(2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date (Animal arrival) 24 June 2020

Experimental completion date: February 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No.422: “Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test” (adopted 29 July 2016).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate

EC Number:

807-159-2

Cas Number:

69701-99-1

Molecular formula:

C10H16O4

IUPAC Name:

(2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

Test item: MEDOL-10
Test item identity (including alternative names): (2-Ethyl-2-methyl-1, 3-dioxolan-4-y)methyl acrylate
Intended use: Industrial Chemical
Appearance: Clear, colorless liquid
Storage conditions: At ambient temperature in the dark (15 to 25C)
Supplier: Sponsor
Batch number: 09892803
Expiry date: 15 February 2023
Purity: 98.8% which excludes optical isomer
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 g representative sample was taken from each batch of test item. This sample was placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Animals
Strain/Species RccHan™;WIST rat.
Supplier Envigo (RMS) UK.
Number of animals ordered 44 males and 48 females.
Spare animals were removed from the study room after treatment commenced.

Duration of acclimatization Males six days before commencement of treatment.
Females 20 days before commencement of treatment.

Age of the animals at the start of treatment Males 84 to 90 days old.
Females 98 to 104 days old.

Weight range of the animals at the start of treatment Males 312 to 367 g.
Females 190 to 227 g.

Allocation and Identification
Allocation On arrival and non-selective allocation to cages.

Estrous cycles were evaluated pre-treatment. After 14 days evaluation, females that failed to exhibit typical 4-5 day cycles were not allocated to the study.

On Day 1 of study all animals were weighed and body weights were reviewed by Study Management before dosing commenced to ensure that variations in the body weight of animals did not exceed ± 20% of the mean for each sex.

Identification of animals Each adult animal was assigned a number and identified uniquely within the study by a microchip before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.
Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Replacement
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.

Replacement before allocation Acyclic estrous cycle Two females

Animal Care and Husbandry
Environmental Control
Animal facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 20-24C and 40-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 12 hours light: 12 hours dark.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
Solid (polycarbonate) bottom cages were used throughout the study except during pairing.
Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily.
Cage distribution The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Number of animals per cage
Pre-pairing up to four animals of one sex
Pairing one male and one female
Males after mating up to four animals
Gestation one female
Lactation one female + litter

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study (except during lactation) and replaced when necessary.
Plastic shelter Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.
Paper shavings Approximately two handfuls of paper shavings were provided to each cage as nesting material from Day 20 after mating and throughout lactation. Shavings were replaced at the same frequency as the bedding.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.
A sample (250 g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30C) until finalization of the report. Samples were discarded after finalization of the report.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.
Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Administration / exposure

Route of administration:

oral: gavage

Details on exposure:

Administration
Dosing was restricted to the F0 generation. Animals of the F1 generation were not dosed directly.
Route Oral gavage using a suitably graduated syringe and a plastic cannula inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose 5 mL/kg body weight.
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as treated groups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of Dose Formulations
The formulations for Week 1, Week 3 and the Final week were sampled. For all Groups, 1 ×
10 mL (accurately weighed) sample was taken from the middle of the formulation by
Pharmacy personnel.

Duplicate aliquots from all groups were analyzed in accordance with the analytical
procedure. During Week 3 additional aliquots were taken for rework purposes. The remaining
samples were retained for contingency. Samples were disposed of once satisfactory results
were achieved.

Details on mating procedure:

Pairing commenced After a minimum of two weeks of treatment.
Male/female ratio 1:1 from within the same treatment groups.
Duration of pairing Up to two weeks.
Daily checks for evidence of mating Ejected copulation plugs in cage tray and sperm in the vaginal smear.
Day 0 of gestation When positive evidence of mating was detected.
Male/female separation Day when mating evidence was detected.
Pre-coital interval Calculated for each female as the time between first pairing and evidence of mating.

Duration of treatment / exposure:

Males were treated daily for two weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated daily for two weeks before pairing, throughout pairing, gestation and until Day 13 of lactation.

