2,3-epoxypropyl isopropyl ether

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction: Screening study oral (gavage), Wistar rat m/f, 0, 100, 300, 600 mg/kg, OECD 422, GLP:

NOAEL = 100 mg/kg (female, general toxicity)

NOAEL = 300 mg/kg (male, general toxicity)

LOAEL = 100 mg/kg (female, reproductive toxicity)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting 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

Justification for study design:

Study type is required under REACH Annex VIII

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

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

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

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)

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: 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.

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

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

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.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

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

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.
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.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

There is a OECD 422 screening study on the registered substance available, the tonnage-driven data requirements are fully met, so the database is of high quality.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

OECD 422, GLP:

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.

Live birth index and offspring viability in litters from females treated with 100 mg/kg bw/day was comparable to controls.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

1.    Problem formulation

 

The present MoAA aims to assess human relevance of effects on reproductive toxicity. There were dose-dependent effects noted in rats. Upon evidence of mating, there were no pregnancies (300 or 600 mg/kg bw/d) or notably reduced fertility at 100 mg/kg bw/day.

 

 

2.    Hypothesised Mode of action Statement

 

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), and all animals mated within the first four days of pairing. In consequence, the lack of fertility cannot be attributed to effects in males, and females only need to be regarded.

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. Consequently, effects during pregnancy can also be excluded, to the impairment must be prior to implantation.

Histopathological examinations of the ovaries revealed the following: 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. So as corpora lutea were seen, and mating was observed in all treated animals within four days, it can be concluded that cycling and ovulation occurred within normal ranges, but no fertilisation / implantation occurred, as rats were found to be in metestrus or diestrus. So it can be concluded that IPGE interferes with reproduction only in females between ovulation and implantation.

Secondary effects due to excessive overall toxicity can be excluded as reproductive effects were noted without any adverse effects related to systemic toxicity. Definite conclusions on a potential endocrine disrupting effect cannot be drawn, as weights of associated organs cannot be compared easily with control as control animals were pregnant, the mid and high dose animals not. Further, histopathology revealed only changes in the ovaries, thymus, male organs etc. were not affected. Only a mild disturbance of the reproductive cycle was noted.

 

 

3.    Summary of data for use in Mode of Action Analysis

 

System	Results
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.
Testes	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)
Histopathology	Organs rather associated with EDC effects, e.g. Pituitary, Testes, Thyroid or Parathyroid showed no effects compared to control

 

 

4.    Listing of key events identified for a specific Mode of Action

Mating	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 Length	Gestation lengths for controls and females treated with 100 mg/kg bw/day were between 22½ and 23½ days. The distribution of gestation lengths for the females treated with 100 mg/kg bw/day was essentially similar to control.
Effects in males	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)
Effects in females	Histopathological examinations of the ovaries revealed the following: 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.

 

 

5.    Bradford Hill Considerations for Weight of Evidence Analysis of available data/information for Mode of Action Analysis in experimental species

 

5a. Dose Response Relationships and Temporal Association

The oral administration of IPGE at dose levels of 100, 300 and 600 mg/kg bw/day, resulted in treatment-related effects in animals of either sex treated with 600 mg/kg bw/day and a reproductive effect in all treatment groups. Reproductive effects occurred in doses below overall systemic toxicity, hence, the substance was identified as reproductive toxicant. All animals at 600 and 300 mg/kg bw/day mated within the first four days of pairing, however no females achieved pregnancy. Four females treated with 100 mg/kg bw/day were also non pregnant and one female from this treatment group had one corpora lutea and one implantation site but failed to give birth to any offspring. In consequence, a dose-related response was observed. The findings (increased corpora lutea, metestrus/diestrus) indicate that IPGE interferes with reproduction only in females between ovulation and implantation.

 

 

5b. Consistency & Specificity – Biological Plausibility

The lack of pregnancy can be associated with the observed mild disturbance of the reproductive cycle, and a dose-response was observed. The effect was specific in females between ovulation and implantation. Hence, the findings are consistent and biologically plausible.

 

 

5c. Qualitative and Quantitative human concordance

Due to the lack of human data, no conclusions on concordance can be drawn. However, there is no indication given that, due to a possible species-specificity, the observed effects may not be relevant for humans. However, as the present OECD 422 study was only designed as a screening study, and no human data is available, only classification as Repro Cat. 2 is triggered, Cat. 1 cannot be justified.

 

 

6.    Other potential Modes of Action

 

None identified due to the absence of effects in other studies

 

 

7.    Uncertainties/Inconsistencies and Identification of Data Gaps

 

None identified. The study was assessed with Klimisch 1 and the tonnage-driven data requirements under REACH are fully met, no data gaps exists. The available data was shown to be consistent.

 

8.    Conclusions in relation to problem formulation

 

It can be concluded that there is no indication given that the effects on reproduction noted in rats (no pregnancies (300 or 600 mg/kg bw/d) or notably reduced fertility at 100 mg/kg bw/day) below the NOAEL for systemic / overall toxicity are not relevant for humans. IPGE should be classified as Repro Cat. 2.

Justification for classification or non-classification

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.