2,3-dihydroxypropyl methacrylate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Experimental start date 25 April 2017. Experimental completion date 05 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

yes

Remarks:

On the first day of treatment, two control animals (No’s 23 and 28) received 4 mL of vehicle and not 3.8 mL, as determined by body weight. This deviation was considered to have not affected the i ntegrity or validity of the study

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,3-dihydroxypropyl methacrylate

EC Number:

227-642-6

EC Name:

2,3-dihydroxypropyl methacrylate

Cas Number:

5919-74-4

Molecular formula:

C7H12O4

IUPAC Name:

2,3-dihydroxypropyl 2-methylprop-2-enoate

Test animals

Species:

rat

Strain:

other: Sprague-Dawley Crl:CD(SD) rat

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague-Dawley (Crl:CD(SD)) strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals
Strain/Species: Sprague-Dawley Crl:CD(SD) rat.
Supplier: Charles River (UK) Ltd.
Number of animals ordered: 44 males and 48 females. Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Males: seven days prior to the commencement of treatment. Females: 21 days prior to the commencement of treatment.
Age of the animals at the start of treatment: Males 70 to 77 days old. Females 84 to 91 days old.
Weight range of the animals at the start of treatment: Males 340 to 407 g. Females 235 to 290 g

Allocation and Identification
Allocation: On arrival and non-selective allocation to cages. Oestrous cycles were evaluated pre-treatment. After 14 day evaluation, animals that failed to exhibit typical 4-5 days 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 variations in body weight of animals did not exceed ±20% of the mean for each sex. Groups were adjusted to reduce inter-/intra-group variation.
Identification of animals: Each adult animal was assigned a number and identified uniquely within the study using 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 occupant(s).

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: Irregular oestrous cycle; Three females

Animal Care and Husbandry
Environmental Control
Rodent 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 20-24ºC and 40-70%. Although conditions were occasionally outside the indicated ranges, these deviations were minor and/or of short duration and were not considered to have influenced the health of the animals and/or the outcome of the study.
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 during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
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 five animals of one sex
Pairing one male and one female
Males after mating up to five 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 pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study [(except during pairing and lactation (returned on Day 13 of lactation, after dispatch of the litter)] and replaced at the same time as the cages.

Diet Supply
Diet: SDS VRF1 Certified pelleted diet. A sample (100 g) of each batch of diet used was retained within Pharmacy (frozen -10 to -30ºC) 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.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Test Item Preparation
The required amount of test item was weighed into a suitable container. Starting with the lowest concentration, approximately 50% of the final volume of vehicle was added to the test item and was magnetically stirred until uniformly mixed. A further amount of vehicle was added to make up to the required volume and further mixing, using a magnetic stirrer, was performed until the formulation was uniformly mixed. A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly.
Storage of formulation Refrigerated: (2-8 °C)
Test item accounting: Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

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 rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg bw/day.
Volume dose: 10 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.
Frequency: Once daily at approximately the same time each day.
Formulation: A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 and 100 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid/diet matrix.
Achieved concentration: Samples of each formulation prepared for administration in the first and last week of treatment were analyzed for achieved concentration of the test item.
The mean concentrations of the substance in test formulations analyzed for the study were within -10.3 to -1.5% of nominal concentrations, confirming accurate formulation. The coefficient of variation results were within 4% and confirmed precise analysis.

Duration of treatment / exposure:

Males: Two weeks before pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 13 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

Once daily at approximately the same time each day.

Details on study schedule:

Age of the animals at the start of treatment
Males 70 to 77 days old.
Females 84 to 91 days old.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
Dose level selection was based on the results of the preliminary study (Envigo Study Number: XV75QS). In that study, treatment to females at 1000 mg/kg bw/day was not tolerated and the sex was terminated early following eight doses. Two of the three animals were killed for animal welfare reasons on Day 7 or Day 8, due to poor clinical condition and the third animal was terminated early on Day 8. All females showed persistent marked body weight loss and markedly low food consumption. Macroscopic examination revealed a few depressions in the glandular mucosa and/or nonglandular mucosa of the stomach in two animals, with or without a small spleen and the medulla region of both kidneys from the third animal had a few pale diffuse areas.
Males treated at 500 or 1000 mg/kg bw/day showed low mean body weight gain during Days 1-4 of treatment and females at 250 or 500 mg/kg bw/day showed marked body weight loss. However, overall body weight gain was unaffected in males treated at 250, 500 or 1000 mg/kg bw/day and body weight stasis was evident in females at 250 mg/kg bw/day; low body weight gain was evident in females at 500 mg/kg bw/day. During the first four days of treatment, food consumption was low in females treated at 500 mg/kg bw/day and correlated with the marked effect on body weight. Following 14 days of treatment, organ weights were unaffected by treatment and there were no macroscopic findings. Based on the results of this preliminary study, 500 mg/kg bw/day was selected as the high dose and doses of 50 or 150 mg/kg bw/day were selected as the low and intermediate doses respectively, to investigate a dose response).