Frequency of treatment:

Daily

Duration of test:

Males Two weeks before pairing up to necropsy after minimum of five weeks.
Females 15 days before pairing, then throughout pairing and gestation until Day 13 of lactation.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle only

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10 males/ 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
The dose levels selected for this OECD 422 study were based on the results of a 14-day preliminary toxicity study (Covance Study No. HV57RL).
In the preliminary study, dose levels of 300, 600 and 1000 mg/kg/day were initially investigated. Severe clinical signs were seen on Day 3 of treatment for animals receiving 1000 mg/kg/day and on Day 6 of treatment for animals receiving 600 mg/kg/day. Signs seen were abnormally cold to touch, piloerection, hunched posture, irregular or slow breathing, decreased activity, etc. Due to adverse signs seen, animals receiving 1000 mg/kg/day were terminated on Day 3 of treatment, and animals receiving 600 mg/kg/day were terminated on Day 6. At macroscopic examination findings such as stomach depressions, stomach perforations, ruptured liver, abnormal contents of stomach and cecum, etc., were seen in animals receiving 600 or 1000 mg/kg/day, after only 6 or 3 days of treatment, respectively. No adverse clinical signs were seen in animals receiving 300 mg/kg/day, and this dose level was tolerated for the entire treatment period.
A further group of animals was added on to study, these animals received a dose level of 450 mg/kg/day. Unfortunately, this dose level was not tolerated and similar signs were seen as witnessed at 600 or 1000 mg/kg/day and the group was prematurely terminated after receiving seven daily doses.
Due to the overt toxicity at 450, 600 or 1000 mg/kg/day, these dose levels were not considered suitable for use on this OECD 422 study. Due to the steep dose response, choosing a high dose level greater than 300 mg/kg/day could lead to an unacceptable level of mortality, therefore the high dose level for this OECD 422 study was restricted to 300 mg/kg/day although no evidence of toxicity was observed in the preliminary study. Low and intermediate dose levels of 30 and 100 mg/kg/day were selected, representing an approximate three-fold interval between dose levels.

Examinations

Maternal examinations:

Clinical and Behavioral Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were performed to establish and confirm a pattern of signs in association with dosing according to the following schedule:

F0 females Week 1 - daily
Week 2 - twice during the week
Gestation phase - Days 0, 4, 7, 10, 14 and 20
Lactation phase - Days 1, 3, 6, 9 and 12

Detailed observations were recorded at the following times in relation to dose administration:
• Pre-dose observation.
• One to two hours after completion of dosing.
• As late as possible in the working day.

Detailed physical examination and arena observations

Before treatment commenced and during each week of treatment and on Days 0, 7, 14 and 20 after mating and Days 1, 6 and 12 of lactation, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities. “Blind” recording was not possible for animals during pairing or for females after mating and during lactation, for logistical reasons, therefore observations were made on these occasions without “blinding”.

After removal from the home cage, animals were assessed for physical condition and behavior during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behavior.

Findings were either reported as "present" or assigned a severity grade - slight, moderate or marked.

Sensory reactivity and grip strength
Sensory reactivity and grip strength assessments were performed (before dosing) on the five lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Animals were moved into individual cages prior to transport to the testing room. Animals were removed and returned to their home cages by an assistant, such that the observer was unaware of the treatment group. The cage labels on these individual cages showed only the study and animal number. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

The following measurements, reflexes and responses were recorded:

Approach response
A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:

1 No reaction or ignores/walks past probe
2 Normal awareness and reaction e.g. approaches and/or sniffs probe
3 Active avoidance, abnormally fearful or aggressive reaction

Pinna reflex
The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
1 No response
2 Normal response e.g. ear twitches/flattens or animal shakes its head
3 Abnormally fearful or aggressive response

Auditory startle reflex
The animal’s response to a sudden sharp noise was assessed and scored as:
1 No response
2 Weak response e.g. ear twitch only
3 Normal response e.g. obvious flinch or startle
4 Exaggerated response e.g. all feet off floor

Tail pinch response
The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
1 No response
2 Weak response e.g. turns around slowly or weak vocalization without moving away
3 Normal response e.g. jumps forward or turns around sharply, usually with vocalization
4 Exaggerated response e.g. excessive vocalization, body movement or aggression

Grip strength
Forelimb and hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.

At any point during the observations, additional comments were made as free text where considered appropriate.
Motor activity
During Week 5 of treatment for males and at Day 7-9 of lactation for females, the first five lactating females in each group was measured (before dosing) using a Rodent Activity Monitoring System (Version 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Covance.

Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not all necessarily tested on the same day, but the numbers of animals and the times of testing were balanced across the groups as far as possible on each day of testing.

Body Weight
The weight of animals was recorded as follows:

F0 females Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Day 1, 4, 7, and 13 of lactation.
On the day of necropsy.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals Weekly, from the day that treatment commenced.
Food consumption was not recorded for females during the period when paired for mating (Week 3).

For females after mating food consumption was performed to match the body weight recording:
Days 0-6, 7-13 and 14-20 after mating
Days 1-3, 4-6 and 7-12 of lactation (see Section 4).

From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase


Hematology, Peripheral Blood
Blood samples were collected at the following occasions:
Occasion Animals
At termination The five lowest numbered surviving males per group.
Day 13 of lactation The first five lactating females with a surviving litter per group.

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyzer:
• Hematocrit (Hct)*
• Hemoglobin concentration (Hb)
• Erythrocyte count (RBC)
• Absolute reticulocyte count (Retic)
• Mean cell hemoglobin (MCH)*
• Mean cell hemoglobin concentration (MCHC)*
• Mean cell volume (MCV)
• Red cell distribution width (RDW)
• Total leucocyte count (WBC)
• Differential leucocyte count:
• Neutrophils (N)
• Lymphocytes (L)
• Eosinophils (E)
• Basophils (B)
• Monocytes (M)
• Large unstained cells (LUC)
• Platelet count (Plt)

* - Derived values calculated in ClinAxys

Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscopy, in the case of flags from the Advia 120 analyzer. Confirmation or a written description from the blood film was made where appropriate.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using a Stago STA Compact Max analyzer and appropriate reagent in respect of:
• Prothrombin time (PT) - using IL PT Fibrinogen reagent.
• Activated partial thromboplastin time (APTT) - using IL APTT reagent.

Blood Chemistry
Blood samples were collected at the following occasions:
Occasion Animals
At termination The five lowest numbered surviving males per group.
Day 13 of lactation The first five lactating females with a surviving litter per group.

Animals were held under light general anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Cobas 6000 Analyzer in respect of:
• Alkaline phosphatase (ALP)
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Total bilirubin (Bili)
• Bile acids (BiAc)
• Urea
• Creatinine (Creat)
• Glucose (Gluc)
• Total cholesterol (Chol)
• Triglycerides (Trig)
• Sodium (Na)
• Potassium (K)
• Chloride (Cl)
• Calcium (Ca)
• Inorganic phosphorus (Phos)
• Total protein (Total Prot)
• Albumin (Alb)

Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analyzed albumin concentration.

Thyroid Hormone Analysis
Blood samples were obtained as follows:
Occasion Animals
At termination All surviving F0 males.
All surviving F0 females (no samples were obtained from animals which failed to litter).
Day 4 of age Offspring: up to two females per litter (where possible; male pups were reserved for nipple retention evaluation):
• one for T4 (serum)#
• one for TSH (serum)
# Priority given to T4 sample.

No offspring were allocated to these procedures on Day 4 of age if:
• the resultant live litter size would fall below eight offspring
• the resultant number of female pups would fall below three offspring
• If only four female offspring were available within a litter but the overall litter size was >eight, one female was selected with priority given to the T4 sample.
Day 13 of age F1 offspring, two males and two females per litter (where possible) (see Section 4)
• two for T4 (serum); where possible one male and one female#
• two for TSH (serum); where possible one male and one female
# Priority given to T4 sample.

