Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Treatment commenced 23 March 2017 Experimental completion date (pathology) 26 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Test material form:
liquid
Specific details on test material used for the study:
Appearance: Clear, brown liquid
Storage conditions: Ambient temperature (15 to 25°C)
Expiry date: 20 July 2017

Test animals

Species:
rat
Strain:
other: RccHan;WIST
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 RccHan™;WIST (Han Wistar) strain was used because of the historical control data available at this laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
Supplier: Envigo RMS (UK).
Number of animals ordered: 44 males and 48 females.

Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Males: eight days prior to the commencement of treatment.
Females: 22 days prior to the commencement of treatment.
Age of the animals at the start of treatment: Males 86 to 92 days old.
Females 100 to 106 days old.
Weight range of the animals at the start of treatment Males 310 to 368 g.
Females 192 to 234 g.

Allocation and Identification
Allocation:
On arrival and non-selective allocation to cages.
Estrous cycles were evaluated pre-treatment. After 14 days 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.
The 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 by a tail tattoo before Day 1 of treatment. The offspring were numbered individually within each litter on Day 1 of age, using a toe tattoo.

Identification of cages
Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupants.

Animal Replacement
Before the commencement of treatment, study allocation was revised to reduce inter/intra group body weight variation by replacement of animals with spares and moving animals within groups. Any individuals rejected during the acclimatization period were replaced with spare animals of suitable weight from the same batch.
Replacement before treatment
Irregular estrous cycle - Two females
Body weight range extremes - One male

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%.
There were no deviations from these ranges.

Lighting A
rtificial 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 polypropylene 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) and replaced when necessary.
Plastic shelter - Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

Diet Supply
Diet
SDS VRF1 Certified pelleted diet.
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Samples (100 g) of the batch of diet used was retained within the Pharmacy department (frozen -10 to -30ºC) until finalization of the report.

Availability
Non-restricted (removed overnight before blood sampling for hematology and blood chemistry investigations).

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:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Method of 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 homogeneous. The remaining concentrations were then formulated in ascending order of concentration.
On Day 5 of treatment, the decision was made to reduce the Group 4 dose concentration to 100 mg/mL, therefore the doses prepared at 200 mg/mL were returned to the Pharmacy department and diluted with vehicle to achieve the required concentration.

Frequency of preparation: Weekly.

Storage of formulation: Refrigerated (2 to 8°C).

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 concentrations of 1 and 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix.
Homogeneity and stability were demonstrated for up to 15 days when stored refrigerated (2 to 8°C) and for one day when stored at ambient temperature (15 to 25°C).

Achieved concentration
Samples of each formulation prepared for administration in Week 1 (for each treatment group), Week 4 of treatment and on Day 12 of lactation (females only) were analyzed for achieved concentration of the test item.

The mean analyzed concentrations of Resorcinol Diacetate in test formulations were within ±5% of the nominal concentrations, confirming accurate formulation. The absence of test item in the control samples was confirmed, demonstrating that no inadvertent cross-contamination had occurred.
Duration of treatment / exposure:
Males - Two weeks before pairing up to necropsy after a minimum of five weeks of treatment.
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 86 to 92 days old.
Females 100 to 106 days old.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
330 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
1000/ 500 - Animals received 1000 mg/kg/day on Day 1 to 4 and 500 mg/kg/day from Day 5
No. of animals per sex per dose:
10 males and females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected based on the results of a 14 day preliminary toxicity study in the Han Wistar rat (Envigo Study No.PB62HG). In that study, dose levels of 250, 500 or 1000 mg/kg/day revealed no clear effect of treatment upon general health, body weight gain, food consumption, water consumption, organ weights or macropathology.
The high dose of 1000 mg/kg/day was selected as the limit dose for the OECD 422 test guideline and the intermediate and low doses of 330 and 100 mg/kg/day were selected to investigate any dose relationship should effects be observed in the longer duration study.
On Day 4 of treatment, two males (Animal Nos. 4 and 10) and two females (Animal Nos. 45 and 48) receiving 1000 mg/kg/day were sacrificed for welfare reasons after suffering continuous convulsions. No observations had been seen on previous days, and no signs were recorded in any other animal. Macroscopically, depressions were observed on the stomach mucosa. 
After discussions with the Sponsor, the decision was made to reduce the high dose level to 500 mg/kg/day (100 mg/mL) from Day 5 of treatment. It was anticipated that this reduction in dose concentration would reduce the risk of any further local effects on the stomach.