Study Design and Identity of Treatment Groups
Some serial observations needed to be performed without the knowledge of the treatment group; therefore the animal numbering system was such that it was not easy to identify a treatment group from the animal number.
The F1 generation received no direct administration of the test item. Any exposure to the test item or metabolites was through the mother to the offspring in utero and/or through the milk.

Examinations

Parental animals: Observations and examinations:

Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary

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: males Week 1 - daily. Week 2 onwards - once each week
F0: females Week 1 - daily. Week 2 - once. Gestation phase - Days 0, 7, 14 and 20. Lactation phase - Days 1, 6 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. Before
the start of observations, male cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. For females, animals were moved
into individual cages prior to transport to the testing room. 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 probe/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 hind limb 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 a ppropriate.

Motor Activity
During Week 5 of treatment for males and at Day 7-9 of lactation for females, the motor activity of the five lowest numbered surviving males and 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 Envigo.
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 males: Weekly during acclimatization. Before dosing on the day that treatment commenced (Day 1) and weekly thereafter. On the day of necropsy.
F0 females: Weekly during acclimatization. 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.
More frequent weighings were instituted, when appropriate, for animals displaying ill-health, so that the progress of the observed condition could be monitored. These data are retained in the study data but are not reported.

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 before pairing, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Day 15-22), but recommenced for males in Week 4.
For females after mating food consumption was recorded as follows: Days 0-6, 7-13 and 14-19 after mating Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase.

Haematology, Peripheral Blood
Blood samples were collected after overnight withdrawal of food at the following occasion:
At termination: The five lowest numbered surviving males and the first five lactating females with a surviving litter, in each dose group.
Animals were held under light general anaesthesia induced by isoflurane. Blood samples (nominally 0. 5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant a nd examined for the following characteristics using a Bayer Advia 120 analyzer:
Haematocrit (Hct)*
Haemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell haemoglobin (MCH)*
Mean cell haemoglobin 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 value 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. A manual count of the differential white blood cell parameters was performed where necessary.
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 after overnight withdrawal of food at the following occasion:
At termination: The five lowest numbered surviving males and the first five lactating females with a surviving litter, in each dose group.
Animals were held under light general anaesthesia 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 Roche P Modular Analyzer in respect of:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acids (Bi Ac)
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 alb umin concentration.

Thyroid Hormone Analysis
Blood samples were collected as follows:
At termination: All surviving adult males and females (no samples were obtained from animals which failed to litter).
Day 4 of age : Offspring: two females selected for sampling per litter (where possible):
If four female pups available - one female was selected for T4 (serum)#.
If five or more female pups available - one female was selected for T4 (serum) and one female for TSH (plasma)
No females were selected for sampling if:
The resultant live litter size would fall below 10 pups.
The resultant number of live females would fall to less than three (for subsequent allocation to the Day 13 procedures) e.g. three female pups in the litter – no pups selected.
# priority was given to serum sample
Day 13 of age: F1 offspring, two males and two females per litter (where possible)
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female)
# priority given to serum sample

Sequence of blood sampling on each occasion: In order to minimize any potential confounding effect of the time of day of blood sampling, the order of blood sampling was controlled to allow satisfactory inter-group comparisons.
Conditions: No overnight deprivation of food.
Anaesthetic: Adults: Isoflurane. Offspring: None.
Blood sample site: Adults: Sublingual vein. Offspring: Decapitation.
Parameter: Thyroid stimulating hormone (TSH).
Anticoagulant: K2 EDTA with no separator gel.
Tubes: Standard Envigo.
Blood volume: Adults: 0.5 mL. Offspring: max possible.
Processing: Samples were kept on wet ice prior to centrifugation. Centrifugation commenced within 30 minutes of sampling.
Samples were kept at ambient
Parameter: Thyroxine (T4)
Anticoagulant: None.
Tubes: Greiner Minicollect - with clot activator.
Blood volume: Adults: 0.5 mL. Offspring: max possible. .
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.
Final storage conditions: Deep frozen (approximately -60°C to -90ºC).
Fate of samples: Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences Envigo.
Thyroid hormone analysis: Performed by the Department of Bioanalysis, Envigo.

