Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-02-04 - 2016-07-13 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-02-04 - 2016-07-13 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Remarks:
preliminary dose range-finding test
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, No.422: “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008
Version / remarks:
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).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Department of Health of the Government of the United Kingdom
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions / Solubility and stability of the test substance in the solvent/vehicle: The stability and homogeneity of the test item formulations were determined by the laboratories' Analytical Services as part of this study. Results show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark. Representative samples of test item formulations were taken and analyzed for concentration of IPGE (Cas No. 4016-14-2 ; EC No. 223-672-9). The results indicate that the prepared formulations were within ± 4% of the nominal concentration, confirming the accuracy and suitability of the formulation procedure.
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST strain rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately twelve weeks
- Weight at study initiation: the males weighed 313 to 348g, the females weighed 187 to 213g
- Fasting period before study: no
- Housing: 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. 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.
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum
- Acclimation period: The animals were acclimatized for seven days during which time their health status was assessed.

DETAILS OF FOOD AND WATER QUALITY:
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. A certificate of analysis of the batch of diet used is available.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 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.

IN-LIFE DATES: The in-life phase of the study was conducted between 10 March 2016 (first day of treatment) and 28 April 2016 (final day of necropsy).
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:
PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP.

VEHICLE
- Concentration in vehicle: 0, 25, 75, 150 mg/ml
- Amount of vehicle (if gavage): 4 ml
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by the laboratories' Analytical Services as part of this study. Results show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark. Representative samples of test item formulations were taken and analyzed for concentration of IPGE (Cas No. 4016-14-2 ; EC No. 223-672-9). The method used for analysis of formulations and the results obtained are given in the attachment. The results indicate that the prepared formulations were within ± 4% of the nominal concentration, confirming the accuracy and suitability of the formulation procedure.
Duration of treatment / exposure:
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.
Males were terminated after 43 or 44 days. At Day 5 post partum, all surviving females and surviving offspring were killed, and females were hence dosed 14 days prior to pairing, during pairing and pregnancy, and 4 days afterwards.
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on preliminary dose range-finding test
- Rationale for animal assignment (if not random): 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.
Positive control:
not required
Observations and examinations performed and frequency:
CAGE SIDE / CLINICAL OBSERVATIONS: Yes
- Time schedule: 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: Yes
- Time schedule for examinations: 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 AND COMPOUND INTAKE:
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:
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 AND COMPOUND INTAKE:
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 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 or Day 25 post coitum 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.
- Animals fasted: No
- How many animals: five males and five females selected from each test and control group
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).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 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 or Day 25 post coitum 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.
- Animals fasted: No
- How many animals: five males and five females selected from each test and control group
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, Glucose, Total protein (Tot.Prot.), Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Total cholesterol (Chol), Total bilirubin (Bili), Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes

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, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behavior, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin color, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation
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, Vocalization, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Blink reflex, Startle reflex

IMMUNOLOGY: No

OTHER: Reproductive performance
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or Day 44. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 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.
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown in bold were weighed from all remaining animals: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary (post-fixation), Prostate and Seminal Vesicles, Spleen, Testes, Thymus, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with Cervix)