Examinations

Parental animals: Observations and examinations:
Mortality
A viability check was performed near the start and end of each working day. Four animals were killed for reasons of animal welfare.

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 and 2 - daily
Week 3 onwards - once each week
F0 females Week 1 and 2 - daily
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 (See Section 4)
One to two hours after completion of dosing
As late as possible in the working day

Due to signs observed during the first week of treatment, additional observations were recorded for Animal Nos. 4F 43 and 47 on Days 5 and 6 to monitor their condition. Post dose observations were also recorded for all animals at the end of dosing each group on Day 8 of treatment.

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 hindlimb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.
At any point during the observations, additional comments were made as free text where considered appropriate.

Motor activity
During Week 5 of treatment for males and at Day 7-9 of lactation for females, the 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.

Food Consumption
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:
F0 animals Weekly, from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating (Week 3), but recommenced for males in Week 4.
For females after mating food consumption was performed to match the body weight recording:
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 weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

Parturition Observations and Gestation Length
Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

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

* Derived 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.
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series 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 anesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a 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 albumin concentration.

Thyroid Hormone Analysis
Blood samples were collected as follows:
At termination: All F0 adult males and females.

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: F0 animals: Following overnight deprivation of food
Anesthetic: F0 animals: Isoflurane
Blood sample site: F0 adults: Sublingual vein
Anticoagulant: Plasma samples: K2EDTA. Microtainers used for collection of samples did not contain separator gel
Serum samples: None (Greiner Minicollect tubes with clotting activators)
Blood volume: F0 animals: 2 x 0.5 mL
Processing: Plasma samples: Samples were kept on wet ice prior to centrifugation and commenced within 30 minutes of sampling.
Serum samples: Samples were kept at ambient temperature for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions: At 2000g for ten minutes at 4°C.
Number of aliquots per sample: All available plasma/serum was transferred to appropriately labelled polypropylene “cryo” tubes using micropipettes.
Final storage conditions: Deep frozen (-60 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):
Estrous 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 estrus smear was seen. If a female showed an estrus 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 during the postmortem examination, 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 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
Blood samples were collected as follows:
Day 4 of age : F1 offspring, two females per litter (where possible, ensuring that the number of female offspring did not fall below three).
No pups were allocated to these procedures if the resultant live litter size would fall below eight pups.
- one for T4 (serum)#
- one for TSH (plasma)
# 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 was 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: F1offspring: No overnight deprivation of food
Anesthetic: F1 offspring : None
Blood sample site: F1 offspring : Decapitation
Anticoagulant: Plasma samples: K2EDTA. Microtainers used for collection of samples did not contain separator gel
Serum samples: None (Greiner Minicollect tubes with clotting activators)
Blood volume: F1 offspring: maximum possible
Processing: Plasma samples: Samples were kept on wet ice prior to centrifugation and commenced within 30 minutes of sampling.
Serum samples: Samples were kept at ambient temperature for a minimum of 30 minutes prior to centrifugation.
Centrifugation conditions: At 2000g for ten minutes at 4°C.
Number of aliquots per sample: All available plasma/serum was transferred to appropriately labelled polypropylene “cryo” tubes using micropipettes.
Final storage conditions: Deep frozen (-60 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 (no exposure to carbon dioxide took place until after the completion of blood sampling for thyroid hormone assays).

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.


Organ Weights
For bilateral organs, left and right organs were weighed together, unless specified above. 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 - In modified Davidson’s fluid.
Eyes - In Davidson’s fluid.

Histology
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: The five lowest numbered surviving males and first five lactating females with a surviving litter in Groups 1 and 4.
Abnormalities only: All remaining 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 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.
Day 13 of age.