Oestrous cyclicity (parental animals):

Oestrous cycles
Dry and wet smears were taken as follows:
Dry smears For 15 days before pairing using cotton swabs.
Wet smears Using pipette lavage during the following phases:
For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
After pairing until mating.
For four days before scheduled termination (nominally Days 11-14 of lactation).
Females showing no evidence of mating - following completion of the pairing period females were separated from the male and vaginal smearing continued for up to five days or until the first oestrus smear was seen. If a female showed an oestrus smear during this period, she was killed as soon as practically possible and subject to macroscopic examination.

Sperm parameters (parental animals):

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.

Litter observations:

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.

Thyroid Hormone Analysis
Day 4 of age : Offspring: two females selected for sampling per litter (where possible):
If four female pups available - one female was selected for T4 (serum)#.
If five or more female pups available - one female was selected for T4 (serum) and one female for TSH (plasma)
No females were selected for sampling if:
The resultant live litter size would fall below 10 pups.
The resultant number of live females would fall to less than three (for subsequent allocation to the Day 13 procedures) e.g. three female pups in the litter – no pups selected.
# priority was given to serum sample
Day 13 of age: F1 offspring, two males and two females per litter (where possible)
- two for T4 (serum): where possible one male and one female#
- two for TSH (plasma): where possible one male and one female)
# priority given to serum sample
Sequence of blood sampling on each occasion: In order to minimize any potential confounding effect of the time of day of blood sampling, the order 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).
Anticoagulant: K2 EDTA with no separator gel.
Tubes: Standard Envigo.
Blood volume: Adults: 0.5 mL. Offspring: max possible. .
Processing: Samples were kept on wet ice prior to centrifugation. Centrifugation commenced within 30 minutes of sampling.
Samples were kept at ambient
Parameter: Thyroxine (T4)
Anticoagulant: None.
Tubes: Greiner Minicollect - with clot activator.
Blood volume: Adults: 0.5 mL. Offspring: max possible.
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.
Final storage conditions: Deep frozen (approximately -60°C to -90ºC).
Fate of samples: Dispatched to the Department of Biomarkers, Bioanalysis and Clinical Sciences Envigo.
Thyroid hormone analysis: Performed by the Department of Bioanalysis, Envigo.

Postmortem examinations (parental animals):

Method of Kill
All adult animals: Carbon dioxide asphyxiation with subsequent exsanguination.

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 males: After Week 5 investigations completed.
F0 females failing to mate: Day 25 after last day of pairing.
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 14 of lactation.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed in tables in the section 'any other information on materials and methods' for F0 animals.

Females
The following were recorded: Each uterine horn The number of implantation sites were counted and confirmed if none were visible at visual inspection.

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: All animals killed or dying prematurely. The five lowest numbered surviving F0 males and first five lactating F0 females with a surviving litter in Groups 1, 3 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:
Premature deaths: F0 males and F0 females in Groups 1 and 4 only. - All tissues listed in tables in section 'any other information on materials and methods'
Scheduled kill: The five lowest numbered surviving F0 males and first five lactating F0 females witha surviving litter in Groups 1 and 4. - All tissues listed in tables in section 'any other information onmaterials and methods'
All remaining F0 animals: Tissue with abnormalities only.
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.

Postmortem examinations (offspring):

Method of Kill
Offspring - selected for thyroid hormone sampling on Day 4 and 13 of age: Decapitation.
Offspring - all other: Intraperitoneal injection of sodium pentobarbitone.

Time of Necropsy
F1 offspring: Selected offspring for thyroid hormone analysis - Day 4 of age.
Scheduled kill - Day 13 of age.
The organs weighed, tissue samples fixed and sections examined microscopically are detailed in tables in the section 'any other information on materials and methods' for F0 animals.
Premature deaths: Where possible, a fresh external macroscopic examination with an assessment of stomach for milk content. Abnormal tissues were retained.
F1 offspring on Day 4 of age: Blood sampling required. Offspring were subject to an external macroscopic examination. Particular attention was paid to external genitalia.
F1 offspring on Day 13 of age: Blood sampling required. All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities were retained pending possible future examination. Thyroid glands were preserved from one male and one female in each litter.

