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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
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 integrity or validity of the study
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
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. Estrous 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
Details on route of administration:
The oral gavage route of administration was chosen to simulate the conditions of potential human exposure.
Vehicle:
water
Details on oral 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.
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 test 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.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
150 mg/kg bw/day (nominal)
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/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/day showed low mean body weight gain during Days 1-4 of treatment and females at 250 or 500 mg/kg/day showed marked body weight loss. However, overall body weight gain was unaffected in males treated at 250, 500 or 1000 mg/kg/day and body weight stasis was evident in females at 250 mg/kg/day; low body weight gain was evident in females at 500 mg/kg/day. During the first four days of treatment, food consumption was low in females treated at 500 mg/kg/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/day was selected as the high dose and doses of 50 or 150 mg/kg/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, BLEMMER GLM. Any exposure to the test item or metabolites was through the mother to the offspring in utero and/or through the milk.

Examinations

Observations and examinations performed and frequency:
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), butrecommenced 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.

Sacrifice and pathology:
Method of Kill
All adult animals: Carbon dioxide asphyxiation with subsequent exsanguination.
Sequence: To allow satisfactory inter-group comparison.

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.

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 with a surviving litter in Groups 1 and 4. - All tissues listed in tables in section 'any other information on materials 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.
Other examinations:
Other examinations
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.
Statistics:
Please see any other information on materials and methods section

Results and discussion

Results of examinations

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.
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, piloerection, 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 aspiration 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 (78% and 42% of Control, respectively); 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 higher than Control 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 at 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
Description (incidence and severity):
The analysis of organ weights performed after five weeks of treatment in males and on Day 14 of lactation in females revealed, in those treated at 500 mg/kg bw/day, marginally but statistically significantly higher body weight adjusted kidney weight in males and females (110% and 118% of Control, respectively), higher body weight adjusted liver weight in males (113% of Control) and higher body weight adjusted brain weight in females (106%).

For details please refer to the document "OECD 422_rat_organ weights.pdf" under "Attached background material".
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed after treatment for 5 weeks in males and for 2 weeks before pairing, throughout gestation and to Day 13 of lactation in females revealed no test item related lesions.
The incidence and distribution of all findings were considered to be unrelated to treatment.
Neuropathological findings:
not examined
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
SerumT4 concentrations, in adult males treated at 50, 150 or 500 mg/kg bw/day were unaffected by treatment.

Effect levels

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Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed.
Key result
Dose descriptor:
LOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
mortality
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
500 mg/kg bw/day (nominal)
Organ:
not specified

Any other information on results incl. tables

Discussion

Oral gavage administration of the test substance to Sprague-Dawley rats, at doses of 50, 150 or 500 mg/kg bw/day for at least five weeks in males and for two weeks before pairing, throughout pairing and gestation to Day 13 of lactation was generally well-tolerated. 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 as demonstrated by neutrophilic inflammation or granulomas within the bronchi and alveoli which surrounded foreign material. There was no evidence of oral gavage catheter placement error (there was no damage to the trachea and oesophagus) and the aspiration pneumonia was likely caused by gastro-oesophageal reflux, or direct aspiration of fluid during or after the dosing procedure, coupled with the known irritant properties of the test substance.

Small mean body weight losses were evident in unpaired females receiving 150 or 500 mg/kg bw/day at the end of the first week of treatment and food intake at 500 mg/kg bw/day was marginally low; however overall body weight gain and food intake were unaffected. Body weight gain at 500 mg/kg bw/day was low during Days 14-20 of gestation to Day 4 of lactation and food intake during lactation was low. These differences likely reflected the low litter size at 500 mg/kg bw/day.

All surviving adults treated at 50,150 or 500 mg/kg bw/day remained in good clinical condition throughout treatment. Sensory reactivity and grip strength investigations and oestrus cycles during the two-week pre-pairing period were unaffected by treatment and all females were in di-oestrus at termination, indicating that oestrus cycles had ceased during lactation. Haematology and blood chemistry in males and females were unaffected and any change in organ weights or macroscopic finding was minor and had no microscopic correlate.

The study design also included an assessment of endocrine disruptor relevant endpoints. This objective was met by including the measurement of the hormone thyroxine (T4) in adult males and in offspring at Day 13 of age, by evaluating changes in adult organ weight and gross organ pathology of endocrine-sensitive organs. No adverse effect of treatment was evident on the circulating levels of thyroxine (T4). No significant changes were identified at the microscopic examination of thyroid, adrenal and pituitary glands and the reproductive organs.

Applicant's summary and conclusion

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 the test substance 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, organ weight and macroscopic pathology and histopathology investigations were undertaken.

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. Mean body weight losses were recorded in unpaired females during the first week of treatment at 150 or 500 mg/kg bw/day (-125% of Control at 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 low during Days 14-20 of gestation to Day 4 of lactation and overall food intake during lactation was low. Clinical condition, sensory reactivity and grip strength, haematology and blood chemistry (males and females), circulating levels of thyroxine (T4) (males), organ weights and macroscopic and microscopic findings were unaffected by treatment.

Conclusion

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, 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. Therefore, the No-Observed-Adverse-Effect-Level (NOAEL) for toxicity was 150 mg/kg bw/day.