Sequence of blood sampling on each occasion (F0 males and F0 females and offspring, where practicable) In order to minimize any potential confounding effect of the time of day of blood sampling, the time of blood sampling was controlled to allow satisfactory inter-group comparisons.
Conditions No overnight deprivation of food.
Anesthetic Adults: Isoflurane.
Offspring: None.
Blood sample site Adults: Sublingual vein.
Offspring: Decapitation.
Parameter Thyroid stimulating hormone (TSH)
Thyroxine (T4)
Anticoagulant None.
Blood volume Adults: 1.0 mL.
Offspring: max possible.
Blood tubes Greiner Minicollect tubes with clotting activator.
Processing Samples were kept at ambient temperature (15 to 25C) for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions At 2000g for ten minutes at 4°C.
Aliquot volumes Adults:
Aliquot 1: T4 - 0.2 mL of serum
Aliquot 2: TSH - residual serum
Offspring:
T4 - all available serum
TSH - all available serum
Final storage conditions Deep frozen (approximately -60°C to -90C).
Fate of samples Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences, Covance.
Thyroid hormone analysis Performed by the Department of Bioanalysis, Covance.
Samples from offspring on Day 13 of age and F0 males were assessed for levels of Thyroxine (T4).
The method of T4 hormone analysis and the results are presented in Attachment 13.3.
As there was considered to be no effect of treatment upon serum T4 concentration, no analysis of T4 samples from F0 females and Day 4 of age offspring or any TSH samples was required.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Time of Necropsy
F0 females failing to produce a viable litter Day 25 after mating.
F0 females Day 13 of lactation.

PLEASE REFER TO TABLE IN ANY OTHER INFORMATION ON RESULTS": "PATHOLOGY PROCEDURES FOR THE FIVE LOWEST NUMBERED SURVIVING MALES AND FEMALES WITH A SURVIVING LITTER PER GROUP AT SCHEDULED TERMINATION"
and
"PATHOLOGY PROCEDURES FOR REMAINING F0 MALES AND FEMALES PER GROUP"

Organ Weights
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.

Fixation
Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes Initially in modified Davidson’s fluid.
Eyes In Davidson’s fluid.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List The five lowest numbered surviving males and the first five lactating females with a surviving litter in Groups 1 and 4 at scheduled termination.
Abnormalities All F0 animals.
Routine staining Sections were stained with hematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.

Light Microscopy
Tissues preserved for examination were examined as follows:
Category Animals Tissues
Scheduled kill The five lowest numbered surviving males and the first five lactating females with a surviving litter in Groups 1 and 4. All specified in Section 3.7.
All F0 animals. Abnormalities.

Findings were either reported as "present" or assigned a severity grade. In the latter case one of the following five grades was used - minimal, slight, moderate, marked or severe. A reviewing pathologist undertook a peer review of the microscopic findings.
For the assessment of the testes, a detailed qualitative examination was made, 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, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.

Parturition Observations and Gestation Length
Duration of gestation Time elapsing between the detection of mating and commencement of parturition.

Parturition observations From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

Ovaries and uterine content:

Each uterine horn Number of implantation sites was counted and confirmed if none were visible at visual inspection.

Fetal examinations:

Records Made During Littering Phase
Clinical observations Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size Daily records were maintained of mortality and consequent changes in litter size from Days 1-13 of age.
Sex ratio of each litter Recorded on Days 1, 4, 7 and 13 of age.
Individual offspring body weights Days 1, 4, 7 and 13 of age.
Ano-genital distance Day 1 - all F1 offspring.
Nipple/areolae count Day 13 of age - male offspring.


Premature deaths Where possible, a fresh macroscopic examination with an assessment of stomach for milk content was performed. Grossly externally abnormal pups were retained.
F1 offspring on Day 4 of age Blood sampling required .
All selected Day 4 offspring had no clinical observations and were discarded without examination.
Offspring at scheduled termination Blood sampling required.
All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.
Thyroid glands were preserved from one male and one female in each litter, where possible.

Offspring Sex Ratio
The percentage of male offspring in each litter was calculated at Day 1, and for live offspring on Days 1, 4 (before and after blood sampling) and 13 of age.

Percentage males = (Number of males in litter / Total number of offspring in litter) x 100

Group mean values were calculated from individual litter values.
Offspring Examinations
Ano-genital distance were presented both as absolute/unadjusted and adjusted for body weight, using the weight recorded on Day 1 of age.
A check was performed to assess for the presence or absence of nipple/areolae in male offspring. No nipples were observed, therefore no data is reported.

Statistics:

PLEASE REFER TO "ANY OTHER INFORMATION ON RESULTS"

Indices:

Survival Indices
The following were calculated for each litter:
Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100

Viability index (%) = (Number of live offspring on Day 4 (before blood sampling) / Number live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 13 after littering / Number of live offspring on Day 4 (after blood sampling)) x 100

Group mean values were calculated from individual litter values.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Signs associated with dose administration were restricted to salivation in animals receiving 300 mg/kg/day, which was seen in two males on Day 32 of treatment and in one female during lactation (Day 1). No signs associated with dose administration were observed for female during gestation.