HISTOPATHOLOGY: Yes
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, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Coagulating glandm, Colon, Duodenum, Epididymides (preserved in Modified Davidsons fluid), Esophagus, Eyes (fixed in Davidson’s fluid), Gross lesions, Heart, Ileum (including peyer’s patches), Jejunum, Kidneys, Liver, Lungs (with bronchi) (inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), Lymph nodes (mandibular and mesenteric), Mammary gland, Muscle (skeletal), Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles, Skin (hind limb), Spinal cord (cervical, mid thoracic and lumbar), Spleen, Stomach, Testes (preserved in Modified Davidsons fluid), Thyroid/Parathyroid, Trachea, Thymus, Urinary bladder, Uterus & Cervix, Vagina
The tissues from five selected control and 600 mg/kg bw/day dose group animals and any animals dying during the study 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 from the remaining control and 600 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 600 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 stomach changes, examination was subsequently extended to include similarly prepared sections of the stomach from animals in the low and intermediate groups.
Other examinations:
Reproductive Indices, Litter Responses
Statistics:
Due to limitations of the free text field, information is provided below.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 600 mg/kg bw/day showed incidences of increased salivation between Days 18 and 25.
No such effects were evident in animals of either sex treated with 300 or 100 mg/kg bw/day.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no unscheduled deaths considered to be associated with treatment.
One female treated with 300 mg/kg bw/day was killed in extremis on Day 4. This female had hunched posture, pilo-erection, a decreased respiratory rate and was lethargic. At necropsy, enlarged adrenals, enlarged and fluid filled kidneys with increased pelvic space and reddened lungs was evident. Histopathological examination of this animal revealed pyelonephritis (both kidneys) which was considered likely to have caused the poor condition of the animal and its subsequent early termination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 600 mg/kg bw/day showed a statistically significant reduction (p<0.01) in body weight gain during the first week of treatment. Recovery was evident thereafter however, a slight reduction in body weight gain was evident in these males during the final week of treatment. Consequently, a 10% reduction in overall body weight gain was evident in these males.
No such effects were detected in males treated with 300 or 100 mg/kg bw/day.
No adverse effects were detected in treated females during maturation. Body weight gain during the first week of gestation for females treated with 100 mg/kg bw/day was comparable to controls however a reduction in body weight gain was evident in these females during the final two weeks of gestation. Consequently, cumulative body weight gain between Days 0 and 20 of gestation was lower in these females. A statistically significant reduction (p<0.01) in body weight gain was also evident in females treated with 100 mg/kg bw/day during lactation.
Although females treated with 600 and 300 mg/kg bw/day were all non-pregnant, body weight gain during the first week post coitum was comparable to control females. Body weight gain for these females during the remaining treatment period was reduced; with the majority of females showing actual body weight losses.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effects were detected in male food consumption. Males treated with 600 mg/kg bw/day showed a reduction in food conversion efficiency during the first week of treatment.
No adverse effects were detected in food consumption for treated females during maturation. Food consumption for 100 mg/kg bw/day females was comparable to controls throughout gestation however it was statistically significantly reduced (p<0.05) during lactation. This lower food consumption was consistent with lower body weight gain compared to control during this phase of the study.
Although females treated with 600 and 300 mg/kg bw/day were all non-pregnant, food consumption during the first week post coitum was comparable to control females and remained fairly consistent throughout the remainder of the treatment period.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 600 mg/kg bw/day showed a reduction in food conversion efficiency during the first week of treatment.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No adverse effects were detected in water consumption.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters measured for males treated with 600 and 300 mg/kg bw/day or both sexes at 100 mg/kg bw/day.
Males treated with 600 and 300 mg/kg bw/day showed statistically significant reductions (p<0.05-0.01) in hemoglobin (600 mg/kg bw/day only), mean corpuscular hemoglobin concentration, total leucocyte count and lymphocytes. All of the individual values were within historical control ranges and in the absence of any associated histopathological correlates or true dose related responses the intergroup differences were considered of no toxicological significance. Females treated with 100 mg/kg bw/day showed a statistically significant reduction (p<0.01) in platelet count. All of the individual values were within the historical control range and in the absence of any associated histopathological correlates the intergroup difference was considered of no toxicological significance.
Assessment of hematology parameters for females at 600 and 300 mg/kg bw/day has to be treated with caution as no females at these dosages were maintaining a litter at the time of blood sampling and all of theses females were non-pregnant. The female animals at these dosages were therefore in a different physiological state in comparison to the other females on the study and also those females contributing to the historical control data.
At 600 and 300 mg/kg bw/day, higher hemoglobin, erythrocyte count and hematocrit and lower neutrophils and platelet count for females attained statistical significance (p<0.05-0.01) when compared with control. Excluding one female treated with 300 mg/kg bw/day for platelet count, all of the remaining individual values for these parameters were within the historical control range. A true dose related response was also not evident in platelet count.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters measured for males treated with 600 or 300 mg/kg bw/day or both sexes at 100 mg/kg bw/day.
Males treated with 600 mg/kg bw/day showed a statistically significant increase in bile acids (p<0.01) and alanine aminotransferase (p<0.05). Males from all treatment groups and females treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in albumin/globulin ratio. All individual values (excluding one alanine aminotransferase value) were within historical control ranges and a true dose related response was not evident for albumin/globulin ratio. In the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Assessment of blood chemistry parameters for females at 600 and 300 mg/kg bw/day has to be treated with caution as no females at these dosages were maintaining a litter at the time of blood sampling and all of theses females were non-pregnant. The female animals at these dosages were therefore in a different physiological state in comparison to the other females on the study and also those females contributing to the historical control data.
For females at 600 and 300 mg/kg bw/day, higher bile acids, inorganic phosphorus and albumin/globulin ratio attained statistical significance (p<0.05) compared to control. For inorganic phosphorus and albumin/globulin ratio, all individual values were within the historical control ranges however for bile acids the majority of individual values were outside of the historical control range. Additionally higher bilirubin and lower creatinine and urea levels for females treated with 600 mg/kg bw/day attained statistical significance (p<0.05-0.01) compared to control but all individual values were within the historical control ranges.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Assessments: There were no treatment-related changes in the behavioural parameters for either sex at 100, 300 or 600 mg/kg bw/day.
Functional Performance Tests: There were no treatment related changes in functional performance considered to be related to treatment at 100, 300 or 600 mg/kg bw/day. Males treated with 600 mg/kg bw/day showed a statistically significant (p<0.05) increase in forelimb grip strength. The intergroup difference was confined to one out of the three tests and in the absence of any associated clinical signs to suggest a neurotoxic effect, the intergroup difference was considered not to be of toxicological importance.
Sensory Reactivity Assessments: There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 100, 300 or 600 mg/kg bw/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the organ weights measured for males treated with 600 or 300 mg/kg bw/day or both sexes at 100 mg/kg bw/day.
Males treated with 600 mg/kg bw/day showed statistically significant increases (p<0.05-0.01) in kidney and liver weight both absolute and relative to terminal body weight and a statistically significant reduction (p<0.05) in absolute and relative testes weight. The majority of individual values for testes weights were within historical control range although the majority of individual values for kidney and liver weights were outside of the historical control ranges. However, in the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological importance. Males treated with 300 mg/kg bw/day showed a statistically significant reduction (p<0.05) in thyroid weight both absolute and relative to terminal body weight. All of the individual values were within historical control range and in the absence of a similar effect at 600 mg/kg bw/day or any associated histopathological correlates the intergroup difference was considered to be of no toxicological significance. Females treated with 100 mg/kg bw/day showed a statistically significant reduction in spleen weight both absolute and relative to terminal body weight. All of the individual values for relative weights and all but one absolute weight were within historical control ranges and in the absence of any associated histopathological correlates the intergroup difference was considered to be of no toxicological significance.
Assessment of organ weights for females at 600 and 300 mg/kg bw/day has to be treated with caution as no females at these dosages were maintaining a litter at the time of blood sampling and all of theses females were non-pregnant. The female animals at these dosages were therefore in a different physiological state in comparison to the other females on the study and also those females contributing to the historical control data.
Females treated with 600 and 300 mg/kg bw/day showed statistically significant reductions (p<0.05-0.01) in absolute and relative liver and spleen weights and a statistically significant increase (p<0.01) in absolute and relative thymus weight. Females treated with 600 mg/kg bw/day also showed a statistically significant reduction (p<0.01) in uterus and cervix weight, both absolute and relative to terminal body weight. The majority of individual values were within historical control ranges and in the absence of any supporting histopathological changes, these findings were considered to be incidental and of no toxicological significance.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At termination, nine males and one female treated with 600 mg/kg bw/day had raised white patches on the non-glandular region of the stomach.
No toxicologically significant effects were detected in the remaining animals.
Two control males, two control females, two males and one female treated with 300 mg/kg bw/day and two females treated with 600 mg/kg bw/day had reddened lungs at necropsy. One female treated with 600 mg/kg bw/day had a fibrous mass (approximately 5mm x 5mm) on the liver and one female treated with 300 mg/kg bw/day had a fluid filled uterus at necropsy. One male treated with 600 mg/kg bw/day had a small and flaccid right testis and a further male from this treatment group had a small and flaccid left testis and epididymis. In the absence of any associated treatment-related histopathological correlates these findings were considered to be incidental.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following treatment-related microscopic abnormalities were detected:
Stomach: hyperplasia of the non-glandular region was present in eleven males and five females treated with 600 mg/kg bw/day.
Ovaries: the majority of females treated with 600 or 300 mg/kg bw/day and in one female treated with 100 mg/kg bw/day had the appearance of increased corpora lutea and was in metestrus or diestrus. This suggests a mild disturbance of the reproductive cycle which may account for the lack of pregnancy.
There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle).
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
At 600 and 300 mg/kg bw/day, pregnancy was not achieved in any female; in view of this, it is not possible to assess effects of the Test Item on females during gestation or lactation at these dosages.
Key result
Dose descriptor:
NOAEL
Remarks:
overall toxicity
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
overall toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
LOEL
Remarks:
reproductive toxicity
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: notably reduced fertility at 100 mg/kg bw/day
Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no