Reproductive indices:

Mating Performance and Fertility
Group values were calculated for males and females separately for the following:
Percentage mating (%) = Number of animals mating / Animals paired x 100
Conception rate (%) = Number of animals achieving pregnancy / Animals mated x 100
Fertility index (%) = Number of animals achieving pregnancy / Animals pairing x 100

Gestation Length and Index
Gestation length was calculated as the number of gestation days up to and including the day on which offspring were first observed, with Day 1 = day of matingfor calculation purposes. Where parturition had started overnight, this value was adjusted by subtracting half of one day. Gestation index was calculated for each group as:
Gestation index (%) = Number of live litters born / Number pregnant x 100

Offspring viability indices:

Survival Indices
The following were calculated for each litter:
Post-implantation survival index (%) = Total number of offspring born / Total number of uterine im plantation 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.
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 culling) 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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs reported at the weekly physical examinations and arena observations in males during at least 5 weeks of treatment, or during the two-week pre-pairing period, throughout gestation to Day 13 of lactation in females, that were considered to be related to treatment.
Signs related to administration of the test substance were only seen in those animals that died

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

One female (No. 81) receiving 500 mg/kg bw/day was killed for welfare reasons at the first animal check on Day 5 of treatment. The animal showed a loss in body weight from Day 1 to Day 5 of 30 g and was unresponsive, cold to touch, hunched and had flattened posture with slow and laboured respiration and was salivating. Macroscopic examination revealed dark areas on the adrenals (correlated microscopically with multifocal haemorrhage within the cortex), small spleen (correlated with a decrease in white pulp cellularity) and depressions on the glandular mucosa of the stomach (correlated with a focal erosion in the glandular region).
One female (No. 87) receiving 500 mg/kg bw/day was found dead at the first animal check on Day 11 of lactation. The animal had been overtly normal throughout the study and had shown good weight gain during gestation (+95 g) and lactation (+18 g; Day 1 to Day 7).The majority of tissues were autolyzed and no significant microscopic lesions were found. The cause of death was undetermined.
One female (No. 84) receiving 500 mg/kg bw/day was killed for welfare reasons on Day 13 of lactation. After dosing on Study Day 6, the animal had rapid/irregular respiration and rales (wet), hunched posture, piloerection, elevated gait and brown stained muzzle and forelimbs that persisted to after dosing on Study Day 7 and partially closed eyelids. Irregular respiration and rales (wet) and elevated gait were seen on Study Day 10. The animal showed body weight loss of 77 g from Day 7 (scheduled recording) to Day 13 (weight immediately before death) of lactation. The animal had thin build, piloe rection, hunched posture and had brown staining of the head and salivation. Macroscopic examination revealed small spleen and pale areas on the lungs.
The major factor contributing to the deterioration in clinical condition of No. 81 and No. 84 was a spiration pneumonia as demonstrated by microscopic findings of neutrophilic inflammation (No. 81) and granulomas (No.84), within the bronchi and alveoli which surrounded foreign material. It is likely that the aspiration was caused by gastro-oesophageal reflux or direct aspiration of fluid during or after the dosing procedure. The lung findings were unlikely to have caused clinical symptoms on their own and it is possible that there was concurrent blockage of the nasal passages. No local irritant effects of the test item were evident; however, the test item is a known irritant. Female No.84, receiving 500 mg/kg bw/day also had a focus of vacuolation within the medulla of the brain. A similar finding has been found in Sprague-Dawley rats (De Jonghe et al. 2015) alongside other lesions and acute clinical signs. The findings were not test item related, however, no known aetiology was determined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall (Days 1-36) body weight gain in males receiving 50, 150 or 500 mg/kg bw/day was unaffected by treatment.
Mean body weight losses were evident in unmated females receiving 150 or 500 mg/kg bw/day at the end of the first week of treatment (-1 g or -5 g, respectively); during Days 1-8 of pre-mating body weight gain at 500 mg/kg bw/day was -125% of control. However, body weight gains during Week 2 were unaffected.
Overall body weight gains were unaffected by treatment at 50 or 150 mg/kg bw/day, during gestation and lactation. Body weight gain at 500 mg/kg bw/day during Days 14-20 of gestation and Days 1-4 of lactation were low ; however, overall body weight gains were not statistically significantly different to Control.

For details please refer to the document "OECD 422_rat_body weights.pdf" under "Attached background material".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption in males receiving 50, 150 or 500 mg/kg bw/day was unaffected by treatment. Food consumption in females was unaffected during the two-week pre-mating period and throughout gestation and lactation at 50 or 150 mg/kg bw/day. At 500 mg/kg bw/day and when compared with Control, food consumption was marginally low (82%) in females during the first week of treatment, and was low during lactation (73% of Control); food intake was unaffected by treatment during gestation.