Premature deaths: Where possible, a fresh macroscopic examination (external and internal) with an assessment of stomach for milk content was performed.
F1 offspring on Day 4 of age: Blood sampling was required.
Externally normal offspring were discarded without examination.
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.
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 mating for 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 implantation sites x 100

Post-implantation survival index was expressed as 100% where the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = Number of live offspring on Day 1 after littering / Total number of offspring born x 100

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

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

Group mean values were calculated from individual litter values.
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:
effects observed, treatment-related
Description (incidence and severity):
Signs associated with dosing were principally recorded during the first week of treatment. On Days 5 and 6, two females receiving 1000/500 mg/kg/day were noted to be tremoring and one of the affected animals also appeared to be nervous and unsteady. Piloerection was recorded in one female received 330 mg/kg/day on Day 10.
Chin rubbing was recorded in one high dose female on Day 4, all animals receiving 330 or 1000/500 mg/kg/day on Day 5 and in three high dose females on Day 8. This finding is commonly observed in studies where the test item is administered by gavage, and as such, was considered to be of no toxicological importance.

At the routine weekly examination, one female receiving 1000/500 mg/kg/day was observed to have muscle tremors and slightly abnormal gait on Day 6. No other signs recorded were considered to be related to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Four animals died on Day 4 of treatment.
Male Animal Nos. 4 and 10, and female Animal Nos. 45 and 48, receiving 1000 mg/kg/day of the test item, were killed on welfare grounds on Day 4 because of the occurrence of convulsions approximately two hours after dosing. In the males, the macroscopic examination revealed the presence of depressions of the forestomach that correlated histologically with erosion/ulceration and inflammatory cell infiltrate in the mucosa. However, these changes did not account for the clinical conditions of these animals and the cause of death was undetermined. In the females decedents, depressions of the forestomach were observed macroscopically but no significant lesions were observed histologically and the cause of death was therefore undetermined.
These deaths were considered likely to be treatment-related due to the occurrence in the high dose group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall group mean body weight gain (Day 1 to 36) was slightly low in males receiving 1000/500 mg/kg/day (90% of control), which was principally attributed to low weight gain during Week 1 of treatment, where males receiving 100, 330 or 1000/500 mg/kg/day gained 90, 90 and 70% of control, respectively, with statistical significance attained at the high dose. During subsequent weeks, the group mean body weight gains of all groups of Resorcinol diacetate-treated males were generally similar to controls.
No similar trend was apparent in the females before mating, where the body weight gain of females receiving 100 mg/kg/day was slightly low (90% of control) but those receiving 330 or 1000/500 mg/kg/day gained marginally more weight than the controls. During gestation, females receiving 1000/500 mg/kg/day gained slightly less weight than the controls (93%) and during lactation there was a trend towards low weight gain in females receiving 330 or 1000/500 mg/kg/day (91 and 75% of control, respectively).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food intake was marginally low, when compared with controls, in females receiving 1000/500 mg/kg/day during Days 4 to 12 of lactation (90% of control). No other effect of treatment upon food consumption was observed.
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 hematological examination of peripheral blood performed after five weeks of treatment for males and on Day 14 of lactation for 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. Such differences included the slightly high hemoglobin concentration in males receiving 100, 330 or 1000/500 mg/kg/day, which, at the high dose, associated with a statistically significant increase in mean cell hemoglobin concentration and the marginal increase in mean cell hemoglobin concentration in females receiving 1000/500 mg/kg/day. However, in the absence of any other changes among the erythrocyte parameters, this was considered unlikely to be related to treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The biochemical examination of plasma performed after five weeks of treatment for males and on Day 14 of lactation for 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. Such differences included the slightly, but statistically significantly low creatinine concentrations in males at all dose levels, where there was no clear dose-response as the group mean value for the high dose was the same as that of the low dose, and the lowest value was recorded for Animal No. 11 (Group 2, 100 mg/kg/day). Alanine amino transferase activity was statistically significantly high among males receiving 1000/500 mg/kg/day, but the high group mean value was principally attributed to one animal (Animal No. 6) and the control group mean was low, due to one animal with an abnormally low value (Animal No. 23). Plasma cholesterol concentrations were high, when compared with controls, in males at all dose levels but there was no clear dose relationship (the highest group mean value was for animals receiving 330 mg/kg/day) and statistical significance was not attained.
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Treatment Related Findings
There were no treatment related changes.
Incidental Findings
All the histological changes were considered to be incidental and unrelated to treatment.
Histopathological findings: neoplastic:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males.

Sensory Reactivity and Grip Strength
Sensory reactivity responses and grip strength were unaffected by treatment.

