was evident thereafter and comparable to controls during gestation and lactation.

There were no effects detected for water consumption and there were no clinical signs of systemic toxicity. However, a number of animals treated with 50 mg/kg bw/day exhibited increase salivation and/or noisy respiration during the treatment period. Observations of this nature are often associated with gavage administration and are normally considered to reflect an unpalatable test formulation rather than test item toxicity.

The histopathological examination revealed foamy macrophages present in the mesenteric lymph node and the spleen these were considered likely to be related to the absorption or clearance of the test item or its metabolites. There were no other associated pathological changes and thus this is considered likely to be a non-adverse change within the confines of this study.

Mating performance and fertility was unaffected by treatment and there were no treatment related effects detected in the offspring parameters observed.

As no specific target organ toxicity was observed and all adverse effects were considered to be a consequence of local irritant type effects, under CLP Regulation (Classification, Labelling and Packaging of substances and mixtures) this study should not be classified for this end point. LA-63P is therefore considered not classified as STOT RE (Specific target organ toxicity - repeat exposure) as the test item did not cause any specific target organ toxicity. However, reduced body weight for males at 50 mg/kg bw/day was considered an adverse effect therefore the data can be used to calculate DNEL (Derived No-Effect Level) to address potential adverse effects.

Applicant's summary and conclusion

Conclusions:
In conclusion, based on the findings of this study, due to the irritant nature of the test item, the prolonged treatment period at 50 mg/kg bw/day (maximum dose) showed an adverse reduction in body weight development throughout the treatment period in males resulting in 71% lower overall body weight gain compared to controls, which accompanied reduction in food consumption. These effects were initially observed in all female treatment groups during Week 1 however recovery was evident thereafter. At 25 mg/kg bw/day males exhibited slight fluctuations in body weight gains during treatment resulting in lower overall body weight (13%) compared to controls. These findings were considered to be associated with the irritant/sensitising properties of the test item as identified in the Fourteen Day Range Finding Study (Envigo Study number: QB15NM), Acute Eye Irritation (Envigo Study number: WB71VG) performed to OECD TG405 and Local Lymph Node Assay (Envigo
Study number: 1824100) performed to OECD TG429. Although there were adverse effects at 50 mg/kg bw/day (maximum dose), there was no evidence of adverse target organ effects,
hence, this may not be indicative of true systemic toxicity. Taking this into account the No Observed Adverse Effect Level (NOAEL) for toxicity could therefore be considered to be at
least 50 mg/kg bw/day (maximum dose) for animals of either sex. The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 50 mg/kg bw/day (maximum dose).
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 10, 25 and 50 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 4 post 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 44, followed by the termination of all females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on Day 26 post 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 during the study.

Clinical Observations

There were no clinical signs of systemic toxicity at 10, 25 or 50 mg/kg bw/day.

Behavioral Assessment

There were no treatment-related effects detected.

Functional Performance Tests

There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

There was adverse effects on body weight gain for males treated with 50 mg/kg bw/day. Statistically significantly lower body weight gains were evident throughout the treatment period. Males treated with 25mg/kg bw/day also showed a reduction in overall body weight gain. No such effects were evident in males treated with 10mg/kg bw/day.

Females from all dose groups initially showed lower body weight gains during the first week of treatment. Recovery thereafter was evident.

Food Consumption

At 50 mg/kg bw/day males showed lower dietary intake throughout the treatment period.

During the pre-pairing phase, all female dose groups showed lower food consumptions when compared to controls. Food consumption during gestation and lactation for treated females was comparable to controls. .

Reductions in food conversion efficiency was observed in both males and females and generally followed the fluctuations seen in body weight gains and food consumptions.

Water Consumption

There were no effects on water consumption during the study.

Reproductive Performance

Mating

There were no treatment-related effects detected in mating performance.

Fertility

There were no treatment-related effects on fertility.

Gestation Lengths

There were no differences in gestation lengths. The distribution for treated females was comparable to controls as gestation lengths were between 22 and 23 ½ days.

Litter Responses

Offspring Litter Size, Sex Ratio and Viability

There was no detrimental effect of treatment with the test item on the mean number of corpora lutea, implantations, pre and post-implantation losses, litter size, sex ratio and subsequent offspring survival to Day 5 of age.

Offspring Growth and Development

There was no detrimental effect of treatment with the test item indicated by offspring body weight or body weight gain and litter weights or clinical signs up to Day 5 of age.

Laboratory Investigations

Hematology

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

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemistry parameters examined.

Pathology

Necropsy

There were no macroscopic findings considered to be related to the administration of the test item.

Organ Weights

There were no toxicologically significant effects detected on the organ weights measured.

Histopathology

There were foamy macrophages present from minimal or mild severity in the mesenteric lymph node and spleen which was considered likely to be a non-adverse change within the confines of this study with no other associated pathological changes.

Conclusion

In conclusion, based on the findings of this study, due to the irritant nature of the test item, the prolonged treatment period at 50 mg/kg bw/day (maximum dose) showed an adverse reduction in body weight development throughout the treatment period in males resulting in 71% lower overall body weight gain compared to controls, which accompanied reduction in food consumption. These effects were initially observed in all female treatment groups during Week 1 however recovery was evident thereafter. At 25 mg/kg bw/day males exhibited slight fluctuations in body weight gains during treatment resulting in lower overall body weight (13%) compared to controls. These findings were considered to be associated with the irritant/sensitising properties of the test item as identified in the Fourteen Day Range Finding Study (Envigo Study number: QB15NM), Acute Eye Irritation (Envigo Study number: WB71VG) performed to OECD TG405 and Local Lymph Node Assay (Envigo Study number: 1824100) performed to OECD TG429. Although there were adverse effects at 50 mg/kg bw/day (maximum dose), there was no evidence of adverse target organ effects, hence, this may not be indicative of true systemic toxicity. Taking this into account the No Observed Adverse Effect Level (NOAEL) for toxicity could therefore be considered to be at least 50 mg/kg bw/day (maximum dose) for animals of either sex. The ‘No Observed Effect Level’ (NOEL) for reproductive and developmental toxicity was considered to be 50 mg/kg bw/day (maximum dose).