For details please refer to the document "OECD 422_rat_food consumption.pdf" under "Attached background material".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The haematological examination of peripheral blood performed after five weeks of treatment in males and on Day 14 of lactation in females did not reveal any toxicologically significant differences from Control.
All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. These included, high eosinophil count, with no relationship to dose, at 50, 150 or 500 mg/kg bw/day (175%, 225% or 175% of Control, respectively) and marginally high lymphocyte and overall white cell counts at 500 mg/kg bw/day (124% and 127% of Control, respectively) in males and marginally low reticulocyte count (78% of Control) in females receiving 500 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The biochemical examination of the plasma performed after five weeks of treatment in males and on Day 14 of lactation in females did not reveal any toxicologically significant differences from Control. All inter-group differences, including those attaining statistical significance, were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. These included in males, marginally low glucose concentration at 500 mg/kg bw/day (80% of Control) and in females at 500 mg/kg bw/day, low alanine amino-transferase activity (58% of Control; high enzyme activity is considered toxicologically significant) and low calcium and phosphorous concentrations (95% and 74% of Control, respectively).

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Sensory Reactivity and Grip Strength
Sensory reactivity and grip strength were unaffected by treatment in males in Week 5 or in females during lactation (Days 7-9).

Motor Activity
When compared with Control, overall motor activity was considered to be unaffected by treatment in males in Week 5 or in females during lactation (Days 7-9).
High beam (rearing) activity in males receiving 50, 150 or 500 mg/kg bw/day was statistically significantly low during the 6 to 12 minute period of the one-hour testing period; however the majority of individual values were within the Control range and low beam activity was unaffected at this time. Low beam activity was marginally statistically significantly high in males receiving 500 mg/kg bw/day during the 37 to 42 minute period; however, it was seen at a single time-point, activity of the Control was also low a t this time-point and there was no relationship with dose and females were unaffected and overall activity was comparable with Control at all doses, therefore it was considered not to be toxicologically significant.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There were no treatment-related findings following treatment for 5 weeks in males and for 2 weeks before pairing, throughout gestation and to Day 13 of lactation in females.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis
Serum T4 concentrations, in adult males treated at 50, 150 or 500 mg/kg bw/day were unaffected by treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

All females allocated to study showed normal 4/5 day oestrous cycles before treatment. Oestrus cycles were unaffected by treatment. Two females receiving 500 mg/kg bw/day showed one or more irregular oestrus cycles during treatment; however, this was also seen in one Control female and was therefore considered not to be attributed to treatment.
All animals were in di-oestrus on Day 14 of lactation; therefore oestrus cycles ceased during lactation.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage-specific abnormalities were noted.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Pre-Coital Interval, Mating Performance, Fertility and Gestation Length, Gestation Index and Number of Implantations:

Pre-coital interval was unaffected by treatment.
Fertility was unaffected by treatment.
Individual gestation lengths were within acceptable limits (22-23 days) with the exception of one animal (No. 88) receiving 500 mg/kg bw/day that had a gestation length of 23.5 days. Due to the isolated occurrence in one single animal, the slightly elongated gestation length is considered as spontaneous finding rather than a treatment-related effect.
Gestation index was unaffected by treatment.

For details please refer to the document "OECD 422_rat_gestation.pdf" under "Attached background material".

There was no effect of treatment on the number of implantations, litter size or the ratio of males to females following treatment at 50 or 150 mg/kg bw/day.
The number of implantations and subsequent litter size at 500 mg/kg bw/day was low (69% or 68% of Control, respectively) compared with the concurrent control and the historical control data range from the testing laboratory. 5 litters contained 10 or fewer pups compared with just 1 litter in the other 3 groups.

For details please refer to the document "OECD 422_rat_litter size.pdf" under "Attached background material".
For the historical control data on mean number of implantations and litter size from the testing laboratory please refer to the document "OECD 422_rat_HCD.pdf" under "Attached background material".

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

2

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

body weight and weight gain

organ weights and organ / body weight ratios

3

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

4

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

no

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Organ:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Description (incidence and severity)
There were no observations seen in offspring considered to be related to parental treatment at 50, 150 or 500 mg/kg bw/day.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no effect of treatment on the number of implantations, litter size or the ratio of males to females following treatment at 50 or 150 mg/kg bw/day.
Two macroscopically normal offspring were found dead in the litter from No. 88 treated at 500 mg/kg bw/day. The gestation length in this animal was prolonged (23.5 days) and the deaths were attributed to possible difficulty during parturition and not to treatment. Survival indices from Day 1 of age was unaffected by treatment.
The number of implantations and subsequent litter size at 500 mg/kg bw/day was low (69% or 68% of Control, respectively) compared with the concurrent control and the historical control data range from the testing laboratory. 5 litters contained 10 or fewer pups compared with just 1 litter in the other 3 groups. However there was no effect of treatment on survival indices or on the ratio of males to females.