There were no findings during the detailed physical examination or arena observations for males that could be attributed to treatment with MEDOL-10. One female receiving 300 mg/kg/day was observed with dry rales and salivation on Day 12 of gestation. This animal refluxed during dose administration on Day 13 of gestation and subsequently showed gasping respiration and rales.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weight gains for males during treatment and females before pairing at all dose levels were unaffected by treatment with MEDOL-10.

Mean body weight for males receiving 100 or 300 mg/kg/day was statistically significantly higher than control on Day 8 but values showed no dose relationship and were consistent with differences already established prior to treatment. In the absence of any obvious effect on overall body weight gain for males during the study, this finding was considered to be incidental and unrelated to treatment.

Mean overall body weight gains for females receiving 100 or 300 mg/kg/day were slightly lower than controls during both gestation (93% and 92% of Control respectively) and lactation (83% and 78% of Control respectively). However, differences did not achieve statistical significance and, at the magnitude observed reflected normal biological variation and were insufficient to be regarded as an adverse effect.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food consumption was generally unaffected by treatment with MEDOL-10.
Higher food consumption, compared to the control, for males receiving 300 mg/kg/day, attained statistical significance during Weeks 2 and 4. Food consumption was also statistically significantly higher than control during Week 4 for males receiving 30 or 100 mg/kg/day. However, food consumption was similar to control for all treated males during Week 5, and similar high food intake was not apparent for treated females. The occasional higher food intake for males was, therefore, considered to be of little biological significance and did not represent an adverse effect.

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Hematological investigation performed in males in Week 5 revealed changes from controls in white blood cell parameters including; low eosinophil and monocyte counts at all dose levels, low neutrophil counts in males receiving 100 or 300 mg/kg/day and low basophil counts in males receiving 300 mg/kg/day. Other statistically significant findings included elevated reticulocyte counts, greater mean cell volume and reduced activated partial thromboplastin time in males receiving 300 mg/kg/day.

Findings in females on Day 13 of lactation were elevated haemoglobin concentrations, elevated mean cell haemoglobin concentrations and reduced prothrombin time in females receiving 300 mg/kg/day. In the absence of any supporting histopathological change these findings were considered to be of little toxicological significance and did not represent an adverse effect.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The biochemical examination following five weeks of treatment revealed, when compared with controls, elevated alanine aminotransferase (ALT) activity in males receiving 300 mg/kg/day and higher calcium concentration at all dose levels.

For females on Day 13 of lactation bile acid concentrations were elevated, compared with controls, at all dose levels as well as elevated cholesterol, phosphorus, total protein and albumin concentrations in females receiving 300 mg/kg/day. In the absence of any supporting histopathological change these findings were considered to be of little toxicological significance and did not represent an adverse effect.

Endocrine findings:

no effects observed

Description (incidence and severity):

The mean serum T4 concentrations in samples obtained from offspring on Day 13 of age and F0 adult terminal male animals across all treatment groups were not significantly increased or reduced when compared with the mean T4 concentration found in the control animals. There was no statistically significant difference observed. Therefore, no effect of treatment is inferred.

PLEASE REFER TO TABLE IN "ANY OTHER INFORMATION ON RESULTS": - "MEAN SERUM T4 CONCENTRATIONS"

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory Reactivity Observations and Grip Strength
Sensory reactivity observations and grip strength were unaffected by treatment at 30, 100 or 300 mg/kg/day.

Motor Activity
Motor activity was considered to be unaffected by treatment.
Activity (low beam) was statistically significantly low in males at the 24-30 minute period and in females at the 54-60 minute period receiving 300 mg/kg/day. However, the overall total score for low beams was similar to Control and these isolated findings for activity were considered to reflect normal biological variation.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males, higher than control absolute and body weight adjusted liver weights were observed at 30, 100 or 300 mg/kg/day with a dose response pattern. These differences achieved statistical significance for body weight adjusted liver weights in males given 300 mg/kg/day. In the absence of any supporting histopathological change these findings were considered to be adaptive in nature and did not represent an adverse effect. All other differences in organ weight parameters, were consistent with normal variation and considered incidental.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings for males that received 30 mg/kg/day.