See attachments

Conclusions:
The study was conducted under GLP according to OECD guideline 422 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as reliable to assess the repeated dose oral toxicity (short-term) in rats, as an OCED 422 study both covers repeated dose and reproductive (screening) toxicity testing.
In general is should be mentioned ahead that study duration, although not yet subchronic, is prolonged compared to an OECD 407 study (43/44 days (males) and ca. 43 days (females) compared to 28 days). Also, females are pregnant, making them in general more susceptible for toxic effects compared to non-pregnant animals. Hence, testing via an OECD 422 may reveal more distinct effects than testing via an OECD 407, and the observed effects may count as worst case short-term toxicity testing.
In the present case, only the effects on general systemic toxicity will be regarded, effects on reproductive toxicity are assessed in the chapter “Toxicity to Reproduction” via a separate entry.
The oral administration of IPGE (CAS No. 4016-14-2; EC No. 223-672-9) to rats by gavage, at dose levels of 100, 300 and 600 mg/kg bw/day, resulted in reduced body weight gain and macroscopic and microscopic changes in the stomach in animals of either sex treated with 600 mg/kg bw/day and minimal disruption of the female reproductive tract (increased corpora lutea, cyclical variation) which most likely caused the complete lack of fertility at 300 mg/kg bw/day and above and the notably reduced fertility at 100 mg/kg bw/day. Increased corpora lutea and cyclical variation account for toxicity to reproduction and should not be regarded when delineating a NOAEL for overall systemic toxicity or when assessing the necessity for classification as STOT RE.
Target organ toxicity (repeated exposure), as defined by the Regulation, “means specific, target organ toxicity arising from a repeated exposure to a substance or mixture. All significant health effects that can impair function, both reversible and irreversible, immediate and/or delayed are included. … Classification for target organ toxicity (repeated exposure) identifies the substance as being a specific target organ toxicant and, as such, it may present a potential for adverse health effects in people who are exposed to it”. Furthermore, “substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement”, and “3.9.2.7. Effects considered to support classification for specific target organ toxicity following repeated exposure...
... (c) any consistent and significant adverse change in clinical biochemistry, haematology, or urinalysis parameters;”
But also: “3.9.2.8. Effects considered not to support classification for specific target organ toxicity following repeated exposure
3.9.2.8.1. It is recognised that effects may be seen in humans and/or animals that do not justify classification. Such effects include, but are not limited to: ...
... (b) small changes in clinical biochemistry, haematology or urinalysis parameters and/or transient effects, when such changes or effects are of doubtful or minimal toxicological importance; ...
... (d) adaptive responses that are not considered toxicologically relevant;”
According to Regulation 1272/2008, Table 3.9.3, Guidance values to assist in Category 2 classification are 10 < C ≤ 100 mg/kg body weight/day derived from a 90 day repeated dose study. For a short-term study, with a safety factor of 3, the boundary value would increase to 300 mg/kg.
Based on the available information, a classification as STOT RE Cat. 2 is not considered necessary as will be outlined below.
The reduced body weight gain does not account for any specific organ, and so only the macroscopic and microscopic changes in the stomach in animals of either sex treated with 600 mg/kg bw/day need to be regarded. At termination, nine males and one female treated with 600 mg/kg bw/day had raised white patches on the non-glandular region of the stomach, which is the only non-incidental finding. The stomach changes were identified as epithelial hyperplasia in animals of either sex treated with 600 mg/kg bw/day and raised white patches on the non-glandular region of the stomach at necropsy. While these microscopic findings represent an adverse effect of treatment they are considered to reflect local irritation rather than any adverse systemic toxicity of the test item. Humans do not have a rat fore-stomach counterpart therefore the macroscopic and microscopic stomach changes detected have limited relevance to human toxicity. So, the LOAEL of 600 mg/kg does not trigger classification as STOT RE as first, the observed effect is less relevant for humans, second, it is rather due to local irritation and not due to a systemic effect of the test item, and third, it is two-fold the guidance value of 300 mg/kg. The observed effects in females treated with 300 or 600 mg/kg in hematology parameters and blood chemistry are difficult to interpret as those females were non-pregnant compared to control, which hence allows no absolute assessment of the toxicological relevance, especially not for humans or STOT RE. Last but not least, as stated above, the notably reduced fertility at 100 mg/kg bw/day and the lack of fertility at doses of 300 and 600 mg/kg including increased corpora lutea and cyclical variation may not account as a trigger for systemic or specific organ toxicity but for an adverse effect on fertility.
Summarizing, the 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was therefore considered to be 300 mg/kg bw/day for males and 100 mg/kg bw/day for females. The observed effects do not suffice for and justify a classification of IPGE as STOT RE Cat. 2.
Executive summary:

Introduction

The GLP 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 approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 600 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 Day 44, followed by the termination of all surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results

Adult Responses

At 600 and 300 mg/kg bw/day, pregnancy was not achieved in any female; in view of this it was not possible to assess effects of the test item on females during gestation or lactation at these dosages.

Mortality:There were no unscheduled deaths considered to be associated with treatment.

One female treated with 300 mg/kg bw/day was killed in extremis on Day 4. This female had hunched posture, pilo-erection, a decreased respiratory rate and was lethargic. At necropsy, enlarged adrenals, enlarged and fluid filled kidneys with increased pelvic space and reddened lungs was evident. Histopathological examination of this animal revealed pyelonephritis (both kidneys) which was considered likely to have caused the poor condition of the animal and its subsequent early termination.

Clinical Observations:Animals of either sex treated with 600 mg/kg bw/day showed incidences of increased salivation between Days 18 and 25. No such effects were evident in animals of either sex treated with 300 or 100 mg/kg bw/day.

Behavioral Assessment:There were no treatment-related changes in the behavioral parameters for either sex at 100, 300 or 600 mg/kg bw/day.

Functional Performance Tests:There were no treatment related changes in functional performance considered to be related to treatment at 100, 300 or 600 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 100, 300 or 600 mg/kg bw/day.

Body Weight:Males treated with 600 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Recovery was evident thereafter, however, a slight reduction in body weight gain was evident in these males during the final week of treatment and consequently a slight reduction in overall gain was also evident in these males. No such effects were detected in males treated with 300 or 100 mg/kg bw/day. No adverse effects were detected in treated females during maturation. Body weight gain during the first week of gestation for females treated with100mg/kg bw/day was comparable to controls however a reduction in body weight gain was evident in these females during the final two weeks of gestation and during lactation. Although females treated with600and300mg/kg bw/day were all non-pregnant, body weight gain during the first week post coitum was comparable to control females. Body weight gain for these females during the remaining treatment period was reduced; with the majority of females showing actual body weight losses.

Food Consumption:No adverse effects were detected in male food consumption. A slight reduction in food conversion efficiency was evident in males treated with600mg/kg bw/day during the first week of treatment. No such effects in food conversion efficiency were evident in males treated with300or100mg/kg bw/day. No adverse effects were detected in food consumption for treated females during maturation. Food consumption for100mg/kg bw/day females was comparable to controls throughout gestation however it was reduced during lactation. Although females treated with600and300mg/kg bw/day were all non-pregnant, food consumption during the first week post coitum was comparable to control females and remained fairly consistent throughout the remainder of the treatment period.

Water Consumption:No adverse effects were detected in water consumption.

 

Reproductive Performance

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

Fertility:All of the females treated with600and300mg/kg bw/day and four females treated with100mg/kg bw/day were non-pregnant following positive evidence of mating. One female treated with100mg/kg bw/day had one corpus luteum and one implantation site but failed to give birth to any offspring.