For details please refer to the document "OECD 422_rat_litter size.pdf" under "Attached background material".
For the historical control data on mean number of implantations and litter size from the testing laboratory please refer to the document "OECD 422_rat_HCD.pdf" under "Attached background material".

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Offspring body weight and weight gain was unaffected by parental treatment at 50, 150 or 500 mg/kg bw/day.

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 specified

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There was no effect of parental treatment on ano-gential distance.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

One male offspring in a Control litter and four males from two litters at 150 mg/kg bw/day had nipples (two) on Day 13 of age; however this was considered not to be toxicologically significant as offspring at 500 mg/kg bw/day were unaffected.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

All offspring were macroscopically normal; in particular no effects were seen on the external genitalia.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Thyroid Hormone Analysis
Serum T4 concentrations in male and female offspring on Day 13 of age following parental treatment, were unaffected by treatment.

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Remarks:

developmental

Generation:

F1

Effect level:

500 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

It is concluded that oral gavage administration of the test substance to Sprague-Dawley rats, at 500 mg/kg bw/day was associated with two females being killed for welfare reasons due to poor clinical condition principally caused by aspiration pneumonia, and low mean numbers of implantations and subsequently litter size. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) for general toxicity and reproductive toxicity was 150 mg/kg bw/day. No effects on in utero development were observed. Thus, a NOAEL of 500 mg/kg bw/day was derived for developmental toxicity.

Executive summary:

The purpose of this study was an assessment of general systemic toxic potential in Sprague-Dawley rats, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, with administration of 2,3-dihydroxypropyl methacrylate (GMMA) by oral gavage administration for at least five weeks. Three groups of ten male and ten female rats received the test substance at doses of 50, 150 or 500 mg/kg bw/day by oral gavage administration. 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. Females were allowed to litter, rear their offspring and were killed on Day 14 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, purified water, at the same volume dose as treated groups. During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, thyroid hormone analysis, oestrous cycles, pre-coital interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

Results

F0 responses: Two females receiving 500 mg/kg bw/day were killed for welfare reasons due to poor clinical condition: the major factor contributing to the deterioration in the clinical condition of both females was aspiration pneumonia. Another female was found dead but the cause of death could not be determined. The mean number of implantations and litter size were decreased at 500 mg/kg bw/day compared to Control. Mean body weight losses were recorded in unpaired females during the first week of treatment at 150 or 500 mg/kg bw/day and food intake was marginally low at 500 mg/kg bw/day. Body weight gain at 500 mg/kg bw/day was decreased during Days 14-20 of gestation to Day 4 of lactation and overall food intake during lactation was reduced. Clinical condition, sensory reactivity and grip strength, oestrus cycles (during the two-week pre-pairing period and during lactation), pre-coital interval, mating performance, fertility and gestation length and gestation index, haematology and blood chemistry (males and females), circulating levels of thyroxine (T4) (males), organ weights and macroscopic and microscopic findings were unaffected by treatment.

F1 responses: The mean number of implantations and litter size were decreased at 500 mg/kg bw/day compared to Control.

There was no effect of parental treatment on offspring sex ratio, survival, growth, ano-genital distance and nipple count (males) or on circulating levels of thyroxine (T4) on Day 13 of age and there were no treatment related macroscopic findings.

Conclusion

It is concluded that oral gavage administration of 2,3-dihydroxypropyl methacrylate to Sprague-Dawley rats, at 500 mg/kg bw/day was associated with two females being killed for welfare reasons due to poor clinical condition principally caused by aspiration pneumonia, decreased body weight gains during pre-mating, gestation and lactation and marginally but statistically significantly higher body weight adjusted kidney weight in males and females, higher body weight adjusted liver weight in males and higher body weight adjusted brain weight in females. In addition, at the high dose level, mean numbers of implantations and subsequently litter size were decreased compared to the control group. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) for toxicity and reproductive toxicity was 150 mg/kg bw/day. No effects on in utero development were observed. Thus, a NOAEL of 500 mg/kg bw/day was derived for developmental toxicity.