The macroscopic examination of males following six weeks of treatment and females at Day 13 of lactation revealed treatment related changes in the stomachs for animals receiving 100 or 300 mg/kg/day. All males receiving 300 mg/kg/day had thickened stomachs with depressions with one male also observed with a distended stomach. Thickened stomachs with depressions were found in eight females receiving 300 mg/kg/day with two further females at this dose level having thickened stomachs. For males, receiving 100 mg/kg/day thickened stomachs with depressions was observed for five males, two males had thickened stomachs and one male had depressions on the stomach.

Other macroscopic necropsy findings were restricted to pale areas on the lungs and bronchi for one female given 30 mg/kg/day, two females given 100 mg/kg/day and one male, and one female given 300 mg/kg/day and depressions in the mucosa of the cecum and enlarged dark lymph nodes for one male given 300 mg/kg/day. The incidence and distribution of these findings did not indicate any association with treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the stomach, slight to moderate hyperplasia of the nonglandular mucosa, minimal erosions and/or slight to moderate ulcerations were observed in males and females given 100 or
300 mg/kg/day. These microscopic changes generally correlated with the macroscopic observations of thickening and/or mucosal depression of the nonglandular region. Inflammatory changes were also seen in the nonglandular mucosa, including minimal to slight mixed inflammatory cell infiltrate and slight to moderate acute/subacute inflammation of the nonglandular mucosa in males given 100 or 300 mg/kg/day and in females given
300 mg/kg/day. The inflammatory changes were generally seen in association with erosions and/or, more often, with ulcerations of the nonglandular mucosa. The changes seen in the stomach were test article-related and considered adverse at 300 mg/kg/day because of their nature (inflammation and ulceration) and severity.

In the cecum marked inflammation mucosal/submucosal associated with marked focal ulceration was seen in one male given 300 mg/kg/day (male 28) and correlated with the macroscopic observations. These changes in the cecum were considered unrelated to treatment in view of the isolated incidence.

All other microscopic findings were considered spontaneous and/or incidental.
In females killed on Day 13 of lactation, either controls or given 300 mg/kg/day, there were no microscopic changes in the ovaries; all the animals were in lactational diestrus and increased mucification of the vagina was observed in one animal given 300 mg/kg/day. The mammary gland showed evidence of secretory activity and/or (physiological) diffuse lobular hyperplasia. These were physiological changes in lactating females and not test article-related.

In males killed after 5 weeks of treatment, the testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

PLEASE REFER TO TABLE IN "ANY OTHER INFORMATION ON RESULTS": - "INCIDENCE AND SEVERITY OF TEST ARTICLE-RELATED MICROSCOPIC FINDINGS - TERMINAL SACRIFICE"

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

not examined

Description (incidence and severity):

Rats do not abort

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean implantation counts were unaffected by treatment at 30, 100 or 300 mg/kg/day.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

post-implantation survival were unaffected by treatment at 30, 100 or 300 mg/kg/day.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Estrous Cycles, Pre-Coital Interval, Mating Performance, Fertility and Gestation Length
Prior to treatment, all females showed a regular four or five day estrous cycle. During the two-week pre-mating period, one Control animal and one animal at 100 or 300 mg/kg/day showed irregular estrous cycles.
As expected, all animals were in diestrus on Day 13 of lactation.
Pre-coital interval was unaffected by treatment, as all animals mated within four days of pairing.

At 100 or 300 mg/kg/day one animal, out of 10 mated, was not pregnant and one Control animal out of ten mated females was not pregnant.

One female receiving 300 mg/kg/day had a slightly extended gestation period (23.5 days), however, this was not associated with any obvious post-natal effect on the litter. The gestation length of females receiving 30 or 100 mg/kg/day was within the expected 22 23 days range.

The gestation index was slightly low (89%) for females receiving 300 mg/kg/day but this represented the failure of only one single pregnant female to maintain her litter.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: changes were considered to be secondary to test article-related local irritation and not systemic toxicity

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Offspring body weight and body weight gain to Day 13 of age were unaffected by parental
treatment.