Gestation Lengths:Gestation lengths for controls and females treated with 100 mg/kg bw/day were between 22.5 and 23.5 days. The distribution of gestation lengths for the females treated with 100 mg/kg bw/day was essentially similar to control.

 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability:Females treated with100mg/kg bw/day showed a reduction in the number of corpora lutea and implantation sites. Pre-implantation loss was also increased in these females however this was considered to be the result of one female with a particularly high value; the remaining females were comparable to controls. Post implantation loss was unaffected by treatment. Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum were lower for females treated with 100 mg/kg bw/day when compared to control litters. Live birth index, offspring viability and sex ratio in litters from females treated with 100 mg/kg bw/day was comparable to controls.

Offspring Growth and Development:As a consequence of the reduced litter size at 100 mg/kg bw/day, litter weights on Days 1 and 4 post partum were reduced when compared to controls. However offspring body weights at birth and subsequently on Days 1 and 4 post partum and offspring body weight gain between Days 1 and 4 post partum exceeded control litters. Surface righting was comparable to controls. The clinical sign apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100 mg/kg bw/day.

 

Laboratory Investigations

Hematology:There were no treatment-related effects detected in the hematological parameters examined.

Blood Chemistry:Assessment of hematology parameters for females at 600 and 300 mg/kg bw/day has to be treated with caution as the female animals at these dosages were in a different physiological state in comparison to the other females on the study. Females treated with 600 and 300 mg/kg bw/day showed an increase in bile acids. No toxicologically significant effects were detected in males treated with 600 or 300 mg/kg bw/day or in animals of either sex treated with 100 mg/kg bw/day.

 

Pathology

Necropsy

Offspring: Necropsy findings did not indicate any obvious effect of maternal treatment on the offspring at 100 mg/kg bw/day.

Adults: At termination, nine males and one female treated with 600 mg/kg bw/day had raised white patches on the non-glandular region of the stomach. No toxicologically significant effects were detected in the remaining terminal kill animals.

Organ Weights:There were no toxicologically significant effects detected in the organ weights measured.

Histopathology:The following treatment-related microscopic abnormalities were detected:

Stomach:hyperplasia of the non-glandular region was present in eleven males and five females treated with 600 mg/kg bw/day.

Ovaries:the majority of females treated with 600 or 300 mg/kg bw/day and in one female treated with 100 mg/kg bw/day had the appearance of increased corpora lutea and was in metestrus or diestrus. This suggests a mild disturbance of the reproductive cycle which may account for the lack of pregnancy.

 

Conclusion

The oral administration of IPGE (CAS No. 4016-14-2,EC No. 223-672-9) to rats by gavage, at dose levels of 100, 300 and 600 mg/kg bw/day, resulted in reduced body weight gain and macroscopic and microscopic changes in the stomach in animals of either sex treated with 600 mg/kg bw/day and minimal disruption of the female reproductive tract (increased corpora lutea, cyclical variation) which most likely caused the complete lack of fertility at 300 mg/kg bw/day and above and the notably reduced fertility at 100 mg/kg bw/day. The reduced body weight gain and food consumption detected in 100 mg/kg bw/day females was considered not to represent an adverse effect of treatment. The 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was therefore considered to be 300 mg/kg bw/day for males and 100 mg/kg bw/day for females. A 'No Observed Effect Level' (NOEL) could not be established for reproductive toxicity.

Reason / purpose for cross-reference:
reference to other study
Remarks:
preliminary dose range-finding test
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
subacute, 14d range-finding test
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2015-12-09 - 2016-01-26 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
study is a range-finding test for an OECD 422 and satisfies the required scientific standards and documentation required for that purpose
Reason / purpose for cross-reference:
reference to other study
Remarks:
present study is the range-finding test for the linked OECD 422 study
Reason / purpose for cross-reference:
reference to other study
Remarks:
present study is the range-finding test for the linked OECD 422 study
Qualifier:
no guideline available
Principles of method if other than guideline:
Range-finding study for an OECD 422 study
- Principle of test: The study was performed according to the Study Plan and was designed to provide information for further repeated dose toxicity studies. The test item was administered to the Wistar Han™:RccHan™:WIST strain rat for a period of fourteen consecutive days at dose levels of 75, 250 and 500 mg/kg bw/day. A control group was dosed with vehicle alone.
- Parameters analysed / observed: Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to gross necropsy examination.
GLP compliance:
yes (incl. QA statement)
Remarks:
Department of Health of the Government of the United Kingdom
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions: The test item was administered within two hours of it being formulated. It is assumed that the formulation was stable for this duration.
Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST strain rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately twelve weeks
- Weight at study initiation: the males weighed 302 to 322g, the females weighed 189 to 213g
- Fasting period before study: no
- Housing: The animals were housed in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum
- Acclimation period: The animals were acclimatized for five days during which time their health status was assessed.

DETAILS OF FOOD AND WATER QUALITY:
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. A certificate of analysis of the batch of diet used is available.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 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.

IN-LIFE DATES: The in-life phase of the study was performed between 12 January 2016 and 26 January 2016.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP.
The test item was administered within two hours of it being formulated. It is assumed that the formulation was stable for this duration.
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

VEHICLE
- Concentration in vehicle: 0, 18.75, 62.5, 125 mg/ml
- Amount of vehicle (if gavage): 4 ml
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
14 days
Frequency of treatment:
daily
Dose / conc.:
75 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In the absence of any toxicity data, preliminary toxicity work was undertaken, treating one male and one female rat at 1000 mg/kg bw/day for up to three days. Treatment-related effects consisted of clinical signs of toxicity (hunched posture, ataxia, lethargy, ptosis, piloerection and emaciation) and actual body weight losses. These animals were terminated on welfare grounds on Day 3.
- Rationale for animal assignment (if not random): The animals were allocated to dose groups using a randomization procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups.
Positive control:
not required
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill health or behavioral change immediately before dosing, up to thirty minutes after dosing and one hour after dosing. Additional observations were also made four hours following dosing (not at weekends). All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Days 1, 4, 8, 11 and 15.