Motor Activity
Motor activity was unaffected by treatment.
Group mean low beam activity scores were statistically significantly low, when compared with controls, at the 42- and 48-minute intervals at all doses in males, but there was no dose-relationship, the total scores were similar to controls, and there was no similar effect in the females, therefore this was considered unrelated to 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 estrous cycles during the acclimatization period.
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Estrous cyclicity, pre-coital interval, gestation length, mating performance and fertility were considered unaffected by treatment with Resorcinol diacetate.
Gestation index was slightly reduced among females receiving 330 or 1000/500 mg/kg/day, when compared with controls, as one female in each group had no live litter.
All females that mated were pregnant, with the exception of one control animal (Animal No. 67).
All females showed diestrus at termination.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
330 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No signs were recorded that were considered to be related to parental treatment with Resorcinol diacetate.

There was no effect of parental treatment upon ano-gential distances.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
One female receiving 330 mg/kg/day (Animal No. 71) and one female receiving 1000/500 mg/kg/day (Animal No. 49) had no live litter.
Offspring survival to Day 13 of age was unaffected by parental treatment with Resorcinol diacetate.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There was considered to be no effect of parental treatment upon offspring group mean body weights or body weight gain.
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There was considered to be no effect of parental treatment upon offspring group mean body weights or body weight gain.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid Hormone Analysis
There was no effect of treatment on the circulating levels of thyroxine (T4) in offspring on Day 13 of age.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 500 - <= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: 1000 mg/kg/day dosed on Day 1 to 4 and 500 mg/kg/day dosed from Day 5

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Formulation Analysis

The mean analyzed concentrations of Resorcinol Diacetate in test formulations were within ± 5% of the nominal concentrations, confirming accurate formulation. The absence of test item in the control samples was confirmed, demonstrating that no inadvertent cross-contamination had occurred.

Thyroid Hormone Analysis

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age.

Group

Treatment

Dose

(mg/kg/day)

Adult terminal males

(pg/mL)

Male offspring on Day 13 of age

(pg/mL)

Female offspring on Day 13 of age

(pg/mL)

1

Resorcinol diacetate

0

Mean

44100

45400

49300

SD

5450

4850

8430

CV

12.4

10.7

17.1

N

10

9

9

2

Resorcinol diacetate

100

Mean

43500

45200

50000

SD

6940

6630

9810

CV

16.0

14.7

19.6

N

10

10

10

3

Resorcinol diacetate

330

Mean

44100

49900

51200

SD

3720

8520

12000

CV

8.4

17.1

23.4

N

10

7

8

4

Resorcinol diacetate

1000/500†

Mean

43800

48000

50000

SD

8370

6930

6010

CV

19.1

14.4

12.0

N

8

7

7

            Animals received 1000 mg/kg/day on Day 1 to 4 and 500 mg/kg/day from Day 5

Applicant's summary and conclusion

Conclusions:
It was concluded that the oral administration of Resorcinol diacetate to parental Han Wistar rats at dose levels of 100, 330 or 500 mg/kg/day (with 1000 mg/kg/day administered as the high dose for the first four days of treatment) for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 14 of lactation, was generally tolerated, however the administration of 1000 mg/kg/day was not tolerated, and resulted in four animals (two males and two females) being euthanized for welfare reasons after the occurrence of continuous convulsions. It was not possible to determine if the adverse signs observed after the reduction of the high dose to 500 mg/kg/day were a result of the initial (1000 mg/kg/day) or subsequent (500 mg/kg/day) treatment, or both.

Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and, in the context of this study, Resorcinol diacetate showed no evidence of being an endocrine disruptor.
The no-observed adverse-effect level (NOAEL) of Resorcinol diacetate for systemic toxicity was considered to be 330 mg/kg/day and the no-observed adverse-effect level (NOAEL) of Resorcinol diacetate for reproductive/developmental toxicity was considered to be 1000/500 mg/kg/day.
Executive summary:

Summary

The purpose of this study was to assess the general systemic toxic potential of Resorcinol diacetate, including a screen for reproductive/developmental effects and assessment of endocrine disruptor relevant endpoints, when administered to Han Wistar rats by oral gavage administration for at least five weeks.