PLEASE REFER TO THE ATTACHED TABLE: "BODYWEIGHT AND BODYWEIGHT CHANGE - GROUP MEAN VALUES FOR OFFPSRING_F1"

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

PLEASE REFER TO THE ATTACHED TABLE: "LITTER SIZES - GROUP MEAN VALUES_F1"

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The ratio of male to female offspring were unaffected by parental treatment throughout, indicating no selective effect on survival for either sex.

PLEASE REFER TO THE ATTACHED TABLE: "OFFSPRING SEX RATIOS - GROUP MEAN VALUES_F1"

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

Ano-genital distances of male and female offspring were unaffected by parental treatment.

PLEASE REFER TO ATTACHED TABLE: "ANO-GENITAL DIFFERENCE DATA"

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Male Offspring Nipple Count
No nipples were observed for male offspring; therefore no data is reported.

Offspring Macropathology
Macroscopic findings in decedent offspring were typical of the age observed and included no milk in stomach and abdominal contents autolyzed. There were no findings in offspring who survived until scheduled termination on Day 13 of Age.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

300 other: 3000 mg/kw bw/day given to maternal animals

Based on:

test mat.

Sex:

not specified

Basis for effect level:

other: Treatment with the test item did not affect the survival of the offspring from the treated mothers, the sex ratio, the total number of live offspring born or the overall offspring body weight gain. Ano-genital distance of the offspring did not indicate an

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

Treatment related:

no

Any other information on results incl. tables

Mean serum T4 concentrations (pg/mL)

Group	Treatment	Dose   (mg/kg/day)	Parameters	F1 male offspring on Day 13 of age	F1 female offspring on Day 13 of age	F0 Adult Terminal Male
1	Control (Vehicle)^a	0	Mean	80700	78900	50000
			SD	20400	17100	8490
			CV%	25.3	21.7	17.0
			N	9	9	10
2	MEDOL-10	30	Mean	68100	68800	57500
			SD	15900	20900	16800
			CV%	23.3	30.4	29.2
			N	10	10	10
3	MEDOL-10	100	Mean	77000	82700	62500
			SD	14200	24800	20500
			CV%	18.4	30.0	32.8
			N	9	9	10
4	MEDOL-10	300	Mean	69800	83500	62800
			SD	17100	19100	14900
			CV%	24.5	22.9	23.7
			N	8	8	10

 

^a - 1% methylcellulose

Incidence and Severity of Test Article-Related Microscopic Findings – Terminal Sacrifice

Sex	MEDOL-10
	Males				Females
Dose Level (mg/kg/day)	0	30	100	300	0	30	100	300
Stomach
Number Examined	5	5	10	10	5	5	5	8
Erosion, Nonglandular Region
Minimal	0	0	1	0	0	0	0	1
Slight	0	0	0	0	0	0	0	0
Total	0	0	1	0	0	0	0	1
Ulceration, Epithelial, Nonglandular Region
Slight	0	0	0	3	0	0	0	1
Moderate	0	0	0	5	0	0	0	3
Total	0	0	0	8	0	0	0	4
Hyperplasia, Epithelial, Nonglandular Region
Slight	0	0	1	0	0	0	1	1
Moderate	0	0	7	3	0	0	0	6
Marked	0	0	0	7	0	0	0	1
Total	0	0	8	10	0	0	1	8
Infiltrate, Inflammatory Cell, Mucosal/Submucosal, Nonglandular Region
Minimal	0	2	1	0	0	0	0	0
Slight	0	0	4	1	0	0	0	1
Total	0	2	5	1	0	0	0	1
Inflammation, Nonglandular Region
Slight	0	0	3	3	0	0	0	5
Moderate	0	0	0	6	0	0	0	1
Total	0	0	3	9	0	0	0	6

 

Applicant's summary and conclusion

Conclusions:

It is concluded that the oral administration of MEDOL-10 to Han Wistar rats at doses of 30, 100 or 300 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated in the adult animals but caused histopathological changes to the non-glandular stomach (slight to moderate hyperplasia, minimal erosions and/or slight to moderate ulcerations) in both sexes receiving 100 or 300 mg/kg/day. These changes were considered to be secondary to test article-related local irritation and not systemic toxicity and therefore, based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity is 300 mg/kg/day.  