FOOD CONSUMPTION AND COMPOUND INTAKE
Food consumption was recorded for each cage group for Days 1 to 4, 4 to 8, 8 to 11 and 11 to 15. Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION AND COMPOUND INTAKE
Water intake was measured and recorded daily for each cage group.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On completion of the dosing period, all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination and subjected to an internal and external macroscopic examination. No tissues were retained.
Statistics:
Data were processed to give summary incidence or group mean and standard deviation values where appropriate. All data were summarized in tabular form.
Clinical signs:
no effects observed
Description (incidence and severity):
Animals of either sex treated with 500 mg/kg bw/day showed a few sporadic instances of increased salivation post dosing from Day 7. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to represent a true effect of systemic toxicity.
No such effects were detected in animals of either sex treated with 250 or 75 mg/kg bw/day.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 500 mg/kg bw/day showed a reduction in body weight gain between Days 1 and 4. A slight reduction was also evident in males treated with 250 mg/kg bw/day. Recovery was evident thereafter.
No such effects were detected in females treated with 250 mg/kg bw/day or in animals of either sex treated with 75 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on overall food consumption was detected in treated animals. A reduction in food conversion efficiency was evident in animals of either sex treated with 500 mg/kg bw/day and in males treated with 250 mg/kg bw/day between Days 1 and 4, however this was considered to be the result of the lower body weight gains seen for these animals during this period.
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Females treated with 500 mg/kg bw/day showed a slight increase in overall water consumption.
No such effects were detected in males treated with 500 mg/kg bw/day or in animals of either sex treated with 250 or 75 mg/kg bw/day.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Remarks on result:
other: While there was a suggestion of an initial effect on body weight performance at 500 mg/kg bw/day, recovery was evident thereafter.

see attachment

Conclusions:
The study was conducted under GLP as is a range-finding test for an OECD 422 and satisfies the required scientific standards and documentation required for that purpose. The method is to be considered scientifically reasonable with no deficiencies in documentation or any deviations. Hence, the results can be considered as reliable to determine the doses for the main study and to give first indications of the repeated dose toxicity of 2,3-epoxypropyl isopropyl ether.
While there was a suggestion of an initial effect on body weight performance at 500 mg/kg bw/day, recovery was evident thereafter. Therefore a dose level of 500 mg/kg bw/day may be considered slightly too low for use as a high dose level on future studies. However, the early termination of the preliminary sighting animals following 3 days of treatment at 1000 mg/kg bw/day indicates that the high dose level must be significantly lower than 1000 mg/kg bw/day. Based on the results from this study, dose levels of 0 (Control), 100, 300 and 600 mg/kg bw/day are recommended for use in the planned OECD 422 study. Further, given data indicate that the substance is relatively non-toxic.
Executive summary:

The study was designed to provide information for further repeated dose toxicity studies, i.e. OECD 422, and performed as a 14 day range-finding study under GLP.

Methods:The test item was administered by gavage to three groups, each of three male and three female Wistar Han™:RccHan™:WIST strain rats, for fourteen consecutive days, at dose levels of 75, 250 and 500 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (Arachis oil BP). Clinical signs, body weight change, dietary intake and water consumption were monitored during the study. All animals were subjected to gross necropsy examination. The results are:

Mortality:There were no unscheduled deaths.

Clinical Observations:Animals of either sex treated with 500 mg/kg bw/day showed increased salivation post dosing from Day 7 onwards. No such effects were detected in animals of either sex treated with 250 or 75 mg/kg bw/day.

Body Weight:Animals of either sex treated with 500 mg/kg bw/day showed a reduction in body weight gain between Days 1 and 4. A slight reduction was also evident in males treated with 250 mg/kg bw/day. Recovery was evident thereafter. No such effects were detected in females treated with 250 mg/kg bw/day or in animals of either sex treated with 75 mg/kg bw/day.

Food Consumption:No adverse effect on overall food consumption was detected in treated animals. A reduction in food conversion efficiency was evident in animals of either sex treated with 500 mg/kg bw/day and in males treated with 250 mg/kg bw/day between Days 1 and 4.

Water Consumption:Females treated with 500 mg/kg bw/day showed a slight increase in overall water consumption. No such effects were detected in males treated with 500 mg/kg bw/day or in animals of either sex treated with 250 or 75 mg/kg bw/day.

Necropsy:No macroscopic abnormalities were detected.

Conclusion:While there was a suggestion of an initial effect on body weight performance at 500 mg/kg bw/day, recovery was evident thereafter. Therefore a dose level of 500 mg/kg bw/day may be considered slightly too low for use as a high dose level on future studies. However, the early termination of the preliminary sighting animals following 3 days of treatment at 1000 mg/kg bw/day indicates that the high dose level must be significantly lower than 1000 mg/kg bw/day. Based on the results from this study, dose levels of 0 (Control), 100, 300 and 600 mg/kg bw/day are recommended for use in the planned OECD 422 study.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD Guidelines for Testing of Chemicals, No.422: “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 22 March 1996).
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Regulation (EC) No 440/2008
Version / remarks:
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).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Department of Health of the Government of the United Kingdom
Limit test:
no
Justification for study design:
Study type is required under REACH Annex VIII

Test material

Constituent 1
Chemical structure
Reference substance name:
2,3-epoxypropyl isopropyl ether
EC Number:
223-672-9
EC Name:
2,3-epoxypropyl isopropyl ether
Cas Number:
4016-14-2
Molecular formula:
C6H12O2
IUPAC Name:
2-[(propan-2-yloxy)methyl]oxirane
Test material form:
liquid
Remarks:
clear colorless liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark under nitrogen
- Stability under test conditions / Solubility and stability of the test substance in the solvent/vehicle: The stability and homogeneity of the test item formulations were determined by the laboratories' Analytical Services as part of this study. Results show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark. Representative samples of test item formulations were taken and analyzed for concentration of IPGE (Cas No. 4016-14-2 ; EC No. 223-672-9). The results indicate that the prepared formulations were within ± 4% of the nominal concentration, confirming the accuracy and suitability of the formulation procedure.

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST strain rats
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately twelve weeks
- Weight at study initiation: the males weighed 313 to 348g, the females weighed 187 to 213g
- Fasting period before study: no
- Housing: 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. 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.
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) ad libitum
- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage ad libitum
- Acclimation period: The animals were acclimatized for seven days during which time their health status was assessed.

DETAILS OF FOOD AND WATER QUALITY:
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. A certificate of analysis of the batch of diet used is available.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 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.

IN-LIFE DATES: The in-life phase of the study was conducted between 10 March 2016 (first day of treatment) and 28 April 2016 (final day of necropsy).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP.

VEHICLE
- Concentration in vehicle: 0, 25, 75, 150 mg/ml
- Amount of vehicle (if gavage): 4 ml
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of successful mating, for a period of up to fourteen days
- Proof of pregnancy: 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)
- After successful mating each pregnant female was caged (how): Mated females were housed individually during the period of gestation and lactation.
- Any other deviations from standard protocol: none stated
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability and homogeneity of the test item formulations were determined by the laboratories' Analytical Services as part of this study. Results show the formulations to be stable for at least twenty-one days. Formulations were therefore prepared fortnightly and stored at approximately 4 ºC in the dark. Representative samples of test item formulations were taken and analyzed for concentration of IPGE (Cas No. 4016-14-2 ; EC No. 223-672-9). The method used for analysis of formulations and the results obtained are given in the attachment. The results indicate that the prepared formulations were within ± 4% of the nominal concentration, confirming the accuracy and suitability of the formulation procedure.
Duration of treatment / exposure:
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.
Males were terminated after 43 or 44 days. At Day 5 post partum, all surviving females and surviving offspring were killed, and females were hence dosed 14 days prior to pairing, during pairing and pregnancy, and 4 days afterwards.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
600 mg/kg bw/day (nominal)
No. of animals per sex per dose:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on preliminary dose range-finding test
- Rationale for animal assignment (if not random): 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.
Positive control:
not required

Examinations

Parental animals: Observations and examinations:
CAGE SIDE / CLINICAL OBSERVATIONS: Yes
- Time schedule: 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: Yes
- Time schedule for examinations: 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 AND COMPOUND INTAKE:
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:
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 AND COMPOUND INTAKE:
- Time schedule for examinations: Water intake was observed daily by visual inspection of water bottles for any overt changes.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 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 or Day 25 post coitum 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.
- Animals fasted: No
- How many animals: five males and five females selected from each test and control group
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).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 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 or Day 25 post coitum 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.
- Animals fasted: No
- How many animals: five males and five females selected from each test and control group
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea, Glucose, Total protein (Tot.Prot.), Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Total cholesterol (Chol), Total bilirubin (Bili), Bile acids

NEUROBEHAVIOURAL EXAMINATION: Yes

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, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behavior, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin color, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation
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, Vocalization, Toe pinch, Tail pinch, Finger approach, Touch escape, Pupil reflex, Blink reflex, Startle reflex
Oestrous cyclicity (parental animals):
Not monitored, estrous secured by sufficient pairing time
Sperm parameters (parental animals):
Parameters examined in male parental generation: testis weight, epididymis weight
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.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
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)


GROSS EXAMINATION OF DEAD PUPS: yes
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.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals on day 43 or 44.
- Maternal animals: All surviving animals on day 5 post partum.

GROSS PATHOLOGY: Yes
Necropsy
Adult males were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 43 or Day 44. Adult females were killed by intravenous overdose of suitable barbiturate agent followed by exsanguination on Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of suitable barbiturate agent. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 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.
The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose group. Tissues shown in bold were weighed from all remaining animals: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Pituitary (post-fixation), Prostate and Seminal Vesicles, Spleen, Testes, Thymus, Thyroid (weighed post-fixation with Parathyroid), Uterus (weighed with Cervix)

HISTOPATHOLOGY: Yes
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, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Caecum, Coagulating glandm, Colon, Duodenum, Epididymides (preserved in Modified Davidsons fluid), Esophagus, Eyes (fixed in Davidson’s fluid), Gross lesions, Heart, Ileum (including peyer’s patches), Jejunum, Kidneys, Liver, Lungs (with bronchi) (inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative), Lymph nodes (mandibular and mesenteric), Mammary gland, Muscle (skeletal), Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles, Skin (hind limb), Spinal cord (cervical, mid thoracic and lumbar), Spleen, Stomach, Testes (preserved in Modified Davidsons fluid), Thyroid/Parathyroid, Trachea, Thymus, Urinary bladder, Uterus & Cervix, Vagina
The tissues from five selected control and 600 mg/kg bw/day dose group animals and any animals dying during the study 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 from the remaining control and 600 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 600 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 stomach changes, examination was subsequently extended to include similarly prepared sections of the stomach from animals in the low and intermediate groups.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
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.
Statistics:
Due to limitations of the free text field, information is provided below.
Reproductive indices:
Pre-coital Interval
Fertility Indices (Mating Index, Mating Index)
Gestation Length
Parturition Index
Offspring viability indices:
Implantation Losses (Pre–implantation loss, Post–implantation loss)
Live Birth and Viability Indices (Live Birth Index, Viability Index)
Sex Ratio (% males)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Animals of either sex treated with 600 mg/kg bw/day showed incidences of increased salivation between Days 18 and 25.
No such effects were evident in animals of either sex treated with 300 or 100 mg/kg bw/day.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were no unscheduled deaths considered to be associated with treatment.
One female treated with 300 mg/kg bw/day was killed in extremis on Day 4. This female had hunched posture, pilo-erection, a decreased respiratory rate and was lethargic. At necropsy, enlarged adrenals, enlarged and fluid filled kidneys with increased pelvic space and reddened lungs was evident. Histopathological examination of this animal revealed pyelonephritis (both kidneys) which was considered likely to have caused the poor condition of the animal and its subsequent early termination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 600 mg/kg bw/day showed a statistically significant reduction (p<0.01) in body weight gain during the first week of treatment. Recovery was evident thereafter however, a slight reduction in body weight gain was evident in these males during the final week of treatment. Consequently, a 10% reduction in overall body weight gain was evident in these males.
No such effects were detected in males treated with 300 or 100 mg/kg bw/day.
No adverse effects were detected in treated females during maturation. Body weight gain during the first week of gestation for females treated with 100 mg/kg bw/day was comparable to controls however a reduction in body weight gain was evident in these females during the final two weeks of gestation. Consequently, cumulative body weight gain between Days 0 and 20 of gestation was lower in these females. A statistically significant reduction (p<0.01) in body weight gain was also evident in females treated with 100 mg/kg bw/day during lactation.
Although females treated with 600 and 300 mg/kg bw/day were all non-pregnant, body weight gain during the first week post coitum was comparable to control females. Body weight gain for these females during the remaining treatment period was reduced; with the majority of females showing actual body weight losses.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effects were detected in male food consumption. Males treated with 600 mg/kg bw/day showed a reduction in food conversion efficiency during the first week of treatment.
No adverse effects were detected in food consumption for treated females during maturation. Food consumption for 100 mg/kg bw/day females was comparable to controls throughout gestation however it was statistically significantly reduced (p<0.05) during lactation. This lower food consumption was consistent with lower body weight gain compared to control during this phase of the study.
Although females treated with 600 and 300 mg/kg bw/day were all non-pregnant, food consumption during the first week post coitum was comparable to control females and remained fairly consistent throughout the remainder of the treatment period.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 600 mg/kg bw/day showed a reduction in food conversion efficiency during the first week of treatment.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No adverse effects were detected in water consumption.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the hematological parameters measured for males treated with 600 and 300 mg/kg bw/day or both sexes at 100 mg/kg bw/day.
Males treated with 600 and 300 mg/kg bw/day showed statistically significant reductions (p<0.05-0.01) in hemoglobin (600 mg/kg bw/day only), mean corpuscular hemoglobin concentration, total leucocyte count and lymphocytes. All of the individual values were within historical control ranges and in the absence of any associated histopathological correlates or true dose related responses the intergroup differences were considered of no toxicological significance. Females treated with 100 mg/kg bw/day showed a statistically significant reduction (p<0.01) in platelet count. All of the individual values were within the historical control range and in the absence of any associated histopathological correlates the intergroup difference was considered of no toxicological significance.
Assessment of hematology parameters for females at 600 and 300 mg/kg bw/day has to be treated with caution as no females at these dosages were maintaining a litter at the time of blood sampling and all of theses females were non-pregnant. The female animals at these dosages were therefore in a different physiological state in comparison to the other females on the study and also those females contributing to the historical control data.
At 600 and 300 mg/kg bw/day, higher hemoglobin, erythrocyte count and hematocrit and lower neutrophils and platelet count for females attained statistical significance (p<0.05-0.01) when compared with control. Excluding one female treated with 300 mg/kg bw/day for platelet count, all of the remaining individual values for these parameters were within the historical control range. A true dose related response was also not evident in platelet count.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There were no toxicologically significant effects detected in the blood chemical parameters measured for males treated with 600 or 300 mg/kg bw/day or both sexes at 100 mg/kg bw/day.
Males treated with 600 mg/kg bw/day showed a statistically significant increase in bile acids (p<0.01) and alanine aminotransferase (p<0.05). Males from all treatment groups and females treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in albumin/globulin ratio. All individual values (excluding one alanine aminotransferase value) were within historical control ranges and a true dose related response was not evident for albumin/globulin ratio. In the absence of any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Assessment of blood chemistry parameters for females at 600 and 300 mg/kg bw/day has to be treated with caution as no females at these dosages were maintaining a litter at the time of blood sampling and all of theses females were non-pregnant. The female animals at these dosages were therefore in a different physiological state in comparison to the other females on the study and also those females contributing to the historical control data.
For females at 600 and 300 mg/kg bw/day, higher bile acids, inorganic phosphorus and albumin/globulin ratio attained statistical significance (p<0.05) compared to control. For inorganic phosphorus and albumin/globulin ratio, all individual values were within the historical control ranges however for bile acids the majority of individual values were outside of the historical control range. Additionally higher bilirubin and lower creatinine and urea levels for females treated with 600 mg/kg bw/day attained statistical significance (p<0.05-0.01) compared to control but all individual values were within the historical control ranges.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Behavioral Assessments: There were no treatment-related changes in the behavioural parameters for either sex at 100, 300 or 600 mg/kg bw/day.
Functional Performance Tests: There were no treatment related changes in functional performance considered to be related to treatment at 100, 300 or 600 mg/kg bw/day. Males treated with 600 mg/kg bw/day showed a statistically significant (p<0.05) increase in forelimb grip strength. The intergroup difference was confined to one out of the three tests and in the absence of any associated clinical signs to suggest a neurotoxic effect, the intergroup difference was considered not to be of toxicological importance.
Sensory Reactivity Assessments: There were no inter-group differences in sensory reactivity scores that were considered to be related to treatment at 100, 300 or 600 mg/kg bw/day.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The following treatment-related microscopic abnormalities were detected:
Stomach: hyperplasia of the non-glandular region was present in eleven males and five females treated with 600 mg/kg bw/day.
Ovaries: the majority of females treated with 600 or 300 mg/kg bw/day and in one female treated with 100 mg/kg bw/day had the appearance of increased corpora lutea and was in metestrus or diestrus. This suggests a mild disturbance of the reproductive cycle which may account for the lack of pregnancy.
There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle).
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no test item related microscopic findings in the testes, including following the qualitative examination of the stages of spermatogenesis in the testes (no test item related abnormalities in the integrity of the various cell types present within the different stages of the sperm cycle).
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
All animals mated within the first four days of pairing (i.e. at their first estrus opportunity) however no females treated with 600 or 300 mg/kg bw/day were pregnant. The pregnancy rate at 100 mg/kg bw/day was also lower than anticipated with four females not pregnant and one female showing one corpora lutea and one implantation site but failing to give birth to any offspring.
Gestation lengths for controls and females treated with 100 mg/kg bw/day were between 22.5 and 23.5 days. The distribution of gestation lengths for the females treated with 100 mg/kg bw/day was essentially similar to control.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
reproductive performance
Remarks on result:
other:
Remarks:
lowest dose tested, no NOAEL could be observed
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
The clinical signs apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100 mg/kg bw/day.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Live birth index and offspring viability in litters from females treated with 100 mg/kg bw/day was comparable to controls.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
As a consequence of the reduced litter size at 100 mg/kg bw/day, litter weights on Days 1 and 4 post partum were reduced when compared to controls, although statistical significance was not achieved. However, offspring body weights at birth and subsequently on Days 1 and 4 post partum and offspring body weight gain between Days 1 and 4 post partum exceeded control litters.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Neither the type, incidence nor distribution of necropsy findings for offspring terminated at Day 5 of age indicated any obvious effect of maternal treatment on the offspring at 100 mg/kg bw/day.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Surface righting was comparable to controls.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
Remarks on result:
other: actual NOAEL not determinable as no litter was produced at dosage levels of 300 and 600 mg/kg bw/d

Overall reproductive toxicity

Key result
Reproductive effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
Treatment related:
yes
Relation to other toxic effects:
reproductive effects in the absence of other toxic effects
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

See attachments

Applicant's summary and conclusion

Conclusions:
The study was conducted under GLP according to OECD guideline 422 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation and performance. Hence, the results can be considered as reliable to assess the repeated dose oral toxicity (short-term) in rats, as an OCED 422 study both covers repeated dose and reproductive (screening) toxicity testing.
Effects of treatment with IPGE were already discussed in the chapter repeated dose toxicity, so this session focuses on effects on reproduction.
Reproductive toxicity includes both adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring. The present OECD 422 study, which suffices for REACH Annex VIII data requirements, does not examine sufficient parameters to unambiguously identify developmental toxicants Category 1, the given information however indicates that developmental effects do not need to be regarded at all. Neither the type, incidence nor distribution of necropsy findings for offspring terminated at Day 5 of age indicated any obvious effect of maternal treatment on the offspring at 100 mg/kg bw/day. Surface righting was comparable to controls.
According to Regulation 1272/2008, Table 3.7.1(a) Hazard categories for reproductive toxicants, “Substances are classified in Category 2 for reproductive toxicity when there is some evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, and where the evidence is not sufficiently convincing to place the substance in Category 1. If deficiencies in the study make the quality of evidence less convincing, Category 2 could be the more appropriate classification.
Such effects shall have been observed in the absence of other toxic effects, or if occurring together with other toxic effects the adverse effect on reproduction is considered not to be a secondary non-specific consequence of the other toxic effects.”
The oral administration of IPGE (CAS No. 4016-14-2; EC No. 223-672-9) to rats by gavage, at dose levels of 100, 300 and 600 mg/kg bw/day, resulted in reduced body weight gain and macroscopic and microscopic changes in the stomach in animals of either sex treated with 600 mg/kg bw/day and minimal disruption of the female reproductive tract (increased corpora lutea, cyclical variation) which most likely caused the complete lack of fertility at 300 mg/kg bw/day and above and the notably reduced fertility at 100 mg/kg bw/day. The reduced body weight gain and food consumption detected in 100 mg/kg bw/day females was considered not to represent an adverse effect of treatment. The 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was therefore considered to be 300 mg/kg bw/day for males and 100 mg/kg bw/day for females. A 'No Observed Effect Level' (NOEL) could not be established for reproductive toxicity.
In consequence, effects on the reproductive system occurred in the absence of other toxic effects, so IPGE needs to be considered as reproductive toxicant. As the available OECD 422 was designed as only a screening study (nevertheless sufficient for the Annex VIII data requirements under REACH), there is no evidence sufficiently convincing to place the substance in Category 1. Consequently, IPGE should be classified as Reproductive toxicant Category 2.
Executive summary:

Introduction

The GLP 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 approximately six weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 100, 300 and 600 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 surviving females and offspring on Day 5 post partum. Any female which did not produce a pregnancy was terminated on or after Day 25 post coitum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Results

Adult Responses

At 600 and 300 mg/kg bw/day, pregnancy was not achieved in any female; in view of this it was not possible to assess effects of the test item on females during gestation or lactation at these dosages.

Mortality:There were no unscheduled deaths considered to be associated with treatment.

One female treated with 300 mg/kg bw/day was killed in extremis on Day 4. This female had hunched posture, pilo-erection, a decreased respiratory rate and was lethargic. At necropsy, enlarged adrenals, enlarged and fluid filled kidneys with increased pelvic space and reddened lungs was evident. Histopathological examination of this animal revealed pyelonephritis (both kidneys) which was considered likely to have caused the poor condition of the animal and its subsequent early termination.

Clinical Observations:Animals of either sex treated with 600 mg/kg bw/day showed incidences of increased salivation between Days 18 and 25. No such effects were evident in animals of either sex treated with 300 or 100 mg/kg bw/day.

Behavioral Assessment:There were no treatment-related changes in the behavioral parameters for either sex at 100, 300 or 600 mg/kg bw/day.

Functional Performance Tests:There were no treatment related changes in functional performance considered to be related to treatment at 100, 300 or 600 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 100, 300 or 600 mg/kg bw/day.

Body Weight:Males treated with 600 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Recovery was evident thereafter, however, a slight reduction in body weight gain was evident in these males during the final week of treatment and consequently a slight reduction in overall gain was also evident in these males. No such effects were detected in males treated with 300 or 100 mg/kg bw/day. No adverse effects were detected in treated females during maturation. Body weight gain during the first week of gestation for females treated with100mg/kg bw/day was comparable to controls however a reduction in body weight gain was evident in these females during the final two weeks of gestation and during lactation. Although females treated with600and300mg/kg bw/day were all non-pregnant, body weight gain during the first weekpost coitumwas comparable to control females. Body weight gain for these females during the remaining treatment period was reduced; with the majority of females showing actual body weight losses.

Food Consumption:No adverse effects were detected in male food consumption. A slight reduction in food conversion efficiency was evident in males treated with 600mg/kg bw/day during the first week of treatment. No such effects in food conversion efficiency were evident in males treated with 300 or 100 mg/kg bw/day. No adverse effects were detected in food consumption for treated females during maturation. Food consumption for 100mg/kg bw/day females was comparable to controls throughout gestation however it was reduced during lactation. Although females treated with 600 and 300 mg/kg bw/day were all non-pregnant, food consumption during the first week post coitum was comparable to control females and remained fairly consistent throughout the remainder of the treatment period.

Water Consumption:No adverse effects were detected in water consumption.

 

Reproductive Performance

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

Fertility:All of the females treated with 600 and 300 mg/kg bw/day and four females treated with 100mg/kg bw/day were non-pregnant following positive evidence of mating. One female treated with100mg/kg bw/day had one corpus luteum and one implantation site but failed to give birth to any offspring.

Gestation Lengths:Gestation lengths for controls and females treated with 100 mg/kg bw/day were between 22.5 and 23.5 days. The distribution of gestation lengths for the females treated with 100 mg/kg bw/day was essentially similar to control.

 

Litter Responses

Offspring Litter Size, Sex Ratio and Viability:Females treated with100mg/kg bw/day showed a reduction in the number of corpora lutea and implantation sites. Pre-implantation loss was also increased in these females however this was considered to be the result of one female with a particularly high value; the remaining females were comparable to controls. Post implantation loss was unaffected by treatment. Of the litters born, litter size at birth and subsequently on Days 1 and 4 post partum were lower for females treated with 100 mg/kg bw/day when compared to control litters. Live birth index, offspring viability and sex ratio in litters from females treated with 100 mg/kg bw/day was comparable to controls.

Offspring Growth and Development:As a consequence of the reduced litter size at 100 mg/kg bw/day, litter weights on Days 1 and 4 post partum were reduced when compared to controls. However offspring body weights at birth and subsequently on Days 1 and 4 post partum and offspring body weight gain between Days 1 and 4 post partum exceeded control litters. Surface righting was comparable to controls. The clinical sign apparent for offspring on the study were typical for the age observed. Neither the incidence nor distribution of these observations indicated any adverse effect of maternal treatment on offspring development at 100 mg/kg bw/day.

 

Laboratory Investigations

Hematology:There were no treatment-related effects detected in the hematological parameters examined.

Blood Chemistry:Assessment of hematology parameters for females at 600 and 300 mg/kg bw/day has to be treated with caution as the female animals at these dosages were in a different physiological state in comparison to the other females on the study. Females treated with 600 and 300 mg/kg bw/day showed an increase in bile acids. No toxicologically significant effects were detected in males treated with 600 or 300 mg/kg bw/day or in animals of either sex treated with 100 mg/kg bw/day.

 

Pathology

Necropsy

Offspring: Necropsy findings did not indicate any obvious effect of maternal treatment on the offspring at 100 mg/kg bw/day.

Adults: At termination, nine males and one female treated with 600 mg/kg bw/day had raised white patches on the non-glandular region of the stomach. No toxicologically significant effects were detected in the remaining terminal kill animals.

Organ Weights:There were no toxicologically significant effects detected in the organ weights measured.

Histopathology:The following treatment-related microscopic abnormalities were detected:

Stomach:hyperplasia of the non-glandular region was present in eleven males and five females treated with 600 mg/kg bw/day.

Ovaries:the majority of females treated with 600 or 300 mg/kg bw/day and in one female treated with 100 mg/kg bw/day had the appearance of increased corpora lutea and was in metestrus or diestrus. This suggests a mild disturbance of the reproductive cycle which may account for the lack of pregnancy.

 

Conclusion

The oral administration of IPGE (CAS No. 4016-14-2,EC No. 223-672-9) to rats by gavage, at dose levels of 100, 300 and 600 mg/kg bw/day, resulted in reduced body weight gain and macroscopic and microscopic changes in the stomach in animals of either sex treated with 600 mg/kg bw/day and minimal disruption of the female reproductive tract (increased corpora lutea, cyclical variation) which most likely caused the complete lack of fertility at 300 mg/kg bw/day and above and the notably reduced fertility at 100 mg/kg bw/day. The reduced body weight gain and food consumption detected in 100 mg/kg bw/day females was considered not to represent an adverse effect of treatment. The 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was therefore considered to be 300 mg/kg bw/day for males and 100 mg/kg bw/day for females. A 'No Observed Effect Level' (NOEL) could not be established for reproductive toxicity.