Three groups of ten male and ten female rats received Resorcinol diacetate, initially at doses of 100, 330 or 1000 mg/kg/day, byoral gavageadministration. From Day 5, Group 4 received Resorcinol diacetate at a dose level of 500 mg/kg/day due to four animals being sacrificed for welfare reasons on Day 4 of treatment. 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, corn oil, at the same dose volume as the treated groups.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food consumption, hematology (peripheral blood), blood chemistry, thyroid hormone analysis, estrous 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

Four animals (two males and two females) given 1000 mg/kg/day were killed for welfare reasons on Day 4 of treatment because of the occurrence of continuous convulsions. Macroscopically, findings were limited to depressions of the forestomach and dark coloration of the stomach corpus, which, in the males, correlated histologically with erosion/ulceration and inflammatory cell infiltrate in the mucosa. No significant histopathological findings were identified in the female decedents. The cause of death of these animals was undermined, but was considered likely to be related to treatment due to the occurrence in the high dose group.

Signs associated with dosing were principally recorded during the first week of treatment and consisted of tremoring, abnormal gait and nervousness in two females receiving 1000/500 mg/kg/day. Chin rubbing was recorded in all animals receiving 330 or 1000/500 mg/kg/day and piloerection was seen in one female receiving 330 mg/kg/day. At the routine weekly examination, one female receiving 1000/500 mg/kg/day was observed to have muscle tremors and abnormal gait.

Group mean body weight gain was low for males receiving 100, 330 or 1000/500 mg/kg/day during the first week of treatment, and before mating, the body weight gain of females receiving 100 mg/kg/day was slightly low but those receiving 330 or 500/1000 mg/kg/day gained marginally more weight than the controls. During gestation, the high dose females gained slightly less weight than the controls and during lactation weight gain was low in females receiving 330 or 1000/500 mg/kg/day.

Group mean food intake was marginally low in females receiving 1000/500 mg/kg/day during Days 4 to 12 of lactation.

Estrous cyclicity, pre-coital interval, gestation length, mating performance and fertility were unaffected by treatment. Gestation index was slightly low in females receiving 330 or 1000/500 mg/kg/day as one female in each group had no live litter and one control female was not pregnant.

 

The hematological examination of blood and the biochemical examination of plasma did not reveal any effect of treatment and there was no effect upon circulating levels of thyroxine (T4) in adult males.

 

The evaluation of organ weights performed after five weeks of treatment for males and on Day 14 of lactation for females revealed statistically significantly low absolute and body weight adjusted seminal vesicles weights in males given 1000/500 mg/kg/day. Prostate weights were low at the high dose level and Cowper’s glands and glans penis weights were marginally low in males at all dose levels. Thymus weights were statistically significantly low in females at all dose levels, but there was no dose-response.

The incidence and distribution of all macroscopic and histopathological findings were considered to be unrelated to treatment.

 

F1 responses

The clinical condition, litter size, sex ratio, survival indices and body weight gain of offspring were unaffected by parental treatment.

 

There was no effect of parental treatment upon circulating levels of thyroxine (T4) in offspring on Day 13 of age.

 

The ano-gential distances of offspring were unaffected by paternal treatment and no nipples were seen on any male offspring on Day 13 of age.

 

No macroscopic findings considered to be related to paternal treatment were recorded.

Conclusion

It was concluded that the oral administration of Resorcinol diacetate to parental Han Wistar rats at dose levels of 100, 330 or 500 mg/kg/day (with 1000 mg/kg/day administered as the high dose for the first four days of treatment) for two weeks before pairing, during pairing and then up to termination of the males after five weeks of treatment and females on Day 14 of lactation, was generally tolerated, however the administration of 1000 mg/kg/day was not tolerated, and resulted in four animals (two males and two females) being euthanized for welfare reasons after the occurrence of continuous convulsions. It was not possible to determine if the adverse signs observed after the reduction of the high dose to 500 mg/kg/day were a result of the initial (1000 mg/kg/day) or subsequent (500 mg/kg/day) treatment, or both.

 

Reproductive performance, fertility, litter size and offspring survival and growth were unaffected by parental treatment and,in the context of this study, Resorcinol diacetate showed no evidence of being an endocrine disruptor.   

The no-observed adverse-effect level (NOAEL) of Resorcinol diacetate for systemic toxicity was considered to be 330 mg/kg/day and the no-observed adverse-effect level (NOAEL) of Resorcinol diacetate for reproductive/developmental toxicity was considered to be 1000/500 mg/kg/day.