Executive summary:

Introduction-

The purpose of this study was the assessment of general systemic toxic potential in Han Wistar rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of MEDOL-10 by oral gavage for at least four weeks.

Results-

The general appearance, behaviour, sensory reactivity, grip strength and motor activity of the animals were unaffected by treatment.

The mean serum T4 concentrations in samples obtained from offspring on Day 13 of age and F0 adult terminal male animals across all treatment groups were unaffected by treatment.

There was no adverse effect of treatment on body weight gain or group mean food intake for either sex, estrous cycles of females were considered to have been unaffected by treatment.

Hematological investigation in males in revealed changes from controls in white blood cell parameters including; low eosinophil and monocyte counts at all dose levels, low neutrophil counts in males receiving 100 or 300 mg/kg/day and low basophil counts in males receiving 300 mg/kg/day. Other statistically significant findings included elevated reticulocyte counts, greater mean cell volume and reduced activated partial thromboplastin time in males receiving 300 mg/kg/day. Findings in females on Day 13 of lactation were elevated haemoglobin concentrations, elevated mean cell haemoglobin concentrations and reduced prothrombin time in females receiving 300 mg/kg/day.

The biochemical examination revealed, when compared with controls, elevated alanine aminotransferase (ALT) activity in males receiving 300 mg/kg/day and higher calcium concentration at all dose levels. For females on Day 13 of lactation bile acid concentrations were elevated, compared with controls, at all dose levels as well as elevated cholesterol, phosphorus, total protein and albumin concentrations in females receiving 300 mg/kg/day. In the absence of any histopathological correlates these findings were not considered to be adverse.

In males, higher than control absolute and body weight adjusted liver weights were observed at 30, 100 or 300 mg/kg/day with a dose response pattern, but statistical significance was only achieved for body weight adjusted liver weights at 300 mg/kg/day. In the absence of any microscopic changes these findings are considered to be adaptive and non-adverse.

The macroscopic examination of males performed following six weeks of treatment revealed changes in the stomachs for males receiving 100 or 300 mg/kg/day. For males, receiving 100 mg/kg/day thickened stomachs with depressions was observed for five males, two males had thickened stomachs and one male had depressions on the stomach. All males receiving 300 mg/kg/day had thickened stomachs with depressions with one male also observed with a distended stomach, for females surviving to Day 13 of lactation thickened stomachs with depressions were found in eight females receiving 300 mg/kg/day with two further females having thickened stomachs at this dose level.

Histopathological changes related to treatment with MEDOL-10 were seen in the stomach. In the stomach, slight to moderate hyperplasia of the nonglandular mucosa, minimal erosions and/or slight to moderate ulcerations were observed in males and females given 100 or

300 mg/kg/day. These microscopic changes generally correlated with the macroscopic observations of thickening and/or mucosal depression of the nonglandular region. Inflammatory changes were also seen in the nonglandular mucosa, including minimal to slight mixed inflammatory cell infiltrate and slight to moderate acute/subacute inflammation of the nonglandular mucosa in males given 100 or 300 mg/kg/day and in females given

300 mg/kg/day. The inflammatory changes were generally seen in association with erosions and/or, more often, with ulcerations of the nonglandular mucosa. The changes seen in the stomach were test article-related and considered adverse at 300 mg/kg/day because of their nature (inflammation and ulceration) and severity.

Three females, including one control animal, was not pregnant and failed to produce a litter. Litter size, post-implantation survival, live birth index, post-natal survival and sex ratio were all considered to be unaffected by treatment.

There was no effect of treatment on ano-genital distance in male or female offspring, and males did not have nipples at any dose level. Offspring body weight and body weight gain to Day 13 of age were unaffected by parental treatment. There were no findings in offspring who survived until scheduled termination on Day 13 of Age.

Conclusion-

It is concluded that the oral administration of MEDOL-10 to Han Wistar rats at doses of 30, 100 or 300 mg/kg/day for five weeks to males and for two weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated in the adult animals but caused histopathological changes to the non-glandular stomach (slight to moderate hyperplasia, minimal erosions and/or slight to moderate ulcerations) in both sexes receiving 100 or 300 mg/kg/day. These changes were considered to be secondary to test article-related local irritation and not systemic toxicity and therefore, based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity is 300 mg/